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International Symposium on Problems of Listeriosis

Ficha Técnica

LIVRODEACTASDOCONGRESSO: ISOPOLXVIIIntrenational Symposium on Problems of Listeriosis

Editor: Universidade Católica Portuguesa – Escola Superior de Biotecnologia

Coordenação e Revisão: Paula Teixeira

Design e Composição Gráfica: Kai Sprecher / Lynn Salt

Impressão: Orgal Impressores

Depósito Legal:

Tiragem: 500 exemplares

WELCOME TO ISOPOL XVII

On behalf of the Organising Committee, I am pleased to welcome you toISOPOL XVII (International Symposium On Problems Of Listeriosis) whichthis year is organized in Porto by the Universidade Católica Portuguesa –Escola Superior de Biotecnologia.

The ISOPOL meetings provide a unique opportunity for interdisciplinarydiscussions concerning various aspects of listeriosis including, but not beinglimited to, food safety and clinical aspects. Since the first edition of thissymposium in Giessen, Germany (1957), much has been discovered andmany more questions have been posed. For this reason the underlying mo-tives for bringing together the evolving ISOPOL community are still verystrong in 2010.

More than 350 delegates from the clinical, veterinary, and public healthareas as well as the food industry, will be present, representing ca. 45 coun-tries. This diversity will certainly provide for a very stimulating and pluralatmosphere which is sure to promote rich discussions and exchanges ofideas.

On this occasion, we would like to thank to the members of the ScientificCommittee for their time and effort in maintaining the high scientific levelof ISOPOL XVII.

We are also thankful to our sponsors, which, in many different ways, havegreatly contributed to the success of the organization of this event.

For the first time ever, ISOPOL will take place in Portugal, namely in Porto– the country’s second city and the capital of the north.

It is easy to see why Porto was designated a World Heritage Site by UN-ESCO in 1996. It is a living city, full of monuments and signs of its rich his-tory. It is best met in person; take the opportunity to stay awhile, to meetthe people, to enjoy the charms of this city of contrasts – you may find theneed to return again to enjoy it all!

Paula Cristina Maia Teixeira

TABLE OF CONTENTS

KEY NOTE SPEECH

Listeria monocytogenes: a multifaceted model 21Cossart, P.

AREA //A // Biology of Listeria monocytogenes

PLENARY LECTURES

A / PL/ 01 The pangenome of Listera spp. 22Chakraborty, T.

A / PL/ 02 Ecology of L. monocytogenes and Listeria spp. in natural 22and food associated environmentsWiedmann, M.

ORAL PRESENTATIONS

A / O/ 01 The cold shock associated proteins (Csps) promote tolerance 33of different environmental stresses and host cell invasionof Listeria monocytogenesTasara, T., Klumpp, J., Loessner, M. J. and Stephan, R.

A / O/ 02 Different levels of flagellin detected during growth of 33Listeria monocytogenes strains at low temperatureCabrita, P., Batista, S., Moes, S., Jenö, P., Trigo, M. J., Boavida Ferreira, R. and Brito, L.

A / O/ 03 Phenotypic and corresponding transcriptomic responses of 34Listeria monocytogenes strains in the presence ofunprotonated organic acidsLee Chang, K. J., Pinfold, T., Koshy, A. and Bowman, J. P.

A / O/ 04 Internalin profiling, multilocus sequence typing and 34virulence assesments suggest evolutionary history of theListeria monocytogenes-Listeria innocua cladeChen, J. and Fang, W.

A / O/ 05 The SOS response of Listeria monocytogenes is involved 35in stress resistance, mutagenesis, and biofilm formationvan der Veen, S. and Abee, T.

A / O/ 06 Clonal diversity of Listeria monocytogenes, a worldwide perspective 35Chenal-Francisque, V., Lopez, J., Cantinelli, T., Caro, V., Tran, C., Leclerq, A.,Lecuit, M. and Brisse, S.

A / O/ 07 Life without a cell wall: Listeria monocytogenes L-form cells 36feature a unique mode of divisionBriers, Y., Dell’Era, S., Schuppler, M. and Loessner, M. J.

A / O/ 08 Pangenomic analysis of Listeria monocytogenes 36Deng, X., Phillippy, A. M., Li, Z., Salzberg, S. L., Tortorello, M. L. and Zhang, W.

A / O/ 09 Evidence for an antiporter-independent glutamate decarboxylase 37(GAD) system in Listeria monocytogenes: Influence of growth mediaon GAD system activityKaratzas, K.-A., Brennan, O., Heavin, S. and O’Byrne, C. P.

A / O/ 10 Role of Listeria monocytogenes tyrosine phosphatases 37in conferring listeriophage resistancePaz, R.-N., Eugster, M. R., Zeiman, E., Loessner, M. J. and Calendar, R.

A / O/ 11 Thiolomics – the thiol: disulfide redox metabolism of Listeria monocytogenes 38Ondrusch, N., Gopal, S., Fuss, A., Hagen, N., Stoll, R., Aharonowitz, Y. and Kreft, J.

A / O/ 12 RNA-structures acting at a distance 38Johansson, J.

A / O/ 13 Deep RNA sequencing of Listeria monocytogenes reveals overlapping 39and extensive stationary phase and sigma B-dependent transcriptomes,including multiple highly transcribed noncoding RNAsOliver, H. F., Orsi, R. H., Ponnala, L., Keich, U., Wang, W., Sun, Q., Cartinhour, S.,Filiatrault, M. J., Wiedmann, M. and Boor, K. J.

POSTER PRESENTATIONS

A / P/ 00 Compartmentalization of IFN-gamma and IL-6 receptors signalling 62in Listeria monocytogenes phagosomes: immune vesiclesRamos-Vivas, J., Carrasco-Marin, E., Madrazo-Toca, F., Fernandez-Prieto, L.,Rodriguez-Del Rio, E., Carranza-Cereceda, C. and Alvarez-Dominguez, C.

A / P/ 01 Antimicrobial susceptibility among Listeria monocytogenes isolates 63from non human sources in France over a ten year periodGranier, S. A., Moubarek, C., Colaneri, C., Roussel, S., Courvalin, P. and Brisabois, A.

A / P/ 02 Five homologous small RNAs are involved in the response 63of Listeria monocytogenes to cell wall acting antibioticsKiil Nielsen, P. and Kallipolitis, B.

A / P/ 03 Phenotypic analysis of selected listerial secretion mutants 64Halbedel, S., Galander, S. and Flieger, A.

A / P/ 04 Distribution of serotypes and pulsotypes of L. monocytogenes 64in pig farms (France 2008)Boscher, E.

A / P/ 05 Comparative phylogenomics of Listeria monocytogenes reveals 65an adaptation profileSilveira Nalério, E., Padilha Silva, W., Stabler, R. and Wren, B. W.

A / P/ 06 Serotyping and PFGE patterns of Listeria monocytogenes isolated 65from poultry meatVasantrao Kurkure, N., Kalorey, D. R., Rodrigues, J., Gunjal, P.1 and Barbuddhe, S. B.

A / P/ 07 Genotypic characterization of Listeria monocytogenes isolated 66from fresh leafy vegetablesWarke, S., Kalorey, D. R., Umap, S., Sonegaonkar, A., Patil, V., Kurkure, N V. and Barbuddhe, S. B.

A / P/ 08 Comprehensive appraisal of the exoproteome of Listeria monocytogenes 66by genomic and proteomic analysesDesvaux, M., Dumas, E., Chafsey, I., Chambon, C. and Hébraud, M.

A / P/ 09 Investigating differences in lineages of Listeria monocytogenes 67using comparative genomicsMcIlwham, S., Farber, J. and Pagotto, F.

A / P/ 10 Autolysis in Listeria monocytogenes – a proteomic approach 67Pinto, E., Marques, N., Andrew, P. W. and Faleiro, M. L.

A / P/ 11 Role of flhA, cheR and motA in growth of Listeria monocytogenes 68at low temperatureMattila, M., Lindström, M., Somervuo, P. and Korkeala, H.

A / P/ 12 The effect of acetic acid (at pH 5.5) or benzoic acid (at neutral pH) on lipid 68composition and fluidity of Listeria monocytogenes membraneIoannis, D., Anita, B., Eleni, S. and Mastronicolis, S.

A / P/ 13 Elucidation of the responses to weak acids in the human pathogen 69Listeria monocytogenes using gene microarraysO’Byrne, C., Heavin, S. and Morrissey, J.

A / P/ 14 The immunogenic surface protein IspC acts as an N-Acetylglucosaminidase 69in Listeria monocytogenes serotype 4bRonholm, J.

A / P/ 15 Listeria monocytogenes EGD chitinolytic activity is regulated 70by carbohydrates but also by the virulence regulatory gene, PrfAHalberg Larsen, M., Leisner, J. J. and Ingmer, H.

A / P/ 16 Infectious dose curves for guinea pigs challenged with a Listeria 70monocytogenes epidemic clone strain and a strain carrying anaturally-occurring virulence-attenuating mutation in inlA show asignificant shift in median infectious doseNightingale, K., Van Stelten, A., Simpson, J. M., Chen, Y., Scott, V. N., Ross, W. H.,Whiting, R. C. and Wiedmann, M.

A / P/ 17 MudPIT based proteomic analysis of alkaline adapted, environmentally 71persistent Listeria monocytogenes strainsNilsson, R. E., Ross, T. and Bowman, J. P.

A / P/ 18 RpoN, the alternative sigma factor, is associated with the growth phase 71transition and pathogenesis in Listeria monocytogenesOkada, Y., Suzuki, H., Monden, S., Igimi, S. and Okada, N.

A / P/ 19 Cellular lipid fatty acid pattern differences between reference 72and ice-cream isolate of Listeria monocytogenes as response to cold stressAnita, B., Ioannis, D. and Mastronicolis, S.

A / P/ 20 Role of the dihydroxyacetone metabolism in the resistance 72of Listeria innocua to pediocinMilohanic, E.

A / P/ 21 Glucose transport system in Listeria monocytogenes 73and their impact on virulence gene expressionMoussan Ake, F.

A / P/ 22 Antimicrobial susceptibilities of Listeria monocytogenes isolated in Japan 73Monden, S., Okutani, A., Suzuki, H., Asakura, H., Nakama, A., Igimi, S., Okada, Y. and Maruyama, T.

A / P/ 23 Influence of sub-lethal concentrations of disinfectants 74on Listeria monocytogenes adhesion and invasion in Caco-2 cellsGaedt Kastbjerg, V., Halberg Larsen, M., Ingmer, H. and Gram, L.

A / P/ 24 The SOS response in Listeria monocytogenes – a stress survival mechanism 74Kiil Nielsen, P., Zahle Andersen, A. and Haahr Kallipolitis, B.

A / P/ 25 Acid shock triggers heavy metal detoxification in Listeria monocytogenes 75Müller, S., Neuhaus, K. and Scherer, S.

A / P/ 26 Listeria monocytogenes mutants defective in growth at 5 °C 75and in high salt environmentBurall, L., Laksanalamai, P. and Datta, A.

A / P/ 27 Revival of 5,000 Listeria strains from Seeliger’s historical collection 76with a semi-automated microbiological pipelineHaase, J., Hof, H. and Achtman, M.

A / P/ 28 Two point mutations are responsible for the lack of glycosidic substition 76in cell wall teichoic acids in Listeria monocytogenes serovar “7”Eugster, M. R., Huwiler, S., Morax, L. and Loessner, M. J.

A / P/ 29 High-throughput genome sequencing of two Listeria monocytogenes 77clinical isolates during a large foodborne outbreakGilmour, M., Graham, M., Van Domselaar, G., Tyler, S., Kent, H., Trout-Yakel, K.,Larios, O., Allen, V., Lee, B., Nadon, C. and Kearney, A.

A / P/ 30 Expression of antimicrobial activity in food 77and clinical Listeria monocytogenes isolatesBarbosa, J., Ferreira, V., Borges, S., Azevedo, I., Magalhães, R., Santos, I, Almeida, G. and Teixeira, P.

A / P/ 31 The Listeria monocytogenes sigma B and sigma H regulons overlap, 78but only sigma B appears to be important for survival of acid,alkaline and oxidative stressChaturongakul, S., Raengpradub, S., Wiedmann, M. and Boor, K. J.

A / P/ 32 Virulence gene expression in Listeria monocytogenes strains isolated 78from different sourcesAlessandria, V., Rantsiou, K. and Cocolin, L.

A / P/ 33 Differentiation of Listeria monocytogenes, Listeria innocua 79and Listeria marthii, a novel Listeria species isolatedfrom the natural environment, Finger Lakes National ForestGraves, L. M., Helsel, L. O., Steigerwalt, A. G., Morey, R. E., Daneshvar, M. I., Roof, S. E.,Orsi, R. H., Fortes, E. D., Milillo, S. R., den Bakker, H. C., Wiedmann, M.,Swaminathan, B. and Sauders, B. D.

A / P/ 34 Differed roles of L,D-carboxypeptidases encoded by lmo0028 79and lmo1638 genesYurov, D., Varfolomeev, A., Kaminskaya, A. and Ermolaeva, S.

A / P/ 35 Antimicrobial susceptibilities of Listeria monocytogenes isolated 80from retail beef, pork and poultry in JapanIda, M., Shimojima, Y., Kaneko, S., Higuchi, Y., Nakama, A. and Kai, A.

A / P/ 36 Genetic basis of two low pathogenic L. monocytogenes strains 80with apparent phospholipase C activityJiang, L., Bai, F., Chen, J., and Fang, W.

A / P/ 37 Molecular genotyping and antimicrobial resistance 81of Listeria monocytogenes from foods and the environmentParisi, A., Miccolupo, A., Fraccalvieri, R., Latorre, L., Normanno, G. and Santagada, G.

A / P/ 38 Virulence transcriptome analysis of Listeria monocytogenes 81by application of microarrays in vitro and in situRantsiou, K., Alessandria, V. and Cocolin, L.

A / P/ 39 Effects of growth conditions on surface properties of Listeria; 82a proposed role for AI-2Wong, H. T. L., Nwaiwu, O. and Rees, C. E. D.

A / P/ 40 Stress behaviour of a Listeria monocytogenes 568 Lmo1634 transposon mutant 82Truelstrup Hansen, L., Holman, D. B. and Ells, T. C.

A / P/ 41 Investigation of the conditions that trigger the activation 83of the alternative sigma factor σB in Listeria monocytogenesUtratna, M., Shaw, I. and O’Byrne, C.

A / P/ 42 Characterization of Listeria monocytogenes 1/2a, 1/2b, 1/2c and 4b 83by amplified fragment length polymorphism and evaluationof their geographical distributions in PortugalMaia, C. H., Goulão, M. M., Santos, M. I., Ferreira, M. A. S. S. and Pintado, C. M. B. S.

A / P/ 43 Global analysis of the Listeria monocytogenes surface proteins 84of the LPXTG familyBotello-Morte, L., Calvo, E., Mariscotti, J., D’Orazio, V., García-del Portillo, F. and Pucciarelli, M. G.

A / P/ 44 Antimicrobial susceptibility of Listeria monocytogenes strains 84derived from food and food-processing bakery plantEusébio, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G., Silva, J. and Teixeira, P.

A / P/ 45 A physiological study to purpose a new formal method 85to obtain L. monocytogenes cells adapted to BACSaá Ibusquiza, P., Cabo, M. L., Herrera, J. J. R., Vázquez, D., Carrera, S. and Eiriz, E.

A / P/ 46 Characterization of a RNA-helicase in the human pathogen 85Listeria monocytogenesNetterling, S. and Johansson, J.

A / P/ 47 Listeria monocytogenes agr system: Quorum sensing, or maybe not? 162Garmyn, D., Révelin, C. and Piveteau, P.

AREA //B // Listeria monocytogenes as a human and animal pathogen

PLENARY LECTURES

B / PL/ 03 How Listeria monocytogenes breaches host barriers 23Lecuit, M.

B / PL/ 04 Secretion of a novel L. monocytogenes cyclic dinucleotide 23into the cytosol of infected host cells activates an innate immune pathwayPortnoy, D. A.

B / PL/ 05 Diagnosis and clinical management of listeriosis in ruminants and camelids 24Poulsen, K. P.

B / PL/ 06 Listeriolysin S – a second haemolysin with a role 24in the virulence of Listeria monocytogenesHill, C.

ORAL PRESENTATIONS

B / O/ 14 Probiotics reduce Listeria monocytogenes-induced tissue invasion 40and stillbirths in pregnant guinea pigsSmith, M. A., Agyekum, K. and Williams, D.

B / O/ 15 Listeriolysin O favors Listeria monocytogenes growth 40in co-culture with the ciliate Tetrahymena pyriformisErmolaeva, S. and Pushkareva, V.

B / O/ 16 Atopy is a risk factor for listeriosis 41Kawamoto, K., Matsubara, S., Da Silva, M. and Makino, S.-I.

B / O/ 17 Cancer immunotherapy using novel Listeria monocytogenes-bacterial 41vectors to target the vasculature of progressive tumorsPaterson, Y., Seavey, M. M., Maciag, P. C. and Sewell, D.

B / O/ 18 The intracellular carbon metabolism of Listeria monocytogenes 42in comparison to that of other intracellular bacterial pathogensreplicating in mammalian host cellsGotz, A., Eylert, E., Stoll, R., Eisenreich, W., and Goebel, W.

B / O/ 19 LIMP2 links late phagosomal trafficking with the onset of the innate 42immune response to Listeria monocytogenes: a role in macrophage activationFernandez-Prieto, L., Carrasco-Marin, E., Madrazo-Toca, F., Rodriguez-Del Rio, E.,Carranza-Cereceda, C. and Alvarez-Dominguez, C.

B / O/ 20 The tetraspanin CD81 is required for entry of Listeria monocytogenes 43in mammalian cellsTham, T. N., Gouin, E., Rubinstein, E., Boucheix, C., Cossart, P. and Pizarro-Cerdá, J.

B / O/ 21 Listeria innate immune evasion by peptidoglycan modification 43Aubry, C.

B / O/ 22 Constitutive activation of the central virulence transcriptional regulator 44PrfA enhances Listeria monocytogenes pathogenesisbut reduces bacterial fitness outside of the hostFreitag, N. E. and Bruno, J. C.

B / O/ 23 The lvfH gene of Listeria monocytogenes encodes a novel 44virulence factor highly activated during infectionCarvalho, F., Camejo, A., Ferreira, P., Sousa, S. and Cabanes, D.


B / P/ 47 A Listeria monocytogenes strain is still virulent despite non-functional 86major virulence genes: Optical Mapping shows a potential mechanismRoche, S. M., Grépinet, O., Corde, Y., Teixeira, A. P., Kerouanton, A., Témoin, S.,Mereghetti, L., Brisabois, A. and Velge, P.

B / P/ 48 Protein expression of lineage I, II, and III Listeria monocytogenes 86strains in murine macrophagesDonaldson, J. R., Nanduri, B., Pittman, J. R., Burgess, S. C. and Lawrence, M. L.

B / P/ 49 Prevalence of L. monocytogenes in bovine mastitic milk samples: 87Possible source of food borne infectionKalorey, D. R., Warke, S., Kurkure, N. V. and Barbuddhe, S. B.

B / P/ 50 Agr-dependent peptide sensing in L. monocytogenes – Effects on 87biofilm formation, virulence and global gene expressionWaidmann, M. S., Monk, I. R., Auchter, M., Preising, N. P., Hill, C. and Riedel, C. U.

B / P/ 51 agrD-deletion affects InlA- and InlB-regulation via 88a temperature-dependent and -independent mechanismWaidmann, M. S., Monk, I. R. and Riedel, C. U.

B / P/ 52 Bovine cranial nerve Schwann cells express E-cadherin, 88a candidate key-player in the brainstem invasionof Listeria monocytogenes in cattleMadarame, H., Seuberlich, T., Vandevelde, M., Zurbriggen, A., and Oevermann, A.

B / P/ 53 Pathogenic potential of Listeria monocytogenes isolates 89from New Zealand seafood premises: implications for controlDurante Cruz, C. and Fletcher, G.

B / P/ 54 Copper homeostasis and virulence in Listeria monocytogenes 89David, C., Schuler, S., Glenn, S., Jen, C., Andrew, P. and Roberts, I. S.

B / P/ 55 Pattern of cytokine production during murine listeriosis 90Dussurget, O.

B / P/ 56 Analysis of the post-translocation chaperone PrsA2 and its unique 90role in facilitating Listeria monocytogenes pathogenesisAlonzo, F. and Freitag, N.

B / P/ 57 CtaP is a multifunctional cysteine-transport associated protein 91required for Listeria monocytogenes pathogenesisXayarath, B.

B / P/ 58 Enzymatic activity of the metalloprotease of Listeria is regulated by pH 91Forster, B. M., Pavinski Bitar, A., Slepkov, E. R. and Marquis, H.

B / P/ 59 Identification of propeptide residues regulating the compartmentalization, 92maturation, and activity of the broad-range phospholipase Cof Listeria monocytogenesSlepkov, E. R., Pavinski Bitar, A. and Marquis, H.

B / P/ 60 A mouse model of fetoplacental Listeria monocytogenes 92infection and abortionPoulsen, K. P., Faith, N., Laura Knoll, L. and Czuprynski, C.

B / P/ 61 Listeria infection of the insect model system Galleria mellonella 93Joyce, S. A. and Gahan, C. G.

B / P/ 62 Construction of a murinised Listeria monocytogenes H7858 (4b) 93strain for improved murine infectionCummins, J. and Gahan, C.

B / P/ 63 The role of a phosphoinositide phosphatase in the intracellular survival 94of Listeria monocytogenesWang, J., Corbett, D. and Roberts, I. S.

B / P/ 64 Virulence gene expression in Listeria monocytogenes strains isolated 94from different sourcesAlessandria, V.

B / P/ 65 Targeted Signature-tagged mutagenesis for phenotype screening 95of Listeria monocytogenes mutantsHenriques, A., Carvalho, F. and Cabanes, D.

B / P/ 66 Listeria monocytogenes cellular infection triggers 95tyrosine-phosphorylation of Myosin IIA, a new proteininvolved in invasionAlmeida, M. T., Cabanes, D. and Sousa, S.

B / P/ 67 Invasion profile of Listeria monocytogenes strains involved 96in invasive and gastroenteritis listeriosis outbreaksLaksanalamai, P., Sahu, S. and Datta, A.

B / P/ 68 Molecular characterization of the Vip-Gp96 interaction 96Martins, M., Cabanes, D. and Sousa, S.

B / P/ 69 Investigation of the molecular mechanisms by which 97Listeria monocytogenes grows in the mammalian gall bladderDowd, G., Joyce, S., Casey, P. G., Hill, C. and Gahan, C. G.

B / P/ 70 Role of cadmium efflux system in Listeria monocytogenes virulence 97Camejo, A. and Cabanes, D.

B / P/ 71 Investigation of chitinase as a potential virulence factor 98in Listeria monocytogenesChaudhuri, S.

B / P/ 72 Sub-lethal concentrations of common disinfectants do not 98influence survival and growth of Listeria monocytogenes in whole bloodHolch, A., Gaedt Kastbjerg, V. and Gram, L.

B / P/ 73 Internalin LRR domain variability in Listeria monocytogenes 99isolated from different hostsZaytseva, E. A., Ermolaeva, S. A. and Somov, G. P.

B / P/ 74 Reduced virulence of an adenylosuccinate lyase transposon mutant 99of a serotype 4b strain of Listeria monocytogenesFaith, N. G., Kim, J.-W., Kathariou, S., Sahaghian, R. and Luchansky, J. B.

B / P/ 75 Manifestations and outcome of listeriosis in adult patients 100Fernández Guerrero, M L., Mancebo Plaza, B., Torres, R., Górgolas, M. and Jusdado, J. J.

B / P/ 76 Model for human Listeriosis: in vivo monitoring of orally infected mice 100using bioluminescent Listeria monocytogenesBergmann, S., Lengeling, A., Pasche, B. and Schughart, K.

B / P/ 77 Galleria mellonella as model system to study Listeria species-specific 101and Listeria monocytogenes serotype-specific pathogenesisMraheil, M. A., Krishnendu, M., Hain, T. and Chakraborty, T.

B / P/ 78 Listeria monocytogenes ActA is a key player in evading autophagic recognition 101Pillich, H., Loose, M., Hain, T. and Chakraborty, T.

B / P/ 79 Non-haemolytic and hypovirulent Listeria monocytogenes became 102haemolytic and virulent after passage through miceSecic, I., Lindbäck, T. and Rørvik, L. M.

B / P/ 80 Mutants of Listeria monocytogenes (Lm) resistant to the polycationic 102peptide protamine appear to be attenuated for virulenceSchlech, W.

B / P/ 81 The role of plasmacytoid dendritic cells in the course 103of Listeria monocytogenes infectionSolodova, E., Lienenklaus, S., Jablonska, J. and Weiss, S.

B / P/ 182 Oxygen restriction increases the infection potential of 103Listeria monocytogenes – a transcriptional analysisAndersen, J. B., Bergstrøm, A., Knudsen, G., Bak Christensen, B., Ebersbach, T.,Boye, M. and Rask Licht, T.

AREA //C // Epidemiologic and clinical aspects of Listeria monocytogenesand listeriosis

PLENARY LECTURES

C / PL/ 07 Update on clinical aspects of listeriosis: What’s new? 25Schlech, W. F.

C / PL/ 08 A Canadian outbreak of listeriosis due to deli-meat: 25Driving change in the food safety systemFarber, J. M., Pagotto, F., Gilmour, M., Nadon, C., Savelli, C., and MacDonald, D.

C / PL/ 09 Listeria monocytogenes phagocytic strategy 26Alvarez-Dominguez, C.

ORAL PRESENTATIONS

C / O/ 24 Human listeriosis due to Listeria ivanovii 45Guillet, C., Join-Lambert, O., Le Monnier, A., Leclercq, A., Mamzer-Bruneel, M. F.,Bielecka, M. K., Scortti, M., Disson, O., Vazquez-Boland, J., Lortholary, O. and Lecuit, M.

C / O/ 25 Foodborne Listeriosis in India: An update 45Barbuddhe, S. B., Malik, S. V. S., Ashok Kumar, J., Kalorey, D. R., Kurkure, N. V.,Rawool, D. B., Swain, B. K., Korikanthimath, V. S., Chakraborty, T.

C / O/ 26 Human listeriosis and co-morbidities in England, 1999 to 2008: 46quantifying the riskMook, P., Grant, K., O’Brien, S. J. and Gillespie, I.

C / O/ 27 Risk factors for death in Listeria monocytogenes infection, 46England and Wales, 1990 to 2008Mook, P., Grant, K. and Gillespie, I.

C / O/ 28 A discrete event model to track Listeria monocytogenes 47in the retail environmentPouillot, R. and Gallagher, D.

C / O/ 29 Clinical and epidemiological aspects of listeriosis in Israel 47Hershko-Klement, A., Eliav, H., Valinsky, L., Schechner, V., Braun, E., Paitan, Y.,Block, C. S. and Nir-Paz, R.

C / O/ 30 Human isolates of Listeria monocytogenes during half a century in Sweden 48Lopez-Valladares, G., Danielsson-Tham, M.-L., Vishal Singh, P. and Tham, W.

C / O/ 31 Estimated incubation periods for listeriosis vary according 48to clinical form of diseaseGoulet, V., King, L., Vaillant, V. and De Valk, H.

C / O/ 32 Listeria monocytogenes in food and animals in the European Union in 2008 49da Silva Felício, M. T., Rizzi, V., Boelaert, F. and Makela, P.

C / O/ 33 Listeria monocytogenes infection in the over 60s in England 49between 2005 and 2008: a retrospective case-control study utilisingmarket research panel dataGillespie, I. A., Mook, P., Little, C. L. and Grant, K.

C / O/ 34 Better and faster typing, MLVA – shall we play together? 50Larsson, J. T., Roussel, S. and Moller Nielsen, E.

C / O/ 35 comK prophage junction fragments in Listeria monocytogenes contain 50SNPs that differentiate subclones of ECII and ECIII that are uniqueto individual meat and poultry processing plants in the U.S.Knabel, S. J.

C / O/ 36 Genetic diversity of Listeria monocytogenes measured by multiple-locus 51variable-number tandem repeat analysis (MLVA)Hyytia-Trees, E., Sabol, A., Graves, L. and Ribot, E.


C / P/ 82 Semi-automated repetitive sequenced-based PCR compared 104to pulsed field gel electrophoresis for Listeria monocytogenes sub-typingRoussel, S., Félix, B., Vignaud, M.-L., Tam Dao, T., Marault, M. and Brisabois, A.

C / P/ 83 Listeriosis: a frequent cause of fatal encephalitis in France 104with high case fatalityMailles, A., Vaillant, V., Lecuit, M. and Stahl, J.-P.

C / P/ 84 Listeria monocytogenes: identification and subtyping 105Favretti, M.

C / P/ 85 Virulotyping of Listeria monocytogenes by high resolution melt analysis 105Amar, C. F., Tamburro, M., Dear, P. and Grant, K.

C / P/ 86 Risk factors for nonperinatal listeriosis mortality in Los Angeles County, 106California, 1992–2004Guevara, R. E., Mascola, L. and Sorvillo, F.

C / P/ 87 Molecular typing of Listeria monocytogenes isolated from ovine sausage 106Nives, M. R., Mele, P., Parisi, A., Latorre, L., Virgilio, S. and Tola, S.

C / P/ 88 A novel phage-PCR assay for the rapid detection of viable 107Listeria monocytogenes in food within 24 hElemam, M. M.

C / P/ 89 Perinatal listeriosis in Los Angeles County, California, 1992–2004 107Guevara, R. E. and Mascola, L.

C / P/ 90 Antimicrobial resistance of Listeria monocytogenes human strains 108isolated since 1926 in FranceMorvan, C., Moubareck, A., Leclercq, M., Herve-Bazin, S., Bremont, M.,Lecuit, P. and Le Monnier Courvalin, A.

C / P/ 91 Today and tomorrow: The molecular epidemiology of listeriosis in the UK 108Grant, K., Amar, C., Matos, J., Mook, P., Little, C. and Gillespie, I.

C / P/ 92 Use of Fluorescent Amplified Fragment Length Polymorphism 109for improved typing of Listeria monocytogenesMatos, J., Amar, C., Desai, M., Cross, L. and Grant, K.

C / P/ 93 Towards an application for field samples of a multipathogen platform 109for molecular detection of raw milk pathogensOmiccioli, E, Amagliani, G., Brandi, G., Tonucci, F., Foglini, M. and Magnani, M.

C / P/ 94 Detection and recovery of Listeria species from stainless steel 110Boone, R., Iugovaz, I, Trottier, Y.-L. and Pagotto, F.

C / P/ 95 Natural carriage of Listeria in fresh-water fish in Russia 110Egorova, I., Voronin, M., Selyaninov, Y. and Kolbasov, D.

C / P/ 96 Prevalence of Listeria in wild fauna in Russia 111Egorova, I., Fertikov, V. and Kolbasov, D.

C / P/ 97 Quantitative assessment of the exposure to Listeria monocytogenes 111from soft-ripened cheese consumption in North America:a joint FDA/Health Canada projectGendel, S., Pouillot, R., Murray, C., Farber, J., Ross, W., Couture, H. and Jean, A.

C / P/ 98 Human listeriosis in England, 2001 – 2007: association with 112neighbourhood deprivationGillespie, I. A., Mook, P., Little, C. L., Grant, K. and McLauchlin, J.

C / P/ 99 Characterisation of antibodies for use in an antibody-based sensor 112for on-site detection of L. monocytogenesGilmartin, N., Hearty, S. and O’ Kennedy, R.

C / P/ 100 Food Investigation of sporadic cases of neuroinvasive listeriosis 113Goulet, V., Leclercq, A., Laurent, E., King, L., Dusch, V., Salem, S., Vaillant, V.,Chenal-Francisque, V., de Valk, H. and Pihier, N.

C / P/ 101 Evaluation of the antimicrobial activity of Vacciniummyrtillus, 113Prunus domestica and Myrtus communis against Listeria monocytogenesSerio, A., Di Pasquale, F. and Paparella, A.

C / P/ 102 High throughput quantitative detection of Listeria monocytogenes 114in food by RealTime PCRCammà, C., Ancora, M., Rizzi, V., Sperandii, A., Prencipe, V. and Migliorati, G.

C / P/ 103 Bibliographic study concerning procedures for preparing 114environmental samples for analyses, regarding to L. monocytogenesBarre, L., Carpentier, B. and Gnanou Besse, N.

C / P/ 104 Persistent L. monocytogenes isolates from Austrian and Irish dairies 115show the same phenotypic and genetic backgroundStessl, B.

C / P/ 105 Epidemiological data on listeriosis in Portugal: 2003 – 2008 115Almeida, G, Magalhães, R., Hogg, T. and Teixeira, P.

C / P/ 106 Diversity among Listeria monocytogenes isolated from humans 116Kalekar, S., Rodrigues, J., D’Costa, D., Malik, S. V. S., Kalorey, D. R.,Chakraborty, T. and Barbuddhe, S. B.

C / P/ 107 Characterization of Listeria isolated from seafood 116Rodrigues, J., Kalekar, S., Bhosle, S. N., Doijad, S. and Barbuddhe, S. B.

C / P/ 108 Characterization of Listeria species isolated from milk 117D’Costa, D., Bhosle, S. N., Dhuri, R. B., Kalekar, S., Rodrigues, J., Doijad, S. P. and Barbuddhe, S. B.

C / P/ 109 Prevalence of Listeria monocytogenes in chicken production chain 117in ThailandKanarat, S., Nijthavorn, N. and Sukhapesna, J.

C / P/ 110 Listeria monocytogenes in Ireland – Epidemiology and Molecular Typing 118Cormican, M. G., DeLappe, N., McKeown, P. and Garvey, P.

C / P/ 111 Characterization of Listeria monocytogenes isolated 118from human cases of listeriosis occurred in Portugal in 2008Magalhães, R., Barbosa, J., Santos, I., Almeida, G. and Teixeira, P.

C / P/ 112 Post-processing environmental contamination of surface-ripened 119soft cheese during affinageD’Amico, D. and Donnelly, C.

C / P/ 113 Genetic diversity of Listeria monocytogenes isolated 119from Portuguese cheesesAlmeida, G., Magalhães, R., Santos, I., Barbosa, J., Hogg, T. and Teixeira, P.

C / P/ 114 Prevalence of Listeria spp. in retail raw ground beef in Izmir, Turkey: 120A comparison of standard cultural method and Fluorescent in situHybridization (FISH) technique for detectionHandan Baysal, A.

C / P/ 115 Using ListexP100™ for Listeria monocytogenes detection in foods 120Flores Lopes, J., Ferreira Leite, I., Azeredo, J., Gibbs, P. and Teixeira, P.

C / P/ 116 Mapping of molecular profiles associated to Listeria monocytogenes 121food isolates circulating in ItalyDe Cesare, A., Parisi, A., Latorre, L. and Manfreda, G.

C / P/ 117 Zoonotic aspects of Listeria monocytogenes isolates 121from zebu dairy animalsParihar, V. S. , Barbuddhe, S. B., Kalorey, D. R., Kotwal, S.,Danielsson Tham, M.-L. and Tham, W.

C / P/ 118 Performance of ALOA and Palcam agars for detection of 122Listeria monocytogenes in naturally contaminated raw meat productsGravato Rowlands, R. E., Asturiano Ristori, C. A., Geraldes Martins, C.,Jakabi, M. and Gombossy de Melo Franco, B. D.

C / P/ 119 Comparison of MOPS-BLEB and Fraser as secondary enrichment broths 122for Listeria monocytogenesUpham, J., Huszczynski, G., Mosher, M., Borza, A., Dorey, M., Bosley, J., Hara, K.,Mutanda, C., Liu, J., Byrne, B. and Douey, D.

C / P/ 120 Comparison of UVM, Palcam and Oxoid Novel Enrichment (ONE) 123broth as primary enrichment broths for Listeria monocytogenesUpham, J., Mosher, M., Borza, A., Huszczynski, G., Dorey, M., Eloranta, K. and Douey, D.

C / P/ 121 Isolation and characterization of Listeria monocytogenes 123from asazuke (Japanese light pickles)Maklon, K., Kusumoto, A., Makino, S.-I. and Kawamoto, K.

C / P/ 122 Grouping of human Listeria monocytogenes isolates 124Lopez-Valladares, G., Danielsson-Tham, M.-L. and Tham, W.

C / P/ 123 Characterization of Listeria monocytogenes strains isolated 124from food and environmental samplesNucera, D. M., Lomonaco, S., Manila Bianchi, D., Decastelli, L., Grassi, M. A. and Civera, T.

C / P/ 124 Development of a multiplex SNP-typing method to identify 125the four epidemic clones of Listeria monocytogenesLomonaco, S., Civera, T., Dalmasso, A., Knabel, S. J. and Bottero, M. T.

C / P/ 125 Listeria monocytogenes incidents reported to the UK 125Food Standards Agency from 2000 to 2008Aish, J.

C / P/ 183 Genetic diversity of Listeria monocytogenes in broiler flocks 126Courtillon, C. Toquin, M.-T., Le Nôtre, Y., Fravalo, P. and Mansour Chemaly, M.

C / P/ 184 Tracing Listeria monocytogenes contaminations throughout 126the processing chain of a typical Italian pork meat productusing Pulsed Field Gel Electrophoresis (PFGE) characterizationAnnunziata Prencipe, V., Acciari, V., Torresi, M., Migliorati, G.,Marfoglia, C. and Valentina Rizzi, V.

C / P/ 185 Production and validation of capture-ELISA kit based on monoclonal 127antibodies specific for Listeria monocytogenes in foodstuffsPortanti, O., Di Febo, T., Luciani, M., Pompilii, C., Armillotta, G., Principe, V.,Lelli, R. and Semprini, P.

C / P/ 186 Production of a reference material for microbiological tests 127containing Listeria innocuaPomilio, F., Ricci, L, Di Giannatale, E., Semprini, P., Candeloro, L. and Migliorati, G.

AREA //D // Strategies for prevention and control of Listeria monocytogenes

PLENARY LECTURES

D / PL/ 10 Risk assessment using the microbiological criteria: arguments for 27zero tolerance (USDA) other viewpoint (Europe):Risk-based microbiological criteria for Listeria monocytogenesin RTE foodsLuber, P.

D / PL/ 11 USDA regulatory approach and considerations for the control 27of Listeria monocytogenesEngeljohn, D.

D / PL/ 12 Listeria monocytogenes, an emergent pathogen in Chile and Latin America 28Hormazábal, J. C.

ORAL PRESENTATIONS

D / O/ 37 Time Temperature Indicators can be used as an effective 52Risk Management Tool for Listeria monocytogenes in Ready-To-Eat foodsKoutsoumanis, K., Vaikousi, H. and Costas, B.

D / O/ 38 Safety of Salad Leaves and Herbs 52Garland, C. D. and Clark, A.

D / O/ 39 Environmental factors affect adhesion of Listeria monocytogenes 53to inert surfaces through flagellum expressionTresse, O.

D / O/ 40 Shelf-life laboratory durability and challenge studies for Listeria 53monocytogenes in ready-to-eat foods: a presentation of theEuropean technical guidance document intended for laboratoriesBeaufort, A., Bergis, H., Cornue, M. and Lardeux, A.-L.

D / O/ 41 Successful strategies against Listeria monocytogenes in Switzerland 54Imhof, R.

D / O/ 42 Absence of Listeria monocytogenes growth during raw milk cheesemaking: 54a modelling approachJordan, K., Schvartzman, S. Butler, F. and Tenenhaus-Aziza, F.

D / O/ 43 A predictive model to set high pressure processing criteria 55for Listeria monocytogenes inactivation on dry-cured hamBover-Cid, S., Belletti, N., Garriga, M. and Aymerich, T.

D / O/ 44 Application of a validated predictive model to prevent growth 55of Listeria monocytogenes in ready-to-eat foods – importancefor product development and risk managementMejlholm, O. and Dalgaard, P.

D / O/ 45 Application of predictive microbiology to control the growth 56of Listeria monocytogenes – dairy products as an exampleLobacz, A.

D / O/ 46 Characterization of Food Alert for Listeria monocytogenes in France in 2008 56Leclercq, A., Dusch, V., Salah, S., Laurent, E., Chenal-Francisque, V.,Thierry-Bled, F., Lecuit, M., Goulet, V. and Pihier, N.


D / P/ 126 Colonization of a newly constructed commercial chicken further 128processing plant with Listeria monocytogenesBerrang, M. E., Meinersmann, R., Frank, J. and Ladely, S.

D / P/ 127 Episcopic differential interference contrast/epifluorescence microscopy 128to characterise in situ Listeria monocytogenes biofilmson stainless steel surfacesGião, M. S. and Keevil, C. W.

D / P/ 128 Temperature dependent defect in biofilm formation 129by Listeria monocytogenesAbdalla, S., Glenn, S., Shama, G. and Andrew, P.

D / P/ 129 Mode of action of Lactococcus lactis sa31 antimicrobial peptide 129on Listeria monocytogenes ½ cBarile, M., Mormile, A., Ceres, C., Pepe, O., Cortesi, M. L. and Murru, N.

D / P/ 130 Tracing the source, epidemiology and persistence of Listeria monocytogenes 130in Irish Farmhouse cheese processing facilitiesJordan, K., Fox, E., O’Brien, M. and Hunt, K.

D / P/ 131 Phenotypic and genotypic characteristics of Listeria monocytogenes 130strains isolated from a convenience food-processing plantBlatter, S., Stephan, R., Tasara, T. and Zweifel, C.

D / P/ 132 Influence of flow direction on the adhesion of Listeria monocytogenes 131to brushed stainless steel surfacesSkovager, A., Whitehead, K., Ingmer, H., Verran, J. and Arneborg, N.

D / P/ 133 Occurrence of Listeria monocytogenes in raw milk and dairy products 131in Kazerun, IranMehdi Mahmoodi, S. M. and Javanmardi, F.

D / P/ 134 Antilisterial mode of action of bacteriocin ST182Gu produced 132by Enterococcus casseliflavus isolated from guavaTodorov, S., Destro, M. T., Chiarini, E. B., Vaz-Velho, M. and Franco, B. D. G. M.

D / P/ 135 Control of Listeria monocytogenes in fresh goat cheese by bacteriocinogenic 132strain Lactococcus lactis subsp. lactis DF4Mi or commercial nisinNader Furtado, D., Todorov, S., Landgraf, M., Destro, M. T. and Franco, B. D. G. M.

D / P/ 136 High pressure processing ensures elimination of Listeria monocytogenes 133in sliced dry cured hamStollewerk, K., Jofré, A., Comaposada, J., Aymerich, T., Ferrini, G., Arnau, J. and Garriga, M.

D / P/ 137 Inhibition of Listeria monocytogenes by Lactococcus sp. EU2241 133in tropical shrimpAbdoulaye Fall, P.

D / P/ 138 Listeria monocytogenes and Listeria innocua in slaughter line of 134a swine meatpacking plant in Rio Grande do Sul State, BrazilSchittler, L. and Padilha Silva, W.

D / P/ 139 Listeria monocytogenes in raw meat products marketed in the city 134of Sao Paulo, Brazil: Incidence and counts data for risk assessmentRistori, C. A., Rowlands, R. E. G., Martins, C. G., Fávero, L. M. and Franco, B. D. G. M.

D / P/ 140 Inhibiton of Listeria monocytogenes by Carnobacterium maltaromaticum 135in combination with extract of Lippia sidoides Cham.in cold-smoked surubim fish brothBarbosa dos Reis, F., de Souza, V. M., Sousa Thomaz, M. R., Pinto Fernandes, L.,Pereira de Oliveira, W. and Pereira De Martinis, E. C.

D / P/ 141 Inhibition of Listeria monocytogenes in cooked ham 135by virulent bacteriophages and protective culturesHolck, A., Schirmer, B. C. and Berg, J.

D / P/ 142 Surface colonization by Listeria monocytogenes: role of the flagella 136in the biofilm formation processDesvaux, M., Briandet, R., Renier, S., Deschamps, J., NCaccia, N., Chafsey, I. and Hébraud, M.

D / P/ 143 The role of sanitizers in controlling Listeria monocytogenes 136on stainless steel surfaces: Lessons learnedfrom the 2008 Listeriosis outbreakHébert, K., Farber, J. and Pagotto, F.

D / P/ 144 Listeriophage ecology and diversity on dairy farms 137Vongkamjan, K., Moreno Switt, A., den Bakker, H. C., Fortes, E. D., and Wiedmann, M.

D / P/ 145 Evaluation of curative and preventive decontamination treatments 137on Listeria monocytogenes biofilms with a new screening systemQuinon, E., Chamot, S., Groelly, J., Chavant, P., Bernardi, T., Desvaux, M. and Hebraud, M.

D / P/ 146 Pulsed Field Gel Electrophoresis, conventional and molecular 138serotyping on Listeria monocytogenes: An European ProficiencyTesting Inter-laboratory TrialFélix, B., Roussel, S., Tam Dao, T., Asséré, A., Lombard, B. and Brisabois, A.

D / P/ 147 Detection of Listeria spp. in raw and pasteurized liquid egg-products 138and in the egg-breaking plants environmentRivoal, K., Fablet, A., Chemaly, M., Salvat, G. and Protais, J.

D / P/ 148 Biofilm formation by Listeria monocytogenes isolates under conditions 139that mimic food and digestive tractKretli Winkelströter, L., Oliveira, M. A. and De Martinis, E. C.

D / P/ 149 Biofilm formation of Listeria monocytogenes EGDe depends 139on temperature and nutrient availabilityAuchter, M., Endres, J., Waidmann, M. S. and Riedel, C. U.

D / P/ 150 Antimicrobial activity of lactococcal and enterococcal strains 140isolated from artisanal products from North West of Italytoward Listeria monocytogenesDal Bello, B., Rantsiou, K., Ambrosoli, R., Zeppa, G. and Cocolin, L.

D / P/ 151 Plant-based strategies for Listeria monocytogenes control in foods 140Paparella, A., Serio, A., Chaves-Lopez, C. and Di Pasquale, F.

D / P/ 152 Scientific studies for survival of Listeria monocytogenes 141in dairy products and its application in practiceCabanova, L., Škuntova, O. and Kantikova, M.

D / P/ 153 The effect of chilling temperatures on the virulence 141of Listeria monocytogenes isolates with different originsNeves, E. M., Silva, A. C., Louro, P., Ferreira-Dias, S. and Brito, L.

D / P/ 154 How to improve a sampling plan in order to better assess 142L. monocytogenes contamination on diced bacon at the plantBergis, H., Commeau, N., Zuliani, V., Cornu, M., Beaufort, A. and Garry, P.

D / P/ 155 Contamination of Listeria monocytogenes in a cold-smoked pork 142processing plant using brining injectionsBerzins, A., Silins, I. and Korkeala, H.

D / P/ 156 Effects of GRAS products on growth of Listeria monocytogenes 143during cold storage of salmon filletsMcCarthy, S. and Johnson, D.

D / P/ 157 Heavy-metal and detergent resistance of Listeria species isolates 143from milk processing environmentsDoijad, S., Garg, S. and Barbuddhe, S. B.

D / P/ 158 Detection of Listeria monocytogenes in lettuce sold at markets 144and supermarkets in Porto, PortugalNoronha, L., Silva, J. and Teixeira, P.

D / P/ 159 Preliminary analysis of structure and chemical composition of 144extracellular polymeric substance produced by Listeria monocytogenesNwaiwu, O., Lad, M., Davis, A., Foster, T. and Rees, C.

D / P/ 160 Ecology and persistence of Listeria monocytogenes strains in fermented 145meat sausage processors from the Northern region of PortugalFerreira, V., Barbosa, J., Vongkamjan, K., Moreno Switt, A., Hogg, T., Gibbs, P.,Wiedmann, M. and Teixeira, P.

D / P/ 161 Biofilm formation and survival of L. monocytogenes and slaughter house 145bacteria on surfaces at relevant environmental conditionsLangsrud, S., Møretrø, T. and Heir, E.

D / P/ 162 Control of L. monocytogenes by lysozyme combined with olive leaf extract 146in edible pullulan film coated on chicken breast filletsHandan Baysal, A.

D / P/ 163 Evaluation of antilisterial activity by lactic acid bacteria 146Borges, S., Barbosa, J., Albano, H., Silva, J. and Teixeira, P.

D / P/ 164 Molecular methods to assess Listeria monocytogenes route 147of contamination in a dairy processing plantCocolin, L., Alessandria, V., Dolci, P. and Rantsiou, K.

D / P/ 165 Persistence of L. monocytogenes in artisanal cheese producing plants 147Almeida, G., Santos, I., Magalhães, R., Barbosa, J., Hogg, T. and Teixeira, P.

D / P/ 166 Occurrence of Listeria monocytogenes in food products collected 148in Portugal from retail establishments and food plantsMena, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G. and Teixeira, P.

D / P/ 167 Listeria monocytogenes biofilms grown at 12 °C showed reduced 148susceptibility to sanitizersAfonso Lourenço, A., Machado, H. and Brito, L.

D / P/ 168 Modelling growth of Listeria monocytogenes in cheese as function 149of environmental variablesSand Rosshaug, P. and Hallberg Larsen, M.

D / P/ 169 Characterization of anti-Listerial bacteriocin produced by 149Lactobacillus plantarum ST8SH, a strain isolated from Bulgarian salamiTodorov, S. D. and Lemos Vaz-Velho, M.

D / P/ 170 EFSA’s proposal for an EU-wide retail survey on Listeria monocytogenes 150in selected categories of ready-to-eat food productsFrank Helena Boelaert, F. H., Felício, T. and Makela, P.

D / P/ 171 Ripening conditions: an asset to control L. monocytognes in cheeses 150Callon, C., Picque, D., Corrieu, G. and Montel, M.-C.

D / P/ 172 Deterministic and stochastic behavior of Listeria monocytogenes 151suspended cells or detached from stainless steel surfacesduring cheese manufacturingBelessi, C.-E. A., Gounadaki, A. S., Arapakh, S., Schvartzman, S., Jordan, K. and Skandamis, P. N.

D / P/ 173 Prevalence of Listeria monocytogenes in game meat 151Atanassova, V.

D / P/ 174 Relationship between pathogenic profile and in vitro biofilm formation 152capacity of Listeria monocytogenes strains isolated from meat,fish and processing plantsMeloni, D., Mazza, R., Marceddu, M., Piras, F., Mureddu, A. and Mazzette, R.

D / P/ 175 Prevalence and molecular characterization of Listeria monocytogenes 152in traditional fermented pork sausages produced in ItalyMazzette, R., Meloni, D., Busia, G., Melillo, R., Mureddu, A. and Piras, F.

D / P/ 176 Minimum Biofilm Eradication Concentration (MBEC) of different 153antimicrobials on Listeria monocytogenes and Salmonella enterica biofilmsRodrigues, D., Teixeira, P., Oliveira, R., Ceri, H. and Azeredo, J.

D / P/ 177 Examination of the ability of adherence, biofilm formation and sensitivity 153to some disinfectants of different Listeria monocytogenes strainsMilanov, D., Vidić, B., Petrović, J., Bugarski, D. and Ašanin, R.

D / P/ 187 Evolution of Listeria monocytogenes contamination in poultry production: 154from the farms to the processing levelsMansour Chemaly, M., Toquin, M.-T., Courtillon, C., Le Nôtre, Y., Rivoal, K. and Fravalo, P.

D / P/ 188 A regular survey of Listeria in ready-to-eat foods (2004 – 2009) 154Furtado, R., Loreto Campos, M., Correia, C., Ferreira, I., Maia, C., Rosa, N., Santos, S.,Santos, M. I. and Saraiva, M.

D / P/ 189 Incidence of Listeria monocytogenes in Queijo Fresco 155Rosa, N., Campos, L., Correia, C., Ferreira, I., Furtado, R., Maia, C., Santos, S.,Cunha, C. I. and Santos, M. I.

D / P/ 190 Risk factors for Listeria monocytogenes contamination 155in French broiler flocksAury, K., Le Bouquin, S., Toquin, M.-T., Petetin, I., Le Nôtre, Y., Allain, V.,Fravalo, P. and Mansour Chemaly, M.

D / P/ 191 Portuguese sushi: is it contaminated with L. monocytogenes? 156Mendes, D., Furtado, R., Maia, C., Correia, C., Campos Cunha, I., Pedroso, L. and Santos, M. I.

D / P/ 192 Effect of the inoculum size on growth of L. monocytogenes 156in dices of poultry breastLardeux, A.-L., Gnanou-Besse, N., Doux, C. and de Courseulles, E.

D / P/ 193 Investigation into the mechanisms of detergent induced changes 157in disinfectant susceptibility of attached Listeria monocytogenesWalton, J., Hayes, R., Protheroe, R., Hill, D. and Gibson, H.

D / P/ 194 Prevalence of Listeria monocytogenes throughout the production process 157of Parma ham: tracing contaminations from slaughterhousesto the final productPrencipe, V. A., Rizzi, V., Iannetti, L., Serraino, A., Calderone, D., Rossi, A., Morelli, D.,Marino, L. and Migliorati, G.

D / P/ 195 Prevalence of Listeria monocytogenes in raw milk sold at vending machines 158in Abruzzo regionPrencipe, V. A., Scattolini, S., Sperandii, A. F. and Migliorati, G.

D / P/ 196 Characterization of Listeria monocytogenes strains isolated from soft 158and semi soft cheeses sampled at retail levelAcciari, V., Torresi, M., Migliorati, G., Di Giannatale, E., Semprini, P. and Prencipe, V.

D / P/ 197 Preliminary report on the organisation of a food microbiology 159proficiency testing program as a tool to guarantee the equivalenceof the US and IT official control systemsDi Giannatale, E., Marfoglia, C., Prencipe, V., Salini, R., Migliorati, G. and Ricci, L.

D / P/ 198 High nisin susceptibility of Listeria spp. wild-type strains isolated 159from dairies with traditional cheese preservation in PortugalPintado, C. M. B. S and Ferreira M. A. S. S.

D / P/ 199 Utilization of Lactococcus lactis M104, a wild nisin-producing 163raw milk isolate, as an antilisterial adjunct in traditionalGreek Graviera cheese processingSamelis, I., Pappa, E., Bogovic-Matijasic, B. and Rogelj, I.

D / P/ 200 The European Project BASELINE “Selection and improving 163of fit-for-purpose sampling procedures for specific foods and risks”Manfreda, G. and De Cesare, A.

AREA //E // Communication, risk perception and consumer practices –Social sciences in Listeria control

PLENARY LECTURES

E / PL/ 13 How can the social sciences help us understand the prevalence of 29listeriosis in the UK?Wadge, A.

E / PL/ 14 Consumer perceptions, behaviour and microbial food safety; 29implications for Listeria controlFrewer, L. J.

ORAL PRESENTATIONS

E / O/ 47 Efforts to update and perform health education on listeriosis in 57Los Angeles County, California, by the County of Los Angeles Departmentof Public HealthGuevara, R. E.

E / O/ 48 Awareness of listeriosis among Portuguese pregnant women 57Mateus, T., Maia, R. L. and Teixeira, P.

E / O/ 49 The development and progress of a risk-based strategy 58for the control of Listeria monocytogenes in New ZealandCastle, M. and Crerar, S.


E / P/ 178 What is an appropriate level of protection for Listeria monocytogenes 160in foodstuffs consumed by vulnerable groups?Little, C., Gillespie, I., Grant, K., Gormley, F., Mook, P. and McLauchlin, J.

E / P/ 179 ILCD: An Interactive Listeria culture diversity knowledgebase 160Ashok Kumar, J., Barbuddhe, S. B., Kalekar, S., Rodrigues, J., Chopade, N. A.,Hain, T. and Chakraborty, T.

E / P/ 180 Listeria spp. and the domestic environment: consumer knowledge, 161attitudes, risk perceptions and food-handling behavioursRedmond, E. C.

E / P/ 181 Do you know the temperature in your refrigerator? 161Røssvoll, E., Jacobsen, E., Ueland, Ø., Einar Granum, P. and Langsrud, S.

Authors Index 165

PLENARY LECTURES

KEY NOTE SPEACH

Listeria monocytogenes: a multifaceted modelCossart, P.*

Institut Pasteur, Unité des Interactions Bactéries cellules, Inserm U604, INRA USC2020

Listeria monocytogenes is an intracellular pathogen responsible for severe humanfood-borne infections characterized by gastroenteritis, materno-fetal infectionsand brain infections with a mortality rate of 30%. The disease is mainly due the ca-pacity of Listeria to cross three host barriers: the intestinal barrier, the materno-fetal barrier and the blood brain barrier. It is also due to the capacity of the or-ganism to survive and replicate in macrophages and to enter and replicate in nonphagocytic cells. We use a combination of approaches to understand the mecha-nisms which allow establishment andmaintenance of a Listeria infection. We theninvestigate if our findings have a general significance. In nearly three decades ofmolecular and cellular investigations, the study of Listeria momocytogenes and oflisteriosis has led to new concepts in infection biology and in several other areas ofbiology including cell biology, microbiology, molecular medicine and genomics.

* Plenary Supported by FCT

21

AREA //A // Biology of Listeria monocytogenesPLENARY LECTURES // PL / 1–2

The pangenome of Listera spp.Chakraborty, T.*Institute of Medical Microbiology, Justus-Liebig University of Giessen, Germany

Listeria monocytogenes is a food-borne pathogen with a high mortality rate thathas served as an invaluable model for intracellular parasitism. We have sequencedrepresentative genomes comprising all species for the genus Listeria as well asstrains representing clonal lineages of the pathogenic species of L. monocytogenes.Comparative genome analysis now provides clear evidence indicating that the var-ious non-pathogenic species of Listeria have been derived by gene loss and/or mu-tational decay of virulence- and niche-adaptive factors from a progenitor strainthat harboured many of the currently known virulence factors. Thus, non-patho-genic Listeria are compromised with regard to their ability to live in the cytoplasmof infected host cells but have acquired genes enabling their growth in soil and de-caying vegetation. Comparative transcriptome analysis of intracellular growth hasalso uncovered additional levels of adaptive evolution in growth among the dif-ferent lineages of L. monocytogenes. Analysis of the pan-genome of L. monocyto-genes strains revealed an extensive, as yet unexplained gene-repertoire in thesegenomes, and provides evidence for the evolution of these strains by gatheringgenes from organisms in different environmental and host niches.


Ecology of L. monocytogenes and Listeria spp. in naturaland food associated environmentsWiedmann, M.*Department of Food Science, Cornell University, Ithaca, NY, USA

Listeria spp., including the pathogen L. monocytogenes, can be isolated from a va-riety of environments, often at considerable frequency. A number of our studieshave specifically shown that Listeria spp. can often be isolated from > 20% of sam-ples collected from natural, urban, and farm environments and can also be com-monly isolates from food associated environments (e.g., processing plants, retailenvironments). Molecular characterization and subtyping tools not only typicallyreveal considerable diversity among Listeria spp. isolates from different environ-ments, but also provide evidence for (i) association between certain subtypes orspecies and specific environments (suggesting existence of specific Listeria eco-types) as well as (ii) long-term persistence of specific Listeria strains in differentenvironments. For example, in one of our studies, L. seeligeri and L. welshimeriwere significantly associated with natural environments (p<0.0001), while L. in-nocua and L. monocytogenes were significantly associated with urban environ-ments (p<0.0001). Increased efforts to characterize Listeria isolates from varioussources have also lead to discovery of novel Listeria spp. (e.g., L. marthii, L. ro-courtii) and have revealed a number of atypical strains within well-known species(e.g., multiple lineages of hemolytic L. innocua strains). Characterization of Liste-ria isolates from various environments also suggests multiple transitions frompathogenic clades to virulence attenuated clades, including a number of distinctclades which contain homologues for some but not all of the genes critical for vir-ulence in L. monocytogenes. The role of these virulence genes in apparently non-pathogenic Listeria strains still remain to be defined though. Persistence of Liste-ria for up to 10 years has been documented in different food associatedenvironments. Strain persistence in these environments appears to be an impor-tant contributor to food contamination and foodborne listeriosis cases. Identifi-cation of persistent strain and their elimination in food associated environmentsthus has become a critical component in efforts to reduce human listeriosis cases.

* Plenary Supported by FLAD

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AREA //B // Listeria monocytogenes as a human and animal pathogenPLENARY LECTURES // PL / 3–6

How Listeria monocytogenes breaches host barriersLecuit, M.*Institut Pasteur, Inserm, Paris, France

Listeria monocytogenes (Lm) is a human foodborne pathogen that causes listerio-sis, a systemic infection leading to meningitis, encephalitis and feto-placental in-fection. To reach its target organs, Lm crosses the intestinal, blood-brain and pla-cental barriers. We will present the results of our investigations regarding themolecular mechanisms underlying Lm crossing of these three barriers.


Secretion of a novel L. monocytogenes cyclic dinucleotide into thecytosol of infected host cells activates an innate immune pathwayPortnoy, D. A.*Department of Molecular and Cell Biology and The School of Public Health, University of California,Berkeley, USA

Listeria monocytogenes has been used for decades as a model to study basic as-pects of intracellular parasitism and cell-mediated immunity. Because L. monocy-togenes induces a robust CD8+ T-cell response, attenuated strains of L. monocy-togenes are being developed as live, attenuated vaccine vectors for infectiousdisease and malignancies. What makes L. monocytogenes such a strong inducer ofcellular immunity? As everyone in this audience appreciates, virulent strains ofL. monocytogenes secrete a pore-forming cytolysin (LLO) that allows bacteria ac-cess the host cell cytosol. We previously discovered that vacuolar and cytosolicbacteria induced distinct host transcriptional responses, the latter leading to theexpression of beta interferon and a host of co-regulated genes. To understand themicrobial components that activate the cytosolic pathway, we used a forward ge-netic screen to identify bacterial mutants that induced an enhanced or diminishedhost transcriptional response to cytosolic bacteria. Most of the mutants identi-fied mapped to genes encoding or regulating multidrug efflux pumps. We now havea series of strains that induce beta interferon over a 60-fold range. These datawere consistent with a model in which a small molecula is either actively or inad-vertently being pumped from cells, and that the host can recognize and respond.Using conventional biochemistry and mass spectrometry, we have identified theL. monocytogenes molecule as a cyclic dinucleotide. The precise structure of thisnovel bacterial molecule, identification of the cyclase and phosphodiesterase willbe presented at the meeting.


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AREA //B // Listeria monocytogenes as a human and animal pathogenPLENARY LECTURES // PL / 3–6

Diagnosis and clinical management of listeriosisin ruminants and camelidsPoulsen, K. P.*School of Veterinary Medicine, University of Wisconsin, Madison, USA

Listeria monocytogenes is a successful intracellular pathogen of domestic and foodproducing animals. Ruminant (cattle, sheep, goats) and camelid (llama and alpaca)species are constantly exposed to this gram-positive pathogen. It is ubiquitous inthe environment and tends to overgrow in poorly prepared silage (pH>5.0). Lis-teriosis manifests in these species as three distinct clinical syndromes includingabortion, neonatal sepsis, andmeningioencephalitis (circling disease). Infection infood producing animals, and subsequent shedding of L. monocytogenes in milk andmeat represents a significant risk to food safety. In this review, clinical signs, di-agnostics, and treatment of listeriosis of ruminant and camelid species will be pre-sented including video clips of clinically affected animals.


Listeriolysin S – a second haemolysin with a role in the virulenceof Listeria monocytogenesHill, C.*Alimentary Pharmabiotic Centre and Microbiology Department, University College Cork, Ireland

Listeria monocytogenes require listeriolysin O (encoded by llo or hlyA) to escapethe vacuole and initiate intracellular growth and intercellular spread. Llo- mu-tants are essentially avirulent and so listeriolysin O is regarded as the primary vir-ulence factor in L. monocytogenes. Given that all strains and serotypes possess thisvirulence factor, it is unlikely to explain the increased incidence of particularserotypes (such as 4b) in listeriosis epidemics. Many laboratories have sought tosolve the Listeria conundrum of why certain strains may be more likely to causedisease in humans. We have identified a second haemolysin, which we have namedlisteriolysin S, which is associated with epidemic strains. Listeriolysin S is verydifferent from listeriolysin O in that it is a highly modified peptide, which resem-bles (in probable structure if not in primary sequence) the streptococcal virulencefactor, streptolysin S. In fact, a family of these modified virulence peptides can beinferred from genomic data of other pathogens, including Staphylococcus aureusand Clostridium botulinum. listeriolysin S is not expressed under laboratory con-ditions, but can be induced with a number of reagents in vitro. In addition, a mu-tant in which listeriolysin S is constitutively expressed is hyper-hemolytic in com-parison to wildtype strains. We have also demonstrated a small, but significant,role for listeriolysin O in murine models of infection and in polymorphonuclearleucocyte survival assays. Listeriolysin S provides a possible explanation for theenhanced virulence of certain strains in human listeriosis, but much remains to bedone to confirm or refute this hypothesis.

* Plenary Supported by FEMS

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AREA //C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosisPLENARY LECTURES // PL / 7–9

Update on clinical aspects of listeriosis: What’s new?Schlech, W. F.*Dalhousie University, Canada

Listeriosis remains a challenging problem for clinicians. Diagnosis may be delayedand treatments prolonged although decreases in mortality have been noted insome surveillance programs. New syndromes, such as necrotizing fasciitis, havebeen described as well as further outbreaks of febrile gastroenteritis. Sepsis andrhomboencephalitis remain the most common presenting syndromes for invasivelisteriosis. Genetic markers for infection have also been uncovered. Infection inthe immunocompetent host still occurs but risk factors for the disease primarilypoints to abnormalities in cell-mediated and innate immunity as major predispo-sitions to listeriosis. Use of TNF-alpha inhibitors and corticosteroids are of par-ticular concern. Some newer antibiotics active against L. monocytogenes have beenstudied but ampicillin and gentamicin remains the treatment of choice. There isfurther evidence that trimethoprim-sulfamethoxazole has a strong protective ef-fect against listeriosis in the compromised host receiving this drug for protectionagainst other pathogens.


A Canadian outbreak of listeriosis due to deli-meat:Driving change in the food safety systemFarber, J. M.1*, Pagotto, F.1, Gilmour, M.2, Nadon, C.2, Savelli, C.3, and MacDonald, D.3

1. Food Directorate, Health Canada2. National Microbiology Laboratory, Public Health Agency Canada3. Centre for Food-borne, Environmental and Zoonotic Infectious Diseases,Public Health Agency Canada

In 2008, Canada experienced the first multi-provincial and largest outbreak of in-vasive listeriosis. During this outbreak, 57 cases of illness, in 7 provinces, resultedin 23 deaths from listeriosis, which was traced back to contaminated deli-meatfrom Company A. The age range was 29 to 98 years, with the median age being 78.All of the cases with a known medical history prior to exposure had underlyingmedical conditions. Fifty (88%) cases reported deli-meat consumption. Addition-ally, 84% of cases had institutional exposure. Investigators confirmed Company Adeli meat was served to 27 cases. The major probable cause of the Listeria contami-nation in Company A’s plant was related to meat slicing equipment. High-through-put genome sequencing of two serotype 1/2a Listeria monocytogenes isolates wascompleted during the outbreak. By screening for genetic traits specific to the out-break genomes, examination of clinical, environmental and food isolates associatedwith the outbreak revealed that three distinct, but highly-related strains may havebeen involved in this nationwide outbreak. As a result of the outbreak, the PublicHealth Agency of Canada (PHAC), the Canadian Food Inspection Agency (CFIA)and Health Canada (HC) conducted independent lessons-learned exercises. In ad-dition, the CFIA developedmandatory newDirectives for federally-registeredmeatand poultry plants, and Health Canada started updating their policy on Listeriamonocytogenes in RTE foods. Furthermore, a federal review by an independent in-vestigator (Ms. Sheila Weatherill) into the outbreak resulted in 57 recommenda-tions directed at food processors, regulators, public health professionals and indi-vidual consumers. Work is currently underway at PHAC, HC and CFIA to directlyaddress the recommendations coming out of theWeatherill report.


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AREA //C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosisPLENARY LECTURES // PL / 7–9

Listeria monocytogenes phagocytic strategyAlvarez-Dominguez, C.*Hospital Santa Cruz de Liencres-IFIMAV and IES Zapaton, Spain

Listeria monocytogenes enters macrophages and resides for a short period withinthe phagosomal compartment before escaping and replicating in the cytosol. Lis-teria has evolved a fine phagosomal strategy to survive the microbicidal machi-nery of macrophages and avoid eliciting a strong innate immune response. First,Listeria-GAPDH enzymatic modification and inactivation of Rab5a caused a delayon phagosome maturation. Second, Listeria interferes with the trafficking of twolysosomal proteins, cathepsin-D and LIMP2. Cathepsin-D enzymatic action blocksthe pore-forming function of Listeria-LLO within the phagosomes. Therefore, in-hibiting the transport of this lysosomal protease to the phagosomes increases Lis-teria viability. Finally, the Listeria interference with LIMP2 trafficking disruptsthe connection between late endosomal events and the onset of innate immunity.In brief, Listeria phagosomal strategy has the purpose to avoid the phagosomaltransformation into fully competent bactericidal and antigen-processing com-partments. Deciphering Listeria phagosomal strategy to subvert the host immuneresponse might help to design better therapies against listeriosis.


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AREA //D // Strategies for prevention and control of Listeria monocytogenesPLENARY LECTURES // PL / 10–12

Risk assessment using the microbiological criteria:Arguments for zero tolerance (USDA) other viewpoint (Europe):Risk-based microbiological criteria for Listeria monocytogenesin RTE foodsLuber, P.*Federal Office of Consumer Protection and Food Safety, Berlin, Germany

The control of Listeria monocytogenes in foods has been a focus activity ever sincethe link between listeriosis in vulnerable populations and exposure via foods be-came clear. Listeria spp. originate from soil and can contaminate many differentfoods during food production or via processing environments. Once Listeria bac-teria get in a food plant, they might survive in the processing environment overlong time periods and could pose a contamination hazard to produced foods. More-over, the ability of L. monocytogenes to multiply under refrigeration temperaturesand without oxygen, for example in vacuum packs or in foods which are packed inmodified atmospheres, makes it challenging to control the bacteria further up inthe food chain at the retail level and once foods have reached consumers’ homes.Risk assessments done in the past years have shown that amongst the many foodswhich can become contaminated with L. monocytogenes, ready-to-eat (RTE) foodswhich are consumed without further heat treatment are associated with the great-est listeriosis risk. Regulators in the European Community and the Codex Alimen-tarius Commission have developed risk-based microbiological criteria for L. mono-cytogenes in foods. In both cases, the microbiological criteria specifically apply toRTE foods and take into consideration if growth of Listeria in the food may occuror not. However, in both approaches the main focus is on controlling L. monocyto-genes during processing of RTE foods and in the production environment. The Eu-ropean Regulation on microbiological criteria (Regulation (EC) no 2073/2005) isembedded in the so called ‘hygiene package’ of legislations, and in the Codex Ali-mentarius microbiological criteria are presented in an Annex of the ‘Guidelineson the Application of General Principles of Food Hygiene to the Control of Listeriamonocytogenes in Ready-to-Eat foods’ (CAC/GL 61-2007), only. In the EuropeanCommunity, food business operators have an obligation to demonstrate compli-ance with microbiological criteria and thereby enable verification of their GMPand HACCP systems. The approach of using risk-based microbiological criteriafor L. monocytogenes as management option for controlling Listeria in foods andthus to reduce the likelihood of listeriosis in vulnerable populations will be criti-cally discussed.


USDA regulatory approach and considerations for the controlof Listeria monocytogenesEngeljohn, D.*U.S. Department of Agriculture, Food Safety and Inspection Service, Deputy Assistant Administrator,Office of Policy and Program Development, USA

An overview of the design of the risk management design and objectives of thecontrol programs for ready-to-eat meat and poultry will be presented. Use of riskassessments to inform the verification and enforcement strategy will be high-lighted. Lessons learned regarding the implementation of a “zero tolerance” ap-proach will be discussed, along with future plans for ensuring that progress to-wards good control are not negated in further processing operations.


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AREA //D // Strategies for prevention and control of Listeria monocytogenesPLENARY LECTURES // PL / 10–12

Listeria monocytogenes, an emergent pathogenin Chile and Latin AmericaHormazábal, J. C.*Public Health Institute of Chile

Food borne diseases are a major health burden in Latin America. In Chile Listeriamonocytogenes is a pathogen under laboratory surveillance since 2004. All the iso-lates from human cases must be confirmed at the National Reference Laboratoryat the Public Health Institute of Chile. Until 2008 L. monocytogenes was an infre-quent pathogen, approximately 45 clinical cases per year were reported in all thecountry. At the ending of 2008 an unusual increase of listeriosis was detected inthe Metropolitan region of Chile. In this scenario, the Reference Laboratory in-cluded for first time molecular tools for the detection of related cases. PFGE in-cluding PulseNet International protocols, was a powerful upgrade for the surveil-lance system. In parallel TheMinistry of health, in absence of a specific regulatoryframework for L. monocytogenes in food industry, made important changes in san-itary food regulations, including specific microbiological criteria for Listeria infood and environment. The integration of surveillance data, clinical, environmen-tal and food industry isolates, allowed the creation of a single database, includingmolecular typing information. This dynamic system allowed the detection of thefirst Chilean outbreak of L. monocytogenes, and made possible the link of clinicalcases to potential sources, including the environment, raw or processed food prod-ucts. After few weeks of an intensive investigation the outbreak source was con-firmed (goat and brie cheese). The inclusion of molecular tools in surveillanceprovides valuable information for the early detection of listeriosis outbreaks.PulseNet Latin America Network gives an important support for the constructionof national and regional databases for the detection of local outbreaks and virulentclones with potential international spread. In Latin America Listeria keeps as aninfrequent and unknown pathogen, major efforts are necessary specially in healthstaff and general population education including a sensitive surveillance systemand a strong regulatory framework in food industry.


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AREA //E // Communication, risk perception and consumer practices – Social sciences in Listeria controlPLENARY LECTURES // PL / 13–14

How can the social sciences help us understand the prevalenceof listeriosis in the UK?Wadge, A.*Chief Scientist, Food Standards Agency, UK

There has been amarked change in the epidemiology of listeriosis in humans in theUK over the past decade. A doubling of reported cases has been seen in England andWales since 2001 and this has occurred largely in people aged 60 years and over andpresentingwith bacteraemia rather than central nervous system infection. The inci-dence of listeriosis among other age ranges has remained unchanged and the inci-dence in pregnant women has been stable since the early 1990s. This presentationwill explore the possible reasons for the increase in the over 60s focussing on severalstrands of work that are being undertaken to address this. Firstly in 2007 the UKAdvisory Committee on the Microbiological Safety of Food (ACMSF) consideredthat the change in the epidemiology seen in the UK was most likely linked to socialfactors rather than changes occurring in themicroorganism and referred the issue oflisteriosis in the elderly to its ad hoc group on vulnerable groups for further consid-eration. The presentationwill highlight some of the group’s findingswhich are avail-able in a report (www.food.gov.uk/multimedia/pdfs/committee/acmsflisteria.pdf ).Amongst their recommendations they recognised the importance of food consump-tion and food handling behaviours in the over 60s including those in vulnerablegroups. One of the report’s recommendations was for this to be considered by theFood Standards Agency’s (FSA) Social Science Research Committee (SSRC). Thepresentation will highlight findings from the SSRC’s report which showed that verylittle is known about food storage and handling practices of over 60s in the home.Studies suggest that older people handle food differently from younger people al-though the reason for this difference is unclear. Specific differences reported in-cluded poor refrigeration and defrosting practices; differences in cooling, storageand reheating of leftovers; variable adherence to ‘use-by’ and ‘best-before’ dates; anddifferences in personal and domestic hygiene such as hand-washing and cleaning ofkitchen surfaces. The report noted that there is relatively little evidence regardingthe current levels of food hygiene knowledge among those aged 60 years and overand no information on whether the level of knowledge differs with generations orhas changed as people age. Emphasising the need for correct storage and handling offood in the home particularly by those over 60swas a key theme of Food Safetyweekin theUK in 2009 and the presentationwill illustrate the approach thatwas taken bythe FSA in this campaign. The presentation will conclude by highlighting some ofthe gaps in our knowledge and the SSRC recommendations concerning research.


Consumer perceptions, behaviour and microbial food safety;implications for Listeria controlFrewer, L. J.*Wageningen University, The Netherlands

Inorder tounderstand fully theproblemof food safety linked to theoccurrenceof liste-riosis, it is important to considerhowconsumers respond to food safety issues, in termsof their psychology andhow this determines their behaviour, for example in relation tofood preparation behaviour. An important research objective relates to consumer ac-tivities after foodpurchase.Riskperceptiondetermineshowconsumers react todiffer-ent types of risks. Very generally, risks which are perceived to be unnatural in originand involuntarily imposedon the individualwho is exposed to themareperceived tobemore threatening.Microbiological risks are perceived to be “naturally occurring” and,in the case of food safety risks, highly controllable, and so are not a focus of consumerconcern. In addition, consumers tend to exhibit an “optimistic bias” in relation tomi-crobiological food risks, which means that food safety information tends to be per-ceivedas applying tomorevulnerable, consumers in thepopulation.Research suggeststhat consumers are reasonably knowledgeable about safe food preparation practices,but that this knowledge isnot alwaysapplied inpractice.Foodpreparation tends tobeahabitual behaviour, which is difficult to change. Introducing food safetymessages dur-ing food preparation (for example, in recipe development) tends to activate existingfood safety knowledge, as does the inclusion ofmaterials designed to elicit affective re-sponses to food safety issues, for example disgust. Some groups within the populationare more vulnerable than others, and targeting risk communication messages to theneeds of these groups is particularly relevant. It is argued that, whilst consumer obser-vation studies, combinedwithmodelling of critical control points in food preparation,might indicate the riskiest behaviours in terms of Listeria, this information should becombined with activation of general food safety knowledge if an effective approach toreducing the incidence of foodbornedisease is to bedeveloped.


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ORAL PRESENTATIONS

AREA //A // Biology of Listeria monocytogenesORAL PRESENTATIONS //O / 01–13

The cold shock associated proteins (Csps) promote tolerance of differentenvironmental stresses and host cell invasion of Listeria monocytogenesTasara, T.1, Klumpp, J.2, Loessner, M. J.2 and Stephan, R.1

1. Institute of Food Safety, University of Zurich, Switzerland2. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland

The food-borne pathogen Listeria monocytogenes has to withstand various stressconditions in order to survive and proliferate on foods and within different typesof host cells. The bacterial cold shock family proteins (Csps) consist of small,highly conserved nucleic acid-binding proteins, and are assumed to have an influ-ence on the expression of various microbial genes. In addition to cold adaptationfunctions, some prokaryotic Csp proteins are also involved in promotion of othercell processes during normal bacterial growth, as well as in adaptation to nutri-ent starvation and stationary growth phase stresses. The possible functional con-tribution of L. monocytogenes CspA, CspB and CspD proteins were investigatedunder food-related environmental stress, and during host cell invasion. We showthat the three L. monocytogenes Csp components, although highly homologous,provide distinct cellular functions during cold, osmotic and oxidative stress adap-tation as well as host cell invasion processes. All three csp genes are constitutivelyexpressed but are dispensable for viability and growth of this organism at optimaltemperature. However, a hierarchy in Csp functional importance during cold(CspA>CspD>CspB) and osmotic (CspD>CspA/CspB) stress adaptation is ob-served. With respect to double (DcspBD) and triple (DcspABD) csp deletion mu-tants, we also observe reduced survival of oxidative stress and a reduced ability toinvade Caco-2 and J744A.1 murine macrophages cells. Overall, our data indicateimportant functional roles of L. monocytogenes Csp proteins in promotion of cel-lular functions which facilitate environmental stress resistance and host cell in-vasion processes of this food-borne pathogen.

Different levels of flagellin detected during growthof Listeria monocytogenes strains at low temperatureCabrita, P.1,2,3*, Batista1, S., Moes, S.4, Jenö, P.4, Trigo, M. J.3, Boavida Ferreira, R.1,2and Brito, L.1

1. CBAA/ Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia,Technical University of Lisbon, Lisbon, Portugal

2. Instituto de Tecnologia Química e Biológica, New University of Lisbon, Oeiras, Portugal3. Instituto Nacional dos Recursos Biológicos, IP, Oeiras, Portugal4. Department of Biochemistry, Biozentrum of the University of Basel, Basel, Switzerland

Listeria monocytogenes can tolerate a wide range of environmental stresses.Growth at low temperatures is a stress that L. monocytogenes often has to facesince refrigeration is present throughout the food chain. This study aimed to in-vestigate whether unique or common proteins are up- or down-regulated undernutritional stress conditions and low temperature. To achieve this, a simplifiedmethodology to analyse the extracellular protein profiles of four L. monocytogenesstrains was used. These strains, belonging to four different serovars, were selectedaccording to differences in virulence. Cultures were incubated in minimal medium(Modified Welshimer Broth) at 10 °C. Proteins present in the supernatants of thecell cultures, in late exponential phase, were precipitated and separated by SDS-PAGE, using equivalent amounts of total secreted proteins. For each bacterialstrain, at least three independent culture assays were set. The most abundantpolypeptide bands and those suggesting the widest range in differential expres-sion among strains were identified by ESI LC / MS-MS. The p60 virulence pro-tein, one of the major proteins previously detected at 37 °C, was still detected inrelatively high amounts at 10 °C in all strains. The virulence proteins listeriolysinO and internalin C expressed at 37°C, were not detected at 10°C in all strains, con-firming previous findings. In serovar 1/2a strain, flagellin was the major proteinidentified. Two other strains, from the rare serovares 4c and 4d/4e, showed lowerlevels of flagellin whereas for the serovar 4b strain no flagellin was detected. Fla-gella have been shown to act as mediators in bacteria attachment to stainless steelsurfaces. Serogroup 1/2a has been reported as prevalent among food isolates.Therefore, the different levels of flagellin detected may be related with the abilityof some strains to colonize food contact surfaces at refrigeration temperatures.This hypothesis will be further investigated.

* Participation Supported by IUFoST

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Phenotypic and corresponding transcriptomic responses of Listeriamonocytogenes strains in the presence of unprotonated organic acidsLee Chang, K. J., Pinfold, T., Koshy, A. and Bowman, J. P.University of Tasmania, Australia

Sodium diacetate-resistant L. monocytogenes food and clinical isolates delineatedthrough a culture-based screening process were found to possess significantly bet-ter survival levels following challenge to pH2.4 acid challenges compared to vari-ous reference strains. Sodium diacetate was found to directly and substantiallyimprove survival and was synergistic with acid tolerance resultant from the shift tothe stationary growth phase. The resistant strains had comparatively lower intra-cellular levels of acetate and K+ ions that corresponded to lower transmembranedelta pH values. Another observed feature was that when grown in the presence ofsodium diacatete cell wall stability was enhanced as revealed by lysis assays, util-ising bead-beating and mutanolysin, suggesting acetate induces various cell wallmodifications that may also influence diffusion of unprotonated organic acid intocells. Transcriptomic analysis of pH5.0-habituated sodium diacetate-resistantstrain FW04/0025 compared with reference strain EGD that were grown with21mM (8mMunprotonated) sodium diacetate at pH 5.0 indicated substantial vari-ation in genetic responses with EGD much more reactionary to the presence ofmineral acid. These differences were reflected in regulon-level gene expressiontrends with EGD strongly activating and repressing the SigB- and CodY regulons,respectively, while in FW04/0025 the response of these regulons were muted. Thetranscriptome of FW04/0025 only becomes more congruent with that of EGDwhen in the presence of sodium diacetate, though several distinct genetic expres-sion differences still occur. Gene expression trends deriving from exposure tosodium diacetate suggest extensive cell wall and membrane modification, alter-ations to branched-chain amino acid/fatty acid metabolism/biosynthesis, induc-tion of an SOS-like DNA repair and thioredoxin-mediated antioxidant responsesoccurs, though strain-level specific responses are quite divergent. Correspondingproteomic analyses are underway to further characterize responses to food pre-servative organic acids by L. monocytogenes.

Internalin profiling, multilocus sequence typingand virulence assesments suggest evolutionary historyof the Listeria monocytogenes-Listeria innocua cladeChen, J. and Fang, W.Zhejiang University, China

The morphological, ecological, biochemical and genetic resemblance, and the cleardifference of virulence between L. monocytogenes and L. innocuamake this bacte-rial clade attractive as models to examine the evolution of pathogenicity. Thisstudy was attempted to examine the population structure of L. monocytogenes andL. innocua, and further to investigate the microevolution in this clade via profilingof 37 internalin genes, MLST analysis of gyrB-sigB-dapE-hisJ-ribC-purM-gap-tuf-betL gene cluster, and detection of 17 virulence genes, together with in vitro and invivo virulence assessments. Results indicate that L. monocytogenes comprises threerecognized lineages I, II and III, including a set of lineage III strains displayingstrong phospholipase activity and subdued virulence. While resembling L. mono-cytogenes in having a nearly identical Listeria pathogenicity island I, and inlA andinlB, these nonpathogenic L. monocytogenes strains harbor notably altered prfA,actA and plcB, and share many similar internalin gene deletions with L. innocua,e.g., inlJ, inlC, inlI and inlGHE. On the other hand, L. innocua represents a youngspecies descending from L. monocytogenes, which comprises four subgroups: twomajor subgroups I and II, and one atypical subgroup IV exhibiting the least ge-netic distance to L. monocytogenes. All L. innocua strains lack 17 virulence genesfound in L. monocytogenes, except for subgroup IV strains harboring inlJ, and arenonpathogenic to mice. As shown by the estimation of the time to the most recentcommon ancestor, L. monocytogene lineages I and II appeared at approximatelythe same time, and this is also the case with L. innocua subgroups I and II. Thenonpathogenic L. monocytogenes strains and L. innocua subgroups IV constitutethe possible evolutionary intermediates between L. monocytogenes and L. innocua.The evolutionary history in the L. monocytogenes-L. innocua clade represents arare example of evolution towards reduced virulence of pathogens.

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The SOS response of Listeria monocytogenes is involvedin stress resistance, mutagenesis, and biofilm formationvan der Veen, S.1,2 and Abee, T.1,2

1. Top Institute Food and Nutrition (TIFN), Wageningen, The Netherlands2. Laboratory of Food Microbiology, Wageningen University and Research Centre, Wageningen,

The Netherlands

The food-borne pathogen Listeria monocytogenes is widely distributed in the en-vironment. As a consequence, raw materials used by the food industry could in-troduce L. monocytogenes to food processing facilities. L. monocytogenes hasevolved various strategies and networks to survive and adapt to changing condi-tions e.g. during food processing. One of the stress response mechanisms we re-cently identified in L. monocytogenes is the SOS response.The SOS response is a conserved inducible pathway that is involved in DNA repairand restart of stalled replication forks.We identified the SOS response regulon of L.monocytogenes and showed that it is important for stress resistance and adaptivemutagenesis. In the present study, we investigated the role of the SOS response inL. monocytogenes biofilm formation. L. monocytogenes static biofilms on poly-styrene and glass consists of a homogeneous layer, while on stainless steel L. mono-cytogenes biofilms consist of single attached cells or microcolonies. Static biofilmscontain the small rod-shaped morphology, which is very similar to the morphologyof planktonic cells. However, L. monocytogenes continuous flow biofilms consist ofball-shaped microcolonies, which are surrounded by a dense network of knittedchains composed of elongated cells. We showed that continuous flow biofilm for-mation and not static biofilm formation is dependent on the SOS response. UsingQ-PCR analysis, promoter reporters, and SOS response mutants, we showed thatthe SOS response is activated during knitted-chain biofilm formation and that dele-tion of its regulonmember yneA, which is involved in cell elongation during SOS re-sponse activation, results in diminished biofilm formation in continuous flow con-ditions. Furthermore, we demonstrated that activation of the SOS response duringcontinuous flow biofilm formation induced mutagenesis: wild-type biofilmsshowed considerably higher rifampicin resistant fractions than ∆recA biofilms orwild-type planktonic cultures. Our results show that the SOS response of L. mono-cytogenes is important for stress resistance, adaptive mutagenesis, and continuousflow biofilm formation, and may therefore contribute to the survival and persist-ence of this pathogen in food processing environments.

Clonal diversity of Listeria monocytogenes, a worldwide perspectiveChenal-Francisque, V.1, Lopez, J.1, Cantinelli, T.1, Caro, V.2, Tran, C.2, Leclerq, A.1,Lecuit, M.1 and Brisse, S.2

1. Institut Pasteur, National ReferenceCenter andWHOCollaborating Centre for LISTERIA, Paris, France2. Institut Pasteur, Genotyping of Pathogens and Public Health Platform, Paris, France

Listeria monocytogenes is a foodborne pathogen that can cause listeriosis, a severeinvasive disease in human with a high fatality rate. L. monocytogenes is widespreadin nature. Molecular typing methods have grouped L. monocytogenes into twomajor genetic lineages (lineages I and II) and an additional minor lineage (lineageIII). They differ according to virulence and ecological origin. We have developed aDNA sequencing-based subtyping method, multilocus sequence typing (MLST) toexamine the epidemiology and population genetics of L. monocytogenes. In thepresent study, we have undertaken the first spatiotemporal analysis of L. monocy-togenes biodiversity by sequencing internal portions of seven housekeeping genesin 300 strains isolated from the six continents. We selected a set of 300 unrelatedstrains of L. monocytogenes collected from 41 countries and 6 continents isolatedbetween 1935 and 2009 from different sources. MLST data based on seven house-keeping genes (3,288 nucleotides) were obtained as described previously (Ragon etal., PLoS Pathogens (2008)). The results show a pattern of biodiversity similar tothe one we had initially reported in France. Indeed, the population structure issimilar to that obtained on 360 strains isolated principally from France by Ragonet al. Sequences and allelic profiles are available on the Internet-accessible data-base (www.pasteur.fr/mlst). L. monocytogenes appears to exhibit a homogeneousclonal diversity across continents, indicating a high dispersal rate at a global scale.

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Life without a cell wall: Listeria monocytogenes L-form cells featurea unique mode of divisionBriers, Y., Dell’Era, S., Schuppler, M. and Loessner, M. J.Institute of Food, Nutrition and Health, ETH Zurich, Switzerland

Cell wall-deficient bacteria referred to as L-forms have lost the ability to main-tain or build a rigid peptidoglycan envelope. Although L-forms had been studiedfor decades and many reports exist on their morphological, serological and bio-chemical properties, very little was known about the basic cell biology and molec-ular mechanisms underlying the transformation process. Of particular interest isthe question how L-form bacteria are able to proliferate in the absence of a maturecell wall. We have investigated the biology of stable, non-reverting Listeriamono-cytogenes L-form cells (native and GFP labelled). Transmission electron mi-croscopy demonstrated that L-form cells are devoid of the typical thick Listeriatype cell wall small, and form small protoplast-like vesicles as well as multi-nu-cleated macro-cells, surrounded only by a cytoplasmic membrane. They lack pep-tidoglycan-bound proteins such as Internalin A, whereas membrane-anchoredproteins such as Internalin B are still present. Monitoring L-form growth by time-lapse confocal laser scanning microscopy revealed the development and matura-tion of vesicles within maternal L-form cells. We propose a novel model for growthand division of L-form bacteria, which may explain their ability to multiply in theabsence of a rigid cell wall. Furthermore, transcriptome analysis of parental and L-form L. monocytogenes was performed using whole genome hybridization arrays.Compared to parental bacteria, L-forms feature downregulated metabolic func-tions correlating with the dramatic shift in surface to volume ratio, whereas up-regulation of stress genes reflects the difficulties in adapting to this unusual, cell-wall deficient lifestyle. We also observed that L-form cells taken up intomacrophages are not killed but seem able to survive for prolonged periods of atleast 48 hours. In conclusion, we show that L. monocytogenes L-forms (i) can ariseand survive in the environment, (ii) are able to multiply and divide, and (iii) showintracellular survival in macrophages.

Pangenomic analysis of Listeria monocytogenesDeng, X.1, Phillippy, A. M.2, Li, Z.1, Salzberg, S. L.2, Tortorello, M. L.3 and Zhang, W.1

1. National Center for Food Safety and Technology, Illinois Institute of Technology, Summit, USA2. Center for Bioinformatics and Computational Biology, University of Maryland, College Park, USA3. National Center for Food Safety and Technology, Food and Drug Administration, Summit, USA

Listeria monocytogenes is well known for its adaptability to diverse environmentand host niches and its high fatality rate among infected immunocompromisedpopulations. Three genetic lineages have been identified in this species. Strainsof genetic lineages I and II account for >90% of human infections in the UnitedStates, whereas strains from genetic lineage III are rarely implicated in humaninfections for unclear reasons. Here we compare the genomes of 26 L. monocyto-genes strains representing the three lineages based on both in silico comparativegenomic analysis and high-density, pan-genomic DNA array hybridizations. Weuncover 86 genes and 8 small regulatory RNAs that likely make L. monocytogeneslineages differ in carbohydrate utilization and stress resistance during their resi-dence in natural habitats and passage through the host gastrointestinal tract. Wealso identify 2,330 to 2,456 core genes in the listerial pan-genome that define thisspecies and assess the impact of lysogenic bacteriophages on genomic diversifi-cation. Phylogenomic reconstructions based on 3,560 homologous groups suggesta polyphyletic population infrastructure and gradual loss of genes as this sapro-phytic species diversified into a rare and probably defective lineage.

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Evidence for an antiporter-independent glutamate decarboxylase(GAD) system in Listeria monocytogenes: Influence of growth mediaon GAD system activityKaratzas, K.-A., Brennan, O., Heavin, S. and O’Byrne, C. P.SFI, Ireland

The GAD system plays an important role in survival of Listeria monocytogenesunder acidic conditions (eg. acidic foods) and in virulence (eg. survival in stom-ach). The GAD system imports extracellular L-glutamate (Glu)e through an an-tiporter, converts it to γ-aminobutyric acid (GABA) resulting in the consumptionof an intracellular proton and thus the increase of the intracellular pH. We showfor the first time that (Glu)e added in Defined Medium (DM) is not used by theGAD system and therefore it cannot increase the ability of this bacterium to growin mild acidic conditions, neither does it enhance acid resistance at lethal pH val-ues as it does in BHI. The rate of GABA export was monitored in BHI at various pHvalues showing that it initiates at pH4.6 and the rate increases as pH values de-crease. We also demonstrate that there are activators of the antiporter-based GADsystem in BHI, while its inactivity in DM is not due to the presence of inhibitors inthis medium. Activation was at the transcription level with the expression ofgadD2T2 being more than 100-fold higher in BHI than in DM where levels ofgadD2 were undetectable. Furthermore we demonstrated for first time that inboth acidified DM and BHI, L. monocytogenes accumulates high levels of intracel-lular GABA (GABA)i (>42mM) and despite the slower rate of accumulation in DMthe final levels were identical in both media. Since DM does not contain any (Glu)ewe have shown for first time that this bacterium converts (Glu)i to (GABA)i, whichis not exported and thus is stored intracellularly. This suggests an alternativemechanism of acid resistance based on the GAD system that circumvents the an-tiporter-based mechanism. We suggest that the (GABA)i accumulation mightbuffer the intracellular pH and thereby contribute in survival under extreme acidicconditions.

Role of Listeria monocytogenes tyrosine phosphatases in conferringlisteriophage resistancePaz, R.-N. 1, Eugster, M. R.2, Zeiman, E.1, Loessner, M. J.2 and Calendar, R.3

1. Hadassah Hebrew University Medical Center, Israel2. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland3. Department of Molecular and Cell Biology, University of California, Berkeley, USA

Protein tyrosine phosphatase (PTP)-like proteins exist in many bacteria and aresegregated into 2 major groups: Low molecular weight, and conventional. ThesePTP are suggested to be involved in many aspects of bacterial physiology includingstress response, DNA binding proteins, virulence and capsule/cell wall produc-tion. By annotation Listeria monocytogenes (LM) possesses 2 potential lowmolec-ular weight and 2 conventional PTPs. Although no tyrosine kinases were identifiedyet in LM, using Immunoprecipitation (IP) on total cell lysate andMS plusMS/MSon the IP products we have identified at least 10 tyrosine phosphorylated proteins.These proteins vary in their physiological function and were associated with car-bohydrate metabolism, DNA and RNA binding, processing and transcription andtransport. Using LMWT strain 10403S, we have created an in-frame deletion mu-tant lacking all 4 PTPs, as well as 4 additional complemented strains harboringeach of the PTPs. No major physiological differences were observed between theWT and the mutant lacking all 4 PTPs. However, the deletion mutant strain wasfound to be resistant to listeriophages A511 and P35, and sensitive to other liste-riophages such as U153 and A118. This phage resistance was attributed to reducedattachment to the cell wall. Additionally, the mutant lacking all PTPs was found tolack N-acetylglucosamine in its teichoic acid. Phage sensitivity was rescued in acomplemented strain harboring a lowmolecular weight PTP (LMO2540). We alsofound that attachment of the phages is partially restored by the same PTP and byone with conventional weight PTP (LMO1800). Thus, it seems that PTPs probablyaffect many processes in LM, but mostly resistance to listeriophages.

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Thiolomics – the thiol: disulfide redox metabolismof Listeria monocytogenesOndrusch, N.1, Gopal, S.2, Fuss, A.1, Hagen, N.1, Stoll, R.1, Aharonowitz, Y.3 and Kreft, J.1

1. University of Würzburg, Biocenter, Germany2. Dept. of Microbiology , University of Mysore, India3. Dept. of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel

The thiol: disulfide redox metabolism (TDRM), found in all living cells, consti-tutes a multi-component network. It counteracts oxidative stress, serves to main-tain the proper intracellular redox potential, assists in protein folding, is essentialfor the formation of deoxyribonucleotides for DNA synthesis and helps to repairdamaged proteins. The best-characterized biological thiols are the tripeptide glu-tathione (GSH) and the small proteins of the thioredoxin (Trx) and glutaredoxin(Grx) family. Oxidized GSH and Trx are recycled by cognate reductases (GSH re-ductase – Gor/GshR; thioredoxin reductase – TrxB). A considerable number ofgenes/gene products putatively involved in the TDRM of L. monocytogenes EGD-e has been identified in the genome sequence. It comprises the unique fused genegshF (GSH synthetase), grx, two putative gor/gshR, six members of the thiore-doxin family, trxB, class I and class III ribonucleotide reductases (nrdAB andnrdDG), gpo/gpx, prx, tpx, ohrA/R (detoxification of organic hydroperoxides),msrA(methionine sulfoxide reductase ) and several regulators, e.g. perR (peroxide reg-ulon), spx & rex (redox-sensing regulators), fur, zur & mntR (metal uptake), nrdRand the redox-sensitive chaperone hsp33. We investigated the regulation and func-tion of these genes/gene products in the TDRM in particular and in the physiologyof L. monocytogenes in general, also with respect to infectivity and virulence, by thefollowing approach: i) construction of selected mutants, ii) study of their multi-plication in vitro and in vivo, and iii) genome-wide transcription profiling of wildtype and mutants under several in vitro conditions (oxidative and disulfide stress)and in eukaryotic host cell models. A major result was that mutants defective in theglutathione/glutaredoxin system showed extensive changes in their transcriptionprofiles. Affected were virulence genes, genes for regulators, transporters, stressresponse factors and also for metabolic enzymes. These results emphasize the cen-tral role of the TDRM, their impact on cellular processes and pathogen-host in-teraction will be discussed.

RNA-structures acting at a distanceJohansson, J.Umeå University, Sweden

Riboswitches are RNA-elements acting in cis, controlling expression of theirdownstream genes through a metabolite-induced alteration of their secondarystructure. Here, we demonstrate that an S-adenosylmethionine (SAM) riboswitch,SreA, in Listeria monocytogenes can also function in trans, and act as a non-codingRNA. We show that SreA controls expression of the virulence regulator PrfA bybinding to the 5´-untranslated region of its mRNA in a temperature-dependentmanner. Absence of the SAM riboswitch increases the level of PrfA. Thus, the im-pact of the SAM riboswitch on PrfA highlights a link between virulence and thenutritional status of the bacterium. Together, our results describe a novel role forriboswitches and a new class of regulatory non-coding RNAs in bacteria.

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Deep RNA sequencing of Listeria monocytogenes reveals overlappingand extensive stationary phase and sigma B-dependent transcriptomes,including multiple highly transcribed noncoding RNAsOliver, H. F.1, Orsi, R. H.1, Ponnala, L.1, Keich, U.2, Wang, W.1, Sun, Q.1, Cartinhour, S.3,Filiatrault, M. J.3, Wiedmann, M.1 and Boor, K. J.1

1. Cornell University, USA2. University of Sydney, Australia3. United States Department of Agriculture-Agricultural Research Service, Robert W. Holley Center for

Agriculture and Health, USA

Identification of specific genes and gene expression patterns important for Liste-ria monocytogenes survival, transmission and pathogenesis is critically needed toenable development of more effective control strategies. The stationary phasestress response transcriptome, which includes many sigma B-dependent genes,was defined in L. monocytogenes using RNA sequencing (RNA-Seq) with the Illu-mina Genome Analyzer. Specifically, transcriptomes were compared between sta-tionary phase cells of L. monocytogenes 10403S and an otherwise isogenic ∆sigBmutant, which does not express the alternative sigma factor σB, a major regulatorof genes contributing to stress response, including stresses encountered uponentry into stationary phase. Overall, 83% of all L. monocytogenes genes were tran-scribed in stationary phase cells; 42% of currently annotated L. monocytogenesgenes showed medium to high transcript levels under these conditions. A total of96 genes had significantly higher transcript levels in 10403S than in ∆sigB, indi-cating σB-dependent transcription of these genes. RNA-Seq analyses indicate thata total of 67 noncoding RNA molecules (ncRNAs) are transcribed in stationaryphase L. monocytogenes, including 7 previously unrecognized putative ncRNAs.Application of a dynamically trained Hidden Markov Model, in combination withRNA-Seq data, identified 65 putative σB promoters upstream of 82 of the 96 σB-dependent genes and upstream of the one σB-dependent ncRNA. The RNA-Seqdata also enabled annotation of putative operons as well as visualization of 5’- and3’-UTR regions. The results from these studies provide powerful evidence thatRNA-Seq data combined with appropriate bioinformatics tools allow quantitativecharacterization of L. monocytogenes transcriptomes, thus providing exciting newstrategies for exploring transcriptional regulatory networks.

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AREA //B // Listeria monocytogenes as a human and animal pathogenORAL PRESENTATIONS //O / 14–23

Probiotics reduce Listeria monocytogenes-induced tissue invasion andstillbirths in pregnant guinea pigsSmith, M. A., Agyekum, K. and Williams, D.Environmental Health Science Department, University of Geórgia, USA

One-third of listeriosis cases are pregnancy-related and can lead to miscarriageor stillbirth, premature delivery, or infection of the newborn. Recently we havepublished a risk assessment based on dose response data from nonhuman pri-mates and guinea pigs. Both animal models estimate LD50s of approximately 107 L.monocytogenes cfu similar to the FAO/WHO estimated human LD50 of 1.9 x 106cfu based on outbreak data. The similarities between the dose response curvesand LD50s suggest these animal models are appropriate for testing therapies andpreventive strategies applicable to humans. Recently, probiotics have been pro-posed to protect hosts from pathogens introduced to the system through inges-tion. Our objective was to determine the efficacy of probiotics in yogurt in pre-venting L. monocytogenes invasion and stillbirths. Using our pregnant guinea pigmodel for listeriosis, 5 ml of yogurt containing Lactobacillus and Bifidobacteriumwas administered orally to pregnant guinea pigs on gestation days (gd) 32–36. Ongd35, guinea pigs were orally fed L. monocytogenes (109cfu) in sterilized whippingcream at four hrs after yogurt feeding. By gd 56 in those animals receiving only L.monocytogenes, the pathogen was isolated from 100%, 75%, 64%, 71% and 71%of maternal livers and spleens, placentas, fetal livers and brains, respectively. How-ever in those that received yogurt and L. monocytogenes, the pathogen was iso-lated from 56%, 14%, 17%, 17% and 22% of maternal livers and spleens, placentas,fetal livers and brains respectively. Also, 75% of pregnant guinea pigs treated with108 L. monocytogenes cfu have stillbirths compared to 14% when treated with yo-gurt and L. monocytogenes. These results present opportunities to develop treat-ment and preventive therapies for listeriosis. In summary, consumption of yogurtcontaining Bifidobacterium and Lactobacillus reduced the invasion and numberof stillbirths after L. monocytogenes exposure in pregnant guinea pigs.

Listeriolysin O favors Listeria monocytogenes growth in co-culture withthe ciliate Tetrahymena pyriformisErmolaeva, S. and Pushkareva, V.Gamaleya Institute of Epidemiology and Microbiology, Russian Federation

Listeria monocytogenes was isolated from soil, water, sewage and sludge. We ex-plored the potential of L. monocytogenes major virulence factor Listeriolysin O(LLO) to promote interactions between L. monocytogenes and the ubiquitous in-habitant of natural ecosystems bacteriovorous free-living ciliate Tetrahymena pyri-formis. Axenic T. pyriformis and the following bacterial strains were used: wildtype L. monocytogenes strains EGDe, VIMVR081, VIMVW039, VIMHA034,VIMVF870, EGDe derivative EGDe::Dhly and wild type L. innocua strainNCTC11288. Experiments were performed in LB broth at 28 °C. Bacteria werecounted bacteriologically or by qPCR. T. pyriformis trophozoites and cysts werecounted using light microscopy. The hly gene was introduced into pTRKH2 vector,and the same plasmid supplemented with prfA* gene was used to express LLO in L.innocua. Guinea pigs were infected intraconjunctivally or per os with L. monocy-togenes culture or with T. pyriformis cysts infected L. monocytogenes. After 7 daysof co-culturing, wild type L. monocytogenes strains reduced trophozoite and in-creased cyst concentrations up to 21.1 and 6.1 times, respectively. EGDe::Dhlyfailed to cause mortality among protozoa and to trigger protozoan encystment. Inconcordance, LLO deficiency deteriorated L. monocytogenes growth in the pres-ence of T. pyriformis. Replenishment of the hly gene in the mutant strain restoredtoxicity towards protozoa. L. innocua transformed with the LLO-expressing plas-mid caused extensive mortality and encystment in ciliates. L. monocytogenes EGDeentrapped in cysts caused infection in guinea pigs upon ocular and oral infection.The L. monocytogenes virulence factor LLO promotes bacterial survival and is re-sponsible for L. monocytogenes toxicity for protozoa and induction of protozoanencystment. Therefore, LLO activity might support bacterial survival in the natu-ral habitat outside of a host.

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Atopy is a risk factor for listeriosisKawamoto, K., Matsubara, S., Da Silva, M. and Makino, S.-I.Obihiro Univ. Agri. Vet. Med., Japan

Listeria monocytogenes is a Gram-positive bacterium that causes meningitis, bac-teremia, and febrile gastroenteritis. The outcome of listeriosis is dependent onhost factors such as age, pregnancy, and HIV infection, which influence host im-munocompetency. A Th-1 type cytokine IFN-gamma; plays an important role inthe innate immune response against intracellular bacterial pathogens. In contrast,Th2-biased immune responses often associate with most atopic diseases. In thisstudy, we examined whether atopy affected the immune response to L. monocyto-genes infection. NC/Nga is a model mouse for human atopic dermatitis, which hasa genetic predisposition to develop atopic skin lesion. We compared the suscepti-bility to listeriosis of NC/Nga mice with BALB/c and C57BL/6 mice. The course ofinfection was characterized by monitoring survival of mice, and determination ofbacterial numbers in the organs. NC/Nga mice were highly susceptible to L. mono-cytogenes infection: the 50% lethal dose was significantly lower and the number ofbacteria in livers, spleens and brains were higher in NC/Nga than those for otherinbred mice. The increased permeability of blood-brain barrier was observed inNC/Nga brains at day 3 post infection, but not in BALB/c and C57BL/6. As com-pared to BALB/c and C57BL/6 mice, plasma IFN-gamma-levels of NC/Nga werecomparative. However, markedly increased IL-10 levels were detected in NC/Ngaplasma. Pretreatment with neutralizing antibodies to IL-10 partially protectedmice but retarded the clearance of bacteria. Yet the molecular mechanisms bywhich the infection of L. monocytogenes induces overproduction of IL-10 inNC/Nga mice remain unknown, our results suggest that the differential cytokineproduction may at least partially underlie the higher susceptibility to L. monocy-togenes in NC/Nga mice. Considering the increased prevalence of atopic diseases,an atopic phenotype may be a potential risk factor for listeriosis.

Cancer immunotherapy using novel Listeria monocytogenes-bacterialvectors to target the vasculature of progressive tumorsPaterson, Y., Seavey, M. M., Maciag, P. C. and Sewell, D.University of Pennsylvania, USA

For nearly 20 years our laboratory has been developing Listeria monocytogenes(Lm) as a vaccine carrier to introduce tumor and viral antigens to the immunesystem for the immunotherapy of cancer and as prophylactic vaccines for infec-tious disease. Given the antigenic instability of tumor cells, we recently turnedour attention to the use of immunotherapy to destroy cells actively involved informing new blood vessels that support the growth and spread of cancer. We con-structed Lm expression systems that would target two central cell types involvedin angiogenesis – endothelial cells and pericytes. Two proteins are highly ex-pressed on each cell during active angiogenesis – Vascular Endothelial GrowthFactor Receptor-2 (VEGFR2) (endothelial cells) and High Molecular WeightMelanoma Associated Antigen (HMWMAA) (pericytes). We selected fragmentsfrom each molecule that included peptides predicted to bind to the HLA-A2 mol-ecule. We fused the genes encoding these fragments to the gene encoding the first420 residues of Listeriolysin-O (LLO) and used Lm to deliver these fusion pro-teins. Even though the immune system should be tolerant to these self-molecules,both vaccines elicited potent anti-tumor CTL responses. Lm-LLO-VEGFR2 wasable to reduce tumor microvascular density (MVD), cause regression of estab-lished breast tumors and protect against tumor re-challenge 100+ days post lastimmunization. The Lm-LLO-HMWMAA vaccine also dramatically reducedMVD,induced regression of established breast tumors, increased CD8+ T cell infiltra-tion, and, in addition, reduced the pericyte coverage of intra-tumoral blood vessels.Interestingly anti-tumor efficacy was dependent on epitope spreading to thetumor-associated antigen Her-2/neu. Neither immunotherapeutic interfered withthe generation of normal vasculature in wound healing or gestation. We are cur-rently testing these vaccines to impact the progression andmetastasis of advancedbreast cancer, we hypothesize that reduced angiogenesis will prevent the forma-tion of distal metastases blunting cancer spread and improving overall survival.

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The intracellular carbon metabolism of Listeria monocytogenesin comparison to that of other intracellular bacterial pathogensreplicating in mammalian host cellsGotz, A.1, Eylert, E.2, Stoll, R.1, Eisenreich, W.2, and Goebel, W.3

1. Biocenter-Microbiology, University of Würzburg, Germany2. Institute of Biochemistry, LMUMünchen, Germany3. Max-von-Pettenkofer Institute, LMUMünchen, Germany

Intracellular bacterial pathogens replicate either in the cytosol (e.g. Listeria mono-cytogenes, Shigella spp. and the closely related enteroinvasive Escherichia coli[EIEC]) or in specialized phagosomal compartments (e.g. Salmonella entericaSerovar Typhimurium) of the infected host cells. Our knowledge on the metabolicadaptation processes between intracellular pathogens and their host cells, allow-ing their efficient intracellular growth, and on the influence of the intracellularbacterial metabolism on the expression of those virulence genes, that are decisivefor the intracellular life cycle of the intracellular pathogens, is still rather limited.We have carried out initial studies concerning these important questions with theabove mentioned intracellular bacteria. For this goal we constructed mutants im-paired in the uptake and/or catabolism of specific carbon sources and studied byNMR- or MS-based 13C-isotopologue profiling their intracellular carbon metabo-lism in comparison to the isogenic wild-type strains after infection of suitablemammalian host cells. The results obtained show that the intracellular carbonmetabolism of L. monocytogenes differs significantly from that of the two otherpathogens. Whereas glucose, but not glucose-6-P is the major carbon substratefor intracellular growth of EIEC and S. typhimurium, L. monocytogenes uses C3-substrates (e.g. glycerol) as major and glucose-6-P as subsidiary carbon sources.The reason for this difference is apparently the lack of high affinity glucose trans-porters in L. monocytogenes. However, the intracellular C-metabolism of all threepathogens in mammalian host cells is surprisingly flexible. Mutants defective inthe uptake of the preferential carbon source switch readily to alternative carbonsources and the lower energy supply of the utilized secondary carbon sourcesseems to be compensated by an increased uptake of anabolic monomers from thehost cells. As shown for L. monocytogenes the intracellular carbon metabolism ap-pears to be adapted to optimal expression of the virulence genes that are essentialfor the intracellular life style.

LIMP2 links late phagosomal trafficking with the onsetof the innate immune response to Listeria monocytogenes:a role in macrophage activationFernandez-Prieto, L., Carrasco-Marin, E., Madrazo-Toca, F., Rodriguez-Del Rio, E.,Carranza-Cereceda, C. and Alvarez-Dominguez, C.Immunology Departament, Hospital Santa Cruz de Liencres and Fundacion Marques deValdecilla – IFIMAV, Spain

The innate immune response to Listeria monocytogenes depends on phagosomalbacterial degradation bymacrophages. Here, we describe the role of LIMP2, a lyso-somal type III transmembrane glycoprotein and scavenger-like protein, in Listeriaphagocytosis. We show here that LIMP2 is not involved in bacterial recognition atthe cell surface but participates in the degradation of Listeriawithin phagosomes.LIMP2 appears linked to Rab5a activation and controls the late-endosomal/lyso-somal fusion machinery. Importantly, LIMP2 deficient mice display amacrophage-related defect in innate immunity. They produce less acute-phasepro-inflammatory cytokines/chemokines, MCP-1, TNF-α and IL-6, but normallevels of IL-12, IL-10 and IFN-γ and a 20-fold increase in susceptibility to Listeriainfection. This macrophage dysfunction results in a low listericidal potential, im-paired phago-lysosome transformation into antigen-processing compartmentsand uncontrolled LM cytosolic growth that fails to induce normal levels of acute-phase pro-inflammatory cytokines. Therefore, the role of LIMP2 appears to beconnected to the activation of Listeria-primed macrophages through internal sig-nals linking the regulation of late trafficking with the onset of the innate Listeriaimmune response.

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The tetraspanin CD81 is required for entry of Listeria monocytogenesin mammalian cellsTham, T. N.1,2,3, Gouin, E.1,2,3, Rubinstein, E.4,5, Boucheix, C.4,5, Cossart, P.1,2,3

and Pizarro-Cerdá, J.1,2,3

1. Institut Pasteur, Unité des Interactions Bactéries-Cellules, Département de Biologie Cellulaireet Infection, Paris, France

2. INSERM, U604, Paris, France3. INRA, USC2020, Paris, France4. Institut André Lwoff, Université Paris-Sud, Villejuif, France5. INSERM, U602, Villejuif, France

Listeria monocytogenes is a facultative intracellular bacterial pathogen that in-vades epithelial cells by subverting signaling cascades associated with its twomaincellular receptors, E-cadherin and Met. We recently identified the type II phos-phatidylinositol 4-kinases (PI4KIIs) α and β as required for bacterial entry down-stream of Met. In this work we investigated whether tetraspanins CD9, CD63 andCD81, which figure among the few described molecular partners of the PI4KIIα,function as molecular adaptors recruiting the PI4KIIα to the bacterial entry site.We observed by fluorescence microscopy that CD9, CD63 and CD81 are expressedand detected at the cellular surface and also within intracellular compartments,particularly in the case of CD63. In resting cells, colocalization between thesetetraspanins and the PI4KIIα is only detectable in restricted areas of the perinu-clear region. Upon infection with Listeria, endogenous CD9, CD63 and CD81 wererecruited at the bacterial entry site but did not colocalize strictly with endoge-nous PI4KIIα. Live cell imaging confirmed that tetraspanins and the PI4KIIα donot follow the same recruitment dynamics to the Listeria entry site. Depletion ofCD9, CD63 and CD81 levels by small interfering RNA (siRNA) demonstrated thatCD81 is required for bacterial internalization, identifying for the first time a rolefor a member of the tetraspanin family in the entry of Listeria within target cells.Moreover, depletion of CD81 inhibits recruitment of PI4KIIα to the bacterial entrysite but not that of the Met receptor, suggesting that this tetraspanin could act asa membrane organizer required for the integrity of signaling events occurring atListeria entry sites.

Listeria innate immune evasion by peptidoglycan modificationAubry, C.Department of Cellular Biology and Interaction, Institut Pasteur, France

Peptidoglycan (PG) plays an important role in host-pathogen interaction becausePG is the site of anchoring of virulence factors and an important target for the in-nate immune system. As reported previously, inactivation of pgdA, encoding a PGN-deacetylase in Listeria monocytogenes, revealed a key role of PG modificationin virulence as survival of the mutant was severely impaired in mice (Boneca etal., PNAS 2007). Deletion of the pgdA gene highly increased sensitivity to the bac-teriolytic activity of hen egg lysozyme in vitro. The pgdA mutant was rapidly de-stroyed within phagosomes and induced a potent pro-inflammatory response bymacrophages. Interestingly, inactivation of the deacetylase induced a strong se-cretion of IFN beta by infected macrophages, through a surprisingly and unchar-acterized TLR2-dependent pathway. We have now shown that TLR9 but not TLR4contribute to this IFN beta secretion. The key adaptors and the new signalingpathway leading to IFN beta production are under investigation. We found thatL. monocytogenes PG is also modified by a putative O-acetyltransferase (OatA) es-sential for virulence. A Listeria oatAmutant shows an increased sensitivity to henegg lysozyme, bacteriocin and cell wall targeting antibiotics in vitro. Its growth isimpaired in macrophages, possibly contributing to immune escape. Modificationof PG is a highly efficient mechanism used by pathogenic Listeria to evade innatehost defenses.

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Constitutive activation of the central virulence transcriptionalregulator PrfA enhances Listeria monocytogenes pathogenesis butreduces bacterial fitness outside of the hostFreitag, N. E.1 and Bruno, J. C.2

1. Department of Microbiology & Immunology, University of Illinois at Chicago, USA2. Department of Global Health, University of Washington, USA

Listeria monocytogenes survives as a saprophyte in soil and decaying vegetationwhile maintaining an ability to invade mammalian cells and cause serious disease.Survival and replication of L. monocytogenes within mammalian hosts requiresthe regulated synthesis and expression of multiple gene products that enable hostcell invasion, bacterial replication within the cytosol, and spread to adjacent cells.The transcriptional regulator PrfA controls the expression of multiple bacterialvirulence factors and is required to facilitate the L. monocytogenes transition fromsaprophyte to pathogen. PrfA has been shown to exist in both low and high activitystates, with the transition to high activity occurring within the cytosol of host cells.A number of mutations within prfA have been described (prfA* mutations) thatserve to lock the protein into a high activity state. We examined the consequencesof constitutive PrfA activation on L. monocytogenes physiology and pathogenesisby assessing the fitness of prfA* strains in a variety of in vitro and in vivo condi-tions. prfA* strains exhibit a competitive advantage over wild strains in animalmodels of infection, with ten-fold higher numbers of prfA* bacteria recovered fromthe livers and spleens of infected mice. However, despite apparently normalgrowth in broth culture, the prfA* mutants exhibited a fitness defect when grownin the presence of wild type bacteria, and this fitness defect was exacerbated whenmixed cultures were placed under various stress conditions. Strains containingprfA* mutations were also defective in their ability to adapt to a long term sur-vival phase following prolonged growth in broth culture. Taken together, these re-sults indicate that L. monocytogenes must maintain a critical balance of PrfA ac-tivity to promote bacterial fitness both inside and outside of infected host cells.

The lvfH gene of Listeria monocytogenes encodes a novel virulence factorhighly activated during infectionCarvalho, F., Camejo, A., Ferreira, P., Sousa, S. and Cabanes, D.IBMC – Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology,Universidade do Porto, Portugal

Listeria monocytogenes is a highly adaptable Gram-positive bacterium that caninduce potentially lethal listeriosis in immunocompromised human hosts. As afacultative intracellular pathogen, it is able to invade and replicate inside differ-ent eukaryotic cell types and, by cell-to-cell spread, disseminate infection. Suchprocesses are highly dependent upon expression of specialized proteins whichact as virulence factors on certain steps of the bacterial infectious cycle. Our re-cent studies on the L. monocytogenes EGD-e transcription profile in infectedmice revealed genes that were highly activated throughout the infection timelineand could be involved in virulence. Among these is lvfH, a serotype 1/2a-specificgene that encodes a protein putatively involved in the L-rhamnose biosynthesispathway, a mechanism that provides substrate for the decoration of cell wall tei-choic acids. In order to confirm and characterize the role of this gene in L. mono-cytogenes virulence, we generated an lvfH deletion mutant and investigated theinfluence of this mutation in the Listeria infectious process. In vitro assaysshowed that the mutant was unaffected in its host cell membrane-adheringproperties, but was significantly impaired in its ability to invade different mam-malian cell lines. Organs of mice intravenously infected with the lvfH mutantdisplayed a significant decrease in bacterial load, as compared to animals in-fected with wild type bacteria. This phenotype is unrelated with an intrinsicgrowth defect of the mutant, as it presented growth profiles similar to the wildtype strain in broth and within murine macrophages. Animal organs infectedwith an lvfH-complemented strain showed wild type-like infection levels, con-firming the specific requirement of LvfH for full virulence in this model.Functional characterization of LvfH and analysis of the relation between tei-choic acid rhamnosylation and virulence could reveal a new role for teichoic aciddecoration in L. monocytogenes pathogenesis.

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AREA //C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosisORAL PRESENTATIONS //O / 24–36

Human listeriosis due to Listeria ivanoviiGuillet, C.1, Join-Lambert, O.1, Le Monnier, A.1, Leclercq, A.2, Mamzer-Bruneel,M. F.1, Bielecka, M. K.3, Scortti, M.3, Disson, O.2,4, Vazquez-Boland, J.3, Lortholary, O.1and Lecuit, M.1,2,4

1. Necker-Enfants Malades hospital, Paris Descartes University, Paris, France2. Institut Pasteur, NRC andWHO-CC for Listeria, Paris, France3. University of Edinburgh, Scotland, UK4. Institut Pasteur, Microbes and host barriers Group, Inserm avenir, Paris, France

The genus Listeria contains two pathogenic species: Listeria monocytogenes (Lm)that infects human and animals, and Listeria ivanovii subps. ivanovii (Lii), consid-ered a ruminant-specific pathogen. We describe here a case of gastroenteritis andbacteremia due to Lii in a kidney transplant recipient. A 55-year-old man was re-ferred to our hospital with a three-week history of non-bloody diarrhoea, vomiting,dehydratation, and low-grade fever. He underwent a renal transplantation forchronic renal failure and was chronically infected with hepatitis C virus. Stool andblood cultures allowed the isolation of Lii. Intravenous amoxicillin (6g/day) andgentamicin (6 mg/kg/day) treatment was associated with resolution of symptoms.The 4 Lii isolates from this patient had indistinguishable ApaI and SmaI PFGEpatterns, phenotypic characters and classical resistance for antibiotics. Thesehuman isolates were indistinguishable from prototypic ruminant strains based on(i) the activation status of the central virulence gene regulator PrfA, (ii) the pres-ence of the L. ivanovii-specific pathogenicity island LIPI-2 by PCR mapping, and(iii) invasion assays usingMadin-Darby Bovine Kidney cells and huma, HeLa cells.So, Lii is pathogenic for humans. As for Lm, Lii route of infection is foodborne. Areview of the literature identified 8 cases, mostly in immunosuppressed patients.The rarity of human Lii infections probably reflects low exposure given the rareoccurrence of this species in nature compared to Lm.

Foodborne listeriosis in India: An updateBarbuddhe, S. B.1*, Malik, S. V. S.2, Ashok Kumar, J.1, Kalorey, D. R.3, Kurkure, N. V.3,Rawool, D. B.4, Swain, B. K.1, Korikanthimath, V. S.1 and Chakraborty, T.4

1. ICAR Research Complex for Goa, India2. Division of Veterinary Public Health, Indian Veterinary Research Institute3. Nagpur Veterinary College, India4. Institute of Medical Microbiology, Justus Liebig University, Giessen, Germany

Listeria monocytogenes is a foodborne pathogen that can cause serious invasiveillness, mainly in certain well-defined high-risk groups, including elderly and im-munocompromised patients, pregnant women, newborns and infants. In India,the pathogen has been isolated from humans, animals, a variety of foods includingmilk and milk products, meat and meat products and vegetables. In India, studieson molecular epidemiological aspects of L. monocytogenes are largely lacking.Therefore, we do not know the genetic variability of strains isolated and their epi-demic potential. The genetic diversity of the Listeria strains from various sourcesnamely, milk and milk products (112), fish and seafood (47), wild life (7), poultrymeat (19), meat and processed meats (37), animal clinical cases (41) and humanclinical cases (21) isolated/collected from different places in the country have beencharacterized biochemically and with in vitro assays before attempting for serotyp-ing and virulence gene profiles. Out of the strains characterized phenotypically,195 strains have been serotyped using multiplex PCR and pulsed field gel elec-trophoresis. The predominant serotype among Indian Listeria isolates is L. mono-cytogenes 4b. Significant variation among the isolates recovered from differentsources has been observed as evident by PFGE analysis. As many as 34 differentPFGE profiles have been observed. An electronic database for the characterizedstrains has been created. This is an interactive web based database so that the datacan be exchanged between laboratories electronically. The molecular characteri-zation of Listeria isolates from India would help in better understanding of thesources of infection and their risk assessment, routes of transmission of the in-fective agent, influencing factors, clinical forms and virulence characteristic ofthe pathogenic isolates of Listeria in order to diagnose the infection rapidly and re-liably but also in instituting the effective control measures.

* Participation Supported by Fundação do Oriente

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Human listeriosis and co-morbidities in England, 1999 to 2008:quantifying the riskMook, P., Grant, K., O’Brien, S. J. and Gillespie, I.Health Protection Agency, UK

Listeria monocytogenes causes a rare but severe food borne disease (listeriosis),commonly affecting pregnant women, the elderly and the seriously ill. The epi-demiology of listeriosis in England and Wales changed between 2001 and 2008,with more patients aged ≥60 years presenting with bacteraemia. We examinedthe risks in this age group, and quantified the role of co-morbidities for listeriosisin all age groups. Case-patients were resident in England between 1999 and 2008and were reported to an enhanced national surveillance scheme by hospital mi-crobiologists using a clinical case questionnaire. We coded co-morbidities of nonpregnancy-related cases of L. monocytogenes infection according to ICD-10. Thesedata were compared with appropriate denominator data (Hospital Episode Sta-tistics finished consultant episodes), to calculate incidence rates per million con-sultations (with appropriate 95% confidence intervals). Between 1999 and 2008,1412 non-pregnancy related cases of listeriosis were reported in England. We re-ceived a clinical questionnaire for 81% of cases (N=1141). Eighty-two percent(N=934) had one or more underlying medical conditions and we recorded 1239ICD-10 codes on co-morbidities from these 934 cases. The ≥60 years age groupcomprised 76% of all cases and 77% of all co-morbidities. Overall, the highest co-morbidity rates were diseases of the liver (192.9 episodes per million [95% CI:150.9-242.9]), systemic connective tissue disorders (163.1 [108.4-235.7]), malig-nancies of the lymphoid and haematopoietic tissue (137.5 [118.5-158.7]), alcoholism(107.5 [80.8-140.3]), renal failure (103.5 [83.3-127.3]), diabetes (98.4 [76.8-124.1]),hypertensive disease (71.7 [44.9-108.5]) and malignancies of the eye, brain & otherparts of CNS (66.3 [35.3-113.3]). We have highlighted several underlying condi-tions not previously thought to be strongly associated with listeriosis. The extentto which these co-morbidities are correlated with each other requires further in-vestigation to enable much better, targeted prevention.

Risk factors for death in Listeria monocytogenes infection, England andWales, 1990 to 2008Mook, P., Grant, K. and Gillespie, I.Health Protection Agency, UK

Listeriosis, caused by the Gram positive bacterium Listeria monocytogenes, is arare but severe food borne disease. The elderly, pregnant and the seriously ill aremost often affected, with high mortality rates in all patient groups. The epidemi-ology of listeriosis in England &Wales has changed in recent years, with an averageof 179 cases reported annually from 2000 to 2008 compared with 110 cases on av-erage between 1990 and 1999. Much of the increase has occurred in patients > 59years presenting with bacteraemia but without central nervous system involve-ment. To examine factors influencing L. monocytogenesmortality, non-pregnancyassociated cases resident in England & Wales reported to national surveillancebetween 1990 and 2008 (N=1832) were analysed further. The overall mortalityrate for this period was 39% (N=719). Univariable and subsequent multivariableanalyses were performed and the role of key factors (season, age, underlying con-ditions and treatment) in this outcome investigated. Our initial findings suggestthat, for a disease which disproportionately affects the elderly and the infirm, mor-tality is greatest in older patients and in those with underlying conditions. Un-derlying conditions themselves (particularly alcoholism and malignancies of thelymphatic/haematopoietic system and the breast) appear to be more importantthan the treatment they were receiving for an underlying condition, even whenthe effect of age is considered. Given that listeriosis is largely a food-borne dis-ease, and therefore preventable, it would seem appropriate to actively target highrisk groups with specific advice on what foods to avoid in order to minimise therisk of listeriosis.

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A discrete event model to track Listeria monocytogenesin the retail environmentPouillot, R.1 and Gallagher, D.2

1. CFSAN/FDA, USA2. Va Tech, USA

A recent comparative Listeria monocytogenes (Lm) risk assessment predicts that ofthe listeriosis cases attributed to deli meat, a majority are associated with delimeat sliced and packaged at retail. There is a need to: 1) identify potential sourcesand practices that contribute to Lm contamination of food at the retail level; and 2)identify retail practices that would reduce or eliminate Lm contamination ofready-to-eat foods prepared and sold to consumers. Within the scope of an inter-agency U.S. Food and Drug Administration (FDA) and U.S. Department of Agri-culture/Food Safety and Inspection Service (USDA/FSIS) risk assessment of Lm atretail, a dynamic discrete-event model that tracks Lm cells at various locationsover time in a retail setting was developed. This model considers i) the transfer ofbacteria from some defined compartment to others (unsliced products, slicedproducts, food contact surfaces, non food contact surfaces, slicer, gloves, hands,etc.); ii) the bacterial inactivation through cleaning and disinfection; and iii) thebacterial growth according to the physic and chemical environment (temperature,pH, water activity, presence of growth inhibitors). Sequences of events were de-rived using specifically acquired data from an observational study of food han-dling practices in retail deli departments. Additional data on the transmission ofLm in the retail environment is being collected through laboratory studies. More-over, this risk assessment is designed to evaluate the relative effectiveness of retailLm control measures along with other risk management scenarios. The major un-certainty is in the potential existence of niches and their interaction with the retailenvironment. This presentation will describe this model, provide an illustrationusing current data and highlight the data gaps that mainly influence the outputs.

Clinical and epidemiological aspects of listeriosis in IsraelHershko-Klement, A.1, Eliav, H.2, Valinsky, L.3, Schechner, V.4, Braun, E.5, Paitan, Y.1,Block, C. S.2 and Nir-Paz, R.2

1. Meir medical center, Kfar-Saba, Israel2. Hadassah Hebrew University Medical Center, Israel3. Israel Ministry of Health, Central Laboratories4. Tel-Aviv Souraski medical Center, Tel Aviv, Israel5. Rambammedical center, Haifa, Israel

Listeria monocytogenes (LM) is a ubiquitous foodborne pathogen. Invasive infec-tions are rare, mainly affecting elderly people, the immunocompromised andneonates. In pregnancy it causes fetal loss, preterm delivery and neonatal mor-bidity. Although the incidence of listeriosis is low compared with other en-teropathogens, it carries a high mortality. Our aim was to characterize LM mor-bidity, risk factors andmolecular epidemiology in Israel to improve understandingof the disease burden in Israel and assist public health policy-making. We per-formed a nationwide retrospective study of all LM cases during 1998–2007. Caseswere actively sought in the records of all hospital-based clinical microbiology lab-oratories, the national reference LM laboratory and LM cases reported to the dis-trict physicians. 481 cases were identified, of which 166 were pregnancy-associ-ated. The yearly incidence peaked in 2006 and 2007 at 9.6 cases/million, which isalmost 5 times than the current rate in the USA. The incidence in pregnancy var-ied between 5 and 25 cases/100,000 pregnancies/year. Annual rates above 15cases/100,000 occurred several times. The perinatal case fatality rate was 47%,comprising a fetal loss of 38.2% and additional 7.9% neonatal mortality. Fetal sur-vival improved as pregnancy age advanced (P<0.05). LM associated late abortionoccurred in 26.6% and preterm labor in 46.8%. A single maternal death wasrecorded. The case fatality in non-pregnancy cases was 18%. Additionally a gradualincrease in elderly occurred during the years and reached 42.7 cases/million/year.Geospatial analysis revealed 2 districts with a higher incidence of listeriosis. Mo-lecular analysis (PFGE) of LM isolates suggested that a third of LM pregnancy as-sociated cases were caused by a single clone. In Israel, Listeria monocytogenes is animportant foodborne pathogen causing appreciable morbidity and mortality. Fur-ther studies of the sources, molecular epidemiology and specific virulence deter-minants should be undertaken.

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Human isolates of Listeria monocytogenes duringhalf a century in SwedenLopez-Valladares, G., Danielsson-Tham, M.-L., Vishal Singh, P. and Tham, W.School of Hospitality, Culinary Arts &Meal Sciences, Örebro University, Grythyttan, Sweden

Over 800 isolates of Listeria monocytogenes have been collected from cases of in-vasive listeriosis in Sweden – the first from 1958 and the latest from spring 2010.All isolates are serotyped and characterized with pulsed-field gel electrophoresis(PFGE) and Asc I restriction enzyme. From 1972 to 1995, serovar 4b was the pre-dominant serovar in human invasive listeriosis in Sweden. During the 1980s, somelisteriosis outbreaks caused by L. monocytogenes serovar 4b, belonging to a spe-cial PFGE type, were diagnosed in the USA and Europe and were due to consump-tion of dairy products. The predominant strain in listeriosis cases in Sweden dur-ing the same time belonged to this type. The hypothesis is that a majority of humanlisteriosis serovar 4b strains in Sweden came from soft cheeses imported fromMediterranean countries. The hunt for L. monocytogenes serovar 4b in food pro-duction plants is intensive in EU and USA and during recent years, cheeses havehad higher microbiological quality due to certified dairy farms and increased mi-crobiological control. This may be the reason for the decrease of serovar 4b casesof listeriosis in Sweden. In 1996, serovar 1/2a became the major serovar in liste-riosis cases in Sweden. The consumption of gravad and cold-smoked salmons hasincreased in Sweden, and in a recent study, 12.9% of ready-to-eat vacuum-packedgravad salmon and 28.0% of cold-smoked salmons harboured L. monocytogenes.The maximum number of L. monocytogenes was 1500 cfu/g product. The mostcommon PFGE types found in human cases of listeriosis in Sweden today are alsothe types frequently encountered in vacuum-packed cold-smoked and gravadsalmon/rainbow trout.

Estimated incubation periods for listeriosis vary according to clinicalform of diseaseGoulet, V., King, L., Vaillant, V. and De Valk, H.Institut de Veille Sanitaire, France

Data on the incubation period of listeriosis are scarce. The incubation period canbe calculated precisely when a patient has had a single exposure to a confirmedsource of contamination. This information can be collected during the investiga-tion of point-source food borne outbreaks. Our study aimed to estimate the liste-riosis incubation period using available outbreak investigation data. Cases of in-vasive listeriosis from confirmed food borne point-source listeriosis outbreakswith precisely documented incubation periods and clinical forms of infection wereidentified during the period 1985–2008 by literature review and from non-pub-lished French outbreaks. Data from 9 published papers and from 6 French out-breaks were analysed. A precise incubation period was documented for 28 cases: 13with central nervous system (CNS) involvement (10 French outbreak and 3 pub-lished cases), 15 pregnancy-associated cases (12 French outbreak and 3 publishedcases). A confirmed food vehicle was identified for all outbreaks (rice salad, ice-cream, cheese, meat-based products). The overall median incubation period was 17days (range: 2–88 days). A longer incubation period was observed for pregnancy-associated cases (median: 28 days (range: 14–88 days)) than for cases with CNS in-volvement (median: 10 days (range: 2– 19 days)). Insufficient data did not allowfor analysis of the incubation period of the bacteraemic form of infection. Our re-sults suggest that the incubation period for listeriosis varies according to the clin-ical form of disease. A longer incubation period was observed for pregnancy-as-sociated cases than for cases with CNS involvement. This information could haveimplications for the investigation of food borne listeriosis outbreaks as the incu-bation period is used to determine the time period for which a food history is col-lected. Adapting this time period according to the clinical form of infection couldfacilitate a more precise identification of food products likely to be the source ofcontamination.

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Listeria monocytogenes in food and animalsin the European Union in 2008da Silva Felício, M. T., Rizzi, V., Boelaert, F. and Makela, P.Scientific Cooperation and Assistance Department, EFSA, Italy

The Directive 2003/99/EC on Zoonoses obligates the European Union (EU)Mem-ber States (MSs) to collect data of zoonoses, zoonotic agents, antimicrobial resist-ance and food-borne outbreaks. These data are reported to the European FoodSafety Authority (EFSA) who publishes the Community Summary Report. In 2008,a large number of investigations (128,000) concerning ready-to-eat (RTE) food-stuffs were reported by 26MSs. The food categories most often covered were RTEmeat products, cheeses and fishery products. L. monocytogenes was seldom de-tected above the legal safety limit of 100 cfu/g from RTE foods and findings overthis limit were most often reported from fishery products, cheeses, meat productsand sandwiches at levels of 0.2–0.5% in the EU. L. monocytogenes was isolatedfrom both cheeses made from raw or low-heat-treated milk and pasteurised milkas well as from soft/semi-soft and hard cheeses. L. monocytogenes was most oftendetected in soft and semi-soft cheeses made from pasteurised milk. L. monocyto-genes was also reported, generally at a relatively low prevalence, from various an-imal species in 2008, demonstrating that animals act as one reservoir of Listeriabacteria although they rarely serve as a direct source of human infections. Thehighest prevalence of L. monocytogenes was found in sheep, goats and cattle. Re-ported data on the findings in RTE foods may be used to guide food controls car-ried in MSs to ensure compliance with L. monocytogenes criteria. Compared toprevious years the quality of the data received improved as regards reporting of thestage of sampling and the use of appropriate test methods that has eased the as-sessment of compliance with Listeria criteria at Community level.

Listeria monocytogenes infection in the over 60s in Englandbetween 2005 and 2008: a retrospective case-control study utilisingmarket research panel dataGillespie, I. A., Mook, P., Little, C. L. and Grant, K.Health Protection Agency, UK

Listeriosis is a rare but life-threatening foodborne disease and, therefore, the in-vestigation of factors whichmight alter people’s risk of infection is important to in-form on prevention and control. The incidence of listeriosis in England has dou-bled since 2001, with a largely unexplained increase in people aged ≥60 yearspresenting with bacteraemia without central nervous system involvement. Stan-dardised epidemiological data has been sought on cases of listeriosis reported inEngland since 2005, but the value of the data accrued is limited without someknowledge of exposure prevalence in the population at risk of listeriosis. The ex-posures of listeriosis cases aged ≥60 years reported in England from 2005–2008were compared to those of market research panel members representing the samepopulation and time period. Exposures were grouped to facilitate comparison.Odds ratios and 95% confidence intervals were calculated. Cases were more likelythan panel members to report cold cooked meats (beef and pork, but not chicken),smoked fish (specifically salmon) and shellfish (prawns), dairy products (milk andcertain cheeses) andmixed salads. They were less likely to report the consumptionof sandwiches and fresh vegetables. To our knowledge this is the first time thatmarket research data has been applied to infectious disease epidemiology in thisway. The diversity of high-risk food exposures reflects the ubiquity of the mi-croorganism in the environment and/or the susceptibility of those at risk, and sug-gests that a wider variety of foods can give rise to listeriosis. Food safety advice onavoiding listeriosis should be adapted accordingly.

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Better and faster typing, MLVA – shall we play together?Larsson, J. T.1, Roussel, S.2 and Moller Nielsen, E.1

1. Statens Serum Institut, Copenhagen, Denmark2. Agence Française de sécurité sanitaire des aliments, Maisons-Alfort, France

In 2007 and 2008 there were four papers published on the subject of MLVA (MultiLocus VNTR [Variable Number of Tandem Repeats] Analysis) in L. monocytogenes.During the same period a protocol with an extensive bioinformatic approach wasdeveloped at SSI. The yet unpublished SSIMLVA protocol was created after analy-sis of 20 genomes. Ten VNTRs were chosen on the basis of both nucleotide andamino acid sequences analysis. The VNTRs have repeat lengths ranging from 6 to15 basepairs. Degenerate primers were designed to match the diversity in thegenomes, taking good care not to include any gaps (which were present in severalloci/genomes). Finally three multiplex PCR groups were created and the VNTRswere analysed by electrophoresis. The SSI method has been evaluated with thelast years Danish human listeriosis cases and the North American ILSI Listeriacollection. Results indicate a very close match to two-enzyme PFGE and an evenbetter separation of isolates. These results in concert with the reduced time andcost for using MLVA for typing started off an incentive for unification and stan-dardisation of MLVA in L. monocytogenes. In a recently started cross laboratorystudy, a comparison of the protocol published by Kate Sperry (2007) and the pro-tocol developed at SSI is under way. The methods are evaluated together with tra-ditional typing methods such as PFGE and agglutination serotyping. The MLVAprotocols are tested using capillary and agarose gel electrophoresis. Strain collec-tions includes; a selection of the WHO subtyping study from 1986 and a selectionfrom food and human isolates samples from France and Denmark respectively.The chosen method might include a reduced set of loci and/or a mixture of thetwo assays. The results from this cooperation will lead to a recommendation on aMLVA protocol for use in Denmark and France.

comK prophage junction fragments in Listeria monocytogenes containSNPs that differentiate subclones of ECII and ECIII that are unique toindividual meat and poultry processing plants in the U.S.Knabel, S. J.Department of Food Science, Penn State University, USA

Many outbreaks of listeriosis in the U.S. are due to epidemic clones II and III, es-pecially those associated with ready-to-eat meat and poultry products. Both ofthese epidemic clones have backbone genomes that are highly conserved, but con-tain a hypervariable 40 Kb comK prophage. All isolates analyzed in the presentstudy that were suspected of being ECII or ECIII were first confirmed as such bymultiplex PCR and multi-virulence-locus sequence typing (Chen and Knabel,2007) and included those from the 1998–1999 hot dog and 2002 turkey deli out-breaks in the U.S., those frommultiple states that were generated as part of USDAFSIS's meat and poultry plant surveillance program, and those that were previ-ously isolated from two turkey processing plants in two different states (Eifert etal. 2005). Upstream and downstream prophage junction fragments in all ECII andECIII isolates were amplified using 1/4 and 2/3 primer sets modified from Loess-ner et al. (2000) and subsequently sequenced. The junction fragments in ECIIIisolates from a sporadic case in 1988 and from an outbreak in 2000 due to turkeydeli meat in the U.S. were also amplified and sequenced. Cluster analysis based onjunction fragment sequences revealed the presence of five subclones of ECII, theECIII subclone and the Canadian 2008 subclone, which were unique to individualmeat and poultry processing plants. However, the ECII subclone that caused the2002 outbreak was found in three different processing plants, two of which wereoriginally associated with this outbreak. We speculate that evolution of these sub-clones may be due to extensive recombination and subsequent niche-specificadaptation acting on the comK prophage, which may represent a “Rapid Adapta-tion Island” in Listeria monocytogenes. Genes that were unique to the comKprophage were identified as putative “Adaptons” and their possible roles in colo-nization, transmission between and within meat and poultry plants, and subse-quent contamination of foods and humans will be discussed.

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Genetic diversity of Listeria monocytogenes measured by multiple-locusvariable-number tandem repeat analysis (MLVA)Hyytia-Trees, E., Sabol, A., Graves, L. and Ribot, E.Centers for Disease Control and Prevention, USA

Listeria monocytogenes is a genetically and phenotypically diverse organism, en-compassing at least three genetically distinct lineages that appear to differ in theirlikelihood of causing human and animal disease. Classification of isolates intothese three lineages has been confirmed by different subtyping methods, and alsocorrelates with serotype classification. In the present study, 250 epidemiologi-cally unrelated isolates from 1981 to 2009 representing serotypes 4b (93), 1/2a(70), 1/2b (57), 3a (4), 4c (4), 4a (2), 1/2c (2) and untypeable (18) were character-ized using multiple-locus variable-number tandem repeat analysis (MLVA). Theisolate set also included one isolate from each of ten well characterized outbreaks.The data was analyzed in BioNumerics software and a dendrogram was con-structed by UPGMA clustering using the categorical coefficient. A minimal span-ning tree was also created using theManhattan coefficient. In the minimum span-ning tree, a clonal complex was defined as a group of related patterns that differedfrom each other at a single locus by one or two repeats. A total of 139 unique pat-terns were detected among the 250 isolates. Thirty one MLVA patterns were as-sociated with more than one isolate, with the most common pattern including 16isolates with isolation years varying from 1983 to 2007. With very few exceptions,the isolates clustered according to their lineage. Nineteen clonal complexes weredetected, encompassing a total of 129 isolates. Two different serotypes (1/2a & 3a,1/2a & 1/2c) shared the same clonal complex in two instances. Five of the ten out-break related isolates were located in three different clonal complexes that in-cluded nine or more isolates with multiple isolation years and origins in differentcontinents. In conclusion, the MLVA scheme used in the present study is in ac-cordance with the currently used lineage classification and indicated the presenceof successful epidemic clones.

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AREA //D // Strategies for prevention and control of Listeria monocytogenesORAL PRESENTATIONS //O / 37–46

Time Temperature Indicators can be used as an effective RiskManagement Tool for Listeria monocytogenes in Ready-To-Eat foodsKoutsoumanis, K., Vaikousi, H. and Costas, B.Department of Food Science and Technology, School of Agriculture,Aristotle University of Thessaloniki, Greece

The objective of the present work was to evaluate the effectiveness of Time Tem-perature Indicators as risk management tools for Listeria monocytogenes in RTEfoods. In a previous study we presented a microbial Time Temperature Indicator(TTI) system based on the growth and metabolic activity of a Lactobacillus sakeistrain. In the latter system, an irreversible color change of a chemical chromaticindicator (from red to yellow) progressively occurs due to the pH decline as a re-sult of L. sakei growth andmetabolism in a selected medium. In this study we showthat both the L. sakei growth and colour change of the microbial TTI system ex-hibit similar kinetic responses with the growth of L. monocytogenes. With an ap-propriate selection of the initial level of L. sakei in the system, the TTI end point(time at which a distinct visual color change to the final yellow was observed) canbe adjusted in order to indicate a certain level of L. monocytogenes growth in thefood during distribution and storage. Thus, the use of the microbial TTI can as-sure a maximum limit in the growth of the pathogen from production to con-sumption time by informing the consumers when this limit is exceeded in a prod-uct unit. The latter limit, which we call Growth Tolerance Criterion (GTC), can beconsidered as a performance criterion for the growth of L. monocytogenes duringdistribution and storage. The applicability of the microbial TTI in reducing con-sumer exposure to L. monocytogenes from the consumption of pate was evaluatedin a simulation study. The TTI was appropriately adjusted to provide a GTC=3 logsCFU/g. The concentration of the pathogen and the colour of the TTI at the time ofconsumption were estimated with a probabilistic approach using Monte Carlosimulation. The simulation results showed that the application of the TTI resultedin a significant reduction of consumer exposure to L. monocytogenes. Assumingthat packages in which the TTI end point has been reached before consumptionare discarded, the predicted percentage of consumed products contaminated withL. monocytogenes concentrations above 103 CFU/g was reduced from 5% to 0.2%with the use of TTI.

Safety of salad leaves and herbsGarland, C. D.1 and Clark, A.2

1. FWE Health, North Hobart, Tasmania, Australia2. Houstons Farm, Cambridge, Tasmania, Australia

Comprehensive HACCP-based procedures have been implemented to prevent Lis-teria monocytogenes contamination of RTE (ready-to-eat) salad leaves and herbs ina commercial facility in Tasmania, Australia. Production has increased from 27tonnes in 1995 to 2000 tonnes in 2009, with cultivation now undertaken at 3 fieldsites and processing in a centralised factory certified to third party audited foodsafety standards. The range of consumers is wide, including typical high-riskgroups. More than 1200 samples of RTE products have been tested since 1995 andcurrently 300 RTE samples are tested at early and end of shelflife annually. To date,L. monocytogenes has been detected in one RTE sample only, an outstanding result.Control Points/Critical Control Points for minimising Listeria contamination infield conditions are implemented for: lettuce transplants; cultivation soil; catch-ment animals and wild life; irrigation water; harvesting; acceptance/rejection cri-teria for raw ingredients; potable water; transport of harvest to factory. CPs/CCPsin the factory relate to: restricted access; strict separation of work spaces into low-medium-high risk zones; temperature control; reduction of microbial loads; prod-uct packing; staff training; sensory monitoring of product; physical and microbio-logical monitoring. CPs/CCPs for transport to wholesaler and retailer include:sealed box packing, visual checking; sealed vehicle; temperature control/data log-ger; microbiological monitoring. Data will be presented to illustrate key microbi-ological and physical CPs/CCPs relating to: animal access to the field, soil addi-tives and composts; surfaces, equipment, potable water, irrigation water, rawingredients and RTE products; factory temperature control (ambient, water, prod-uct); removal of organisms by washing, centrifugation and infrared drying, anddisinfection by chlorination (with auto adjustment of free available chlorine andpH); transport.

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Environmental factors affect adhesion of Listeria monocytogenesto inert surfaces through flagellum expressionTresse, O.INRA, France

Listeria monocytogenes could adhere to inert surfaces and form biofilms. Biofilmsof L. monocytogenes constitute eventually a reservoir for cross- or re-contamina-tions of food products in food-processing plants. Biofilm initiation includes an ir-reversible attachment of cells (adhesion) to inert surfaces. Adhesion is thereforecrucial for the biofilm development and subsequently for dissemination and per-sistence of food pathogens. Adhesion assays from four to 101 L. monocytogenesstrains from diverse origins (food, food-processing environment and clinical cases)were conducted in polystyrene and stainless steel 96-wells microtiter plates. Ad-hesion was also assessed after cell cultivation at different pHs, NaCl concentra-tions and temperatures. In parallel, flagella were observed after cultivation in thedifferent environmental conditions using optical microscopy. Three naturally afla-gellated strains, five environmental flaA mutant strains and one flagellum-para-lyzed strain were also tested to verify the contribution of flagella to adhesion. Re-sults indicated that attached cells were significantly higher at pHs greater or equalto 6, at 8 °C and 20°C or at 0% and 6% NaCl than at pH 5, at 37 °C or at 11% NaCl.Correlatively, flagella were not detected at pH 5, at 37 °C and at 11% NaCl. Thisresult indicates that the temperature is not the only environmental factor thatcould regulate flagellum expression in L. monocytogenes. NaCl concentrations andpHs closed to non-growing conditions inhibit flagellum expression. Naturally afla-gellated strains and ∆flaA mutants adhered at the same level of strains cultivatedin flagellum inhibiting conditions. In addition, ∆flaA mutants adhered equallywhile adhesion capability of the respective parental strains varied according tothe strain. Furthermore, adhesion was also affected in the flagellum-paralyzedstrain indicating that motility plays an important role in adhesion capability. Inconclusion, conditions of food conservation have an effect on adhesion capabilityof L. monocytogenes strains through flagellum regulation and flagella are respon-sible for adhesion variation among strains.

Shelf-life laboratory durability and challenge studies forListeria monocytogenes in ready-to-eat foods: a presentation of theEuropean technical guidance document intended for laboratoriesBeaufort, A., Bergis, H., Cornue, M. and Lardeux, A.-L.Agence Française de Sécurité Sanitaire des Aliments (AFSSA), France

Listeria monocytogenes is a foodborne pathogen that may grow at refrigerationtemperatures and may be present in ready-to-eat (RTE) foods. In annex I of regu-lation (EC) No 2073/2005 on microbiological criteria for foodstuffs, a specific at-tention to food safety criteria for L. monocytogenes in RTE foods is given and annexII of this regulation specifies that food business operators (FBO’s) shall conduct, asnecessary, studies to evaluate the growth of L. monocytogenes that may be pres-ent in the product during the shelf-life under reasonably foreseeable storage con-ditions. But this annex does not describe the procedure to conduct such shelf-lifestudies to ensure that the criterion of 100 L. monocytogenes/g is met over the entireintended shelf-life of the product. The European technical guidance document,intended for laboratories conducting shelf-life studies for L. monocytogenes in RTEfoods in collaboration with the FBO’s, provides recommendation on how to select,how to implement and how to perform the test(s) required. It describes the mi-crobiological procedures for determining growth of L. monocytogenes using chal-lenge tests and durability studies. Challenge tests, defined as tests providing in-formation on the behaviour of L. monocytogenes artificially inoculated in the food,can be performed with two different objectives: (i)Classify whether a food does ordoes not support growth of L. monocytogenes; (ii) Quantify the growth of L. mono-cytogenes by assessing either the growth potential (δ) or the maximum growth rate(∝max). Durability studies allow an evaluation of the growth of L. monocytogenes ina naturally contaminated food during its storage according to reasonably foresee-able conditions. This technical document, prepared by the EU Community Refer-ence Laboratory for L. monocytogenes in collaboration with a working group con-sisting of 10 laboratories, including nine National Reference Laboratories for L.monocytogenes, is available since 15 December 2008 on the website of the Euro-pean Commission, DG Health and Consumers.

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Successful strategies against Listeria monocytogenes in SwitzerlandImhof, R.Agroscope Liebefeld-Posieux Research Station ALP, Switzerland

In the aftermath of the massive Listeriosis outbreak in 1987 due to a soft cheesemade out of raw milk, the Swiss government decreed the creation of appropriatemeans to prevent a repetition of such a case. Agroscope Liebefeld-Posieux Re-search Station ALP was given the order to maintain a laboratory for the detectionof Listeria and to develop a nationwide Listeria monitoring programme (LMP) incooperation with the Swiss dairy industry. LMP covers about 70 to 80% of thecheese production of Switzerland. The aims are to detect contamination in cheeseproduction sites and ripening centres as early as possible, to stop the spreadingof Listeria by means of appropriate measures and above all to prevent contami-nated products to be placed on the market. The programme is legally based on pri-vate law and guaranties confidentiality to its members. Although the results arenot open to the public, ALP closely works together with the concerned public au-thorities in cases of enterprises with problems. ALP also created a special taskforce: By incidence of problems with Listeria the ALP consulting team can be de-manded from any enterprise for analysis and advice as well as for direct partici-pation in the process of complete redevelopments. Following defined proceedingsthe ALP team helps in cooperation with the enterprises own emergency team tofind individually designed solutions. Periodical controls in the following yearsproof the success and the sustainability of interventions and worked out securityconcepts. The history of outbreaks and the European RASFF notifications of thelast 15 years concerning Switzerlandmanifest the success of this specific approach.Despite many ingenious efforts and product developments, there exists – to ourknowledge after 20 years of specific experience – no single measure or method tosolve a problem within the dairy food chain caused by L. monocytogenes. But ob-servance of the principles of GoodManufacturing Practice, a specifically designedconcept of security – which has to be lived by staff and personnel – besides to pe-riodical controls guarantee that every production site is capable to produce anddistribute safe and healthy dairy products.

Absence of Listeria monocytogenes growth during raw milkcheesemaking: a modelling approachJordan, K.1, Schvartzman, S.1,2, Butler, F.2 and Tenenhaus-Aziza, F.3

1. Teagasc, Moorepark Food Research Centre, Ferrmoy, Cork, Ireland2. Biosystems Engineering, School of Agriculture, Food Science and Veterinary Medicine,

University College Dublin, Ireland3. CNEIL, National Interprofessionnal Center for Dairy Economy, Paris, France

The presence of Listeria monocytogenes in certain foods and the risk that this posesto public health and food quality is still a problem. Currently, the field of food mi-crobiology focuses on obtaining data on the behaviour of microorganisms in food,but the responses obtained provide little insight into the relationship betweenphysiological processes and growth or survival. This link can be made throughmathematical models. In a simple form, a mathematical model is a simple mathe-matical description of a process. The application of mathematical models to foodmicrobiology has been developed in recent years and now constitutes a new disci-pline named as Predictive Microbiology. However, most predictive models arebased on laboratory experiments in microbiological media under static conditions.As such models tend to be inaccurate, we have undertaken our experiments in afood system under dynamic conditions. Cheese was made with raw and pasteurisedmilk deliberately contaminated with L. monocytogenes. Listeria was monitoredthrough the manufacture and ripening period of the cheese. The results showedthat L. monocytogenes did not grow during manufacture of raw milk cheese, butdid grow during manufacture of pasteurised milk cheese. The data obtained forgrowth, survival and inactivation was modelled. The application of models thatcan explain the behaviour of Listeria in cheese and the further predictions thatcan be obtained from these models are useful for the improvement of ongoing re-search on biotraceability and for the better understanding of the general behaviourof these microorganisms under dynamic conditions, such as in dairy products.

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A predictive model to set high pressure processing criteria forListeria monocytogenes inactivation on dry-cured hamBover-Cid, S.1, Belletti, N.2, Garriga, M.1 and Aymerich, T.1

1. IRTA. Food Technology, Spain2. Università di Bologna, Italy

The aim of the work was to validate and use a predictive mathematical modelfor the inactivation of Listeria monocytogenes on dry cured ham by high hydro-static pressure (HHP) processing, as a function of the technological parameters:intensity, length and fluid temperature. The model was previously developedfrom the inactivation data obtained after processing sliced dry-cured ham,spiked with L. monocytogenes, at different HHP conditions according to a centralcomposite design: at 347 –852MPa; for 138 to 945 seconds; at 7.6 to 24.4 °C. Thebest fitting and most significant polynomial equation indicated that pressureand time were the most important factors determining the inactivation extent.By contrast, temperature showed no significant influence within the assayedrange. The statistically significant quadratic terms of pressure and time indicatethe little effect observed below 450MPa. Similarly, holding time longer than10min did not result in a meaningful reduction of L. monocytogenes counts. Themathematical model was validated with data obtained from further experimentswithin the assayed experimental domain, including data from international sci-entific literature. The accuracy and bias factor were within the proposed ac-ceptable values demonstrating the suitability of the model for predictive pur-poses. As a practical application of the validated model, the mathematicalequation was used to predict the HHP process conditions needed to ensure thesafety performance criteria established by different authors and organisations, interms of logarithmic reductions of L. monocytogenes loads. The general per-formance criteria of 6Log reduction, as equivalent to pasteurization, suggestedby the Food and Drug Administration would be achieved with treatments above600MPa, for instance applying 807MPa for 5min or 746MPa for 10min. Moreproduct-specific performance criteria proposed, i.e. 4D and 2.39D, would beachieved with 5min treatments at 703MPa and 613MPa, respectively.

Application of a validated predictive model to prevent growthof Listeria monocytogenes in ready-to-eat foods – importancefor product development and risk managementMejlholm, O. and Dalgaard, P.Seafood & Predictive Microbiology, Technical University of Denmark

Predictive microbiology is a most active research area within food microbiologyand many successfully validated models are today available and useful for assess-ment and management of microbial food safety and quality. Recently, an exten-sive growth and growth boundary model for Listeria monocytogeneswas developedand successfully validated for ready-to-eat meat, seafood, poultry and non-fer-mented dairy products. This model is flexible and includes the effect of 12 envi-ronmental parameters (temperature, NaCl/aw, pH, smoke intensity, nitrite, CO2,acetic acid, benzoic acid, citric acid, diacetate, lactic acid and sorbic acid) as well astheir interactive effects. It can predict the growth boundary and thereby combi-nations of product characteristics and storage conditions that prevent growth of L.monocytogenes and this is most important in relation to EU regulations and codexguidelines. Nevertheless, growth boundary predictions alone are insufficient forformulation of safe foods because the likely variability in product characteristicsand storage conditions must be taken into account. For this, stochastic models canbe applied together with distributions of the relevant environmental conditions.Important parameters to control growth of L. monocytogenes in ready-to-eat foods,however, are not yet included in those models. We here present a simple alterna-tive that allow variability in product characteristics and storage conditions to betaken into account when conditions that prevent growth of L. monocytogenes arepredicted. This approach relies on validated cardinal parameter growth models.The effect of interaction between environmental parameters is used to predictboth the growth boundary and interface conditions within the no-growth region.Data from ready-to-eat foods will be used to show how these no-growth interfacescan be predicted in a sufficient distance from the growth boundary to account forvariability in the environmental conditions.

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Application of predictive microbiology to control the growthof Listeria monocytogenes – dairy products as an exampleLobacz, A.Faculty of Food Sciences, University of Warmia and Mazury in Olsztyn, Poland

Predictive microbiology is a relatively new scientific discipline which is used formodelling behaviour ofmicroorganisms,mainly food borne pathogens, as a responseto environment. It is based on assumption that the reaction of bacteria on particularenvironment factors is reproducible, and on the basis of researches and observationsmade in past, it is possible to predict the microbiological behaviour in defined envi-ronment. Many mathematical models, with regard to Listeria monocytogenes, havebeen generated, which describe survival, inactivation or growth of this food bornepathogen as a response to environmental parameters. Models are generated usuallyinmicrobiological liquidmedia, althoughmore often data are obtained in food prod-ucts. In the following paper usefulness of predictivemodels describing the growth ofL. monocytogenes is presented. Application of tertiary models – ComBase Predictor(www.combase.cc) and PathogenModeling Program (www.ars.usda.gov) is also dis-cussed. Moreover, microbiological experiment was performed in order to evaluatebehaviour of L. monocytogenes in Camembert type cheese during ripening and stor-age at following temperatures: 3, 6, 9, 12, 15 and 21°C. Primary and secondarymodelswere generated and their performance was checked by mathematical and graphicalvalidation. Predictivemicrobiology is a new tool tomicrobiological risk assessment.As a subdiscipline of food microbiology, it is an intensively developing field. Gener-ated and validated predictivemodels becomemore accurate andmore often used infood industry in order to eliminate food borne outbreaks.

Characterization of Food Alert for Listeria monocytogenes in France in2008Leclercq, A.1, Dusch, V.2, Salah, S.2, Laurent, E.3, Chenal-Francisque, V.1, Thierry-Bled, F.4,Lecuit, M.1, Goulet, V.3 and Pihier, N.2

1. Institut Pasteur, NRC andWHO-CC for Listeria, Paris, France2. DirectionGénérale de l’Alimentation,Ministère de l’Alimentation, de l’Agriculture et de la Pêche, France3. Institut de Veille Sanitaire (InVS), France4. Direction Générale de la Concurrence, de la Consommation et de la Répression des Fraudes,

Ministère de l’Economie, de l’Industrie et de l’Emploi, France

Among measures established to assure a high level of protection of human healthand consumers, the European regulation EC 178/2002 indicates that food businessoperators shall notify all foodstuffs not in compliance with the food safety criteriafor Listeria monocytogenes (Lm) described in regulation EC 2073/2005. A food alertis established when a foodstuff presenting a risk is on themarket, or when rapid ac-tion (withdrawal or recall) is required. In the present study, our aim was to charac-terize all these French food alerts (FAs) for Lm in 2008. In case of FA, competentauthorities asked the food business operators or/and inspection services to sendLmfood strains from own-checks and official control to the NRC for Listeria to obtaintheir genoserotype and combined AscI/ApaI patterns by PFGE. Weekly, compari-son ofmicrobiological characteristics of food and human strainswere performed ona period of 6 months and were sent to involved competent authorities and FrenchInstitute of Public Health Surveillance (InVS) with the aim of possible complemen-tary investigation, if necessary. In 2008, 280FAs forLm have been registered,mainlyat the retail level (184), fromown-checks (215) and official control (65). Among these280 FAs, Lm enumerations on sample(s) of FAs have been performed in 260 FAs[Range: 10-740,000 CFU/g]. Incriminated foods were mainly in decreasing order:meat products (134 FAs), raw milk cheeses (47 FAs) and seafood/fish products (40FAs). 179FAswere only followed by food strains invoice [Range: 1-52 strains/FA; 100FAswith a single strain associated], representing 673 food strains. Genoserotypes offood strains were at 55% IIa, 21% IVb, 19% IIb and 4% IIc. Combined AscI/ApaIpatterns of strains varied from 1 to 27 per FA [137 showed single combinedAscI/ApaI pattern]. 60% of combined AscI/ApaI patterns for FAs food strains havebeen also observed for human strains in 2008. 84FAs have food strains foundmicro-biologically similar to human strains but no FA was linked with human cases in2008.Work is in progress for comparison of these data to 2007 and 2009 data. Evenif high food contamination were sometimes observed, FAs were not at the origin orfollowed by human cases in 2008. In addition with the measures performed by thefood business operators in the context with their sanitary control plan, food man-agementmeasures linked toFAsmay have contributed in the French situation of noLm outbreak since 2003.

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AREA //E // Communication, risk perception and consumer practices – Social sciences in Listeria controlORAL PRESENTATIONS //O / 47–49

Efforts to update and perform health education on listeriosisin Los Angeles County, California, by the County of Los AngelesDepartment of Public HealthGuevara, R. E.*County of Los Angeles Department of Public Health, USA

Listeriosis became a relatively well-known disease in Los Angeles County after thelarge southern California listeriosis outbreak in 1985. However, 20 years later, thetasks of keeping the general population and medical community properly educatedhave not been easy with public health priorities shifting across topics such as bioter-rorismpreparedness,MRSA,WestNileVirus, pandemic influenza, andmost recentlyH1N1 influenza. In 2005, health education efforts on listeriosis were specific to onlycertain high-risk groups and needed a broader audience. Understanding the Countyof Los AngelesDepartment of PublicHealth’s (DPH) health educationmission to en-compass the general community, amulti-disciplinary teamcomposed of epidemiolo-gists, public health nurses, and health educators produced new listeriosis educationmaterials. Public health messages were developed by those who conducted case in-vestigations by interviewing patients, relatives, and providers of listeriosis cases, andby thosewhoperformed epidemiologic surveillance. A standard of attractivenesswasset before recruiting health educators to help design the new education materials.Finding various images and pictures, health educators drafted different designs forposters, flyers, and brochures. Public health nurses formed focus groups to help re-fine health educationmessages, particularly formaterials translated into Spanish.Nosystematic measurement for success of the health education materials was estab-lished; however, the materials continue to be used by DPH as various health careproviders and organizations request them, and even when H1N1 influenza becamethe focus of public health inApril 2009, listeriosiswas themost common search termin theDPHwebsite. This presentation describes the approach theDPHused tomoreeffectively communicate the risks of listeriosis not just to thosewho are vulnerable tothe disease, but also to thosewhomight know or even take care of them.


Awareness of listeriosis among Portuguese pregnant womenMateus, T.1,3, Maia, R. L.2 and Teixeira, P.1

1. CBQF/Escola Superior de Biotecnologia – Universidade Católica Portuguesa, Porto, Portugal2. CECLICO/Centro de Estudos Culturais, da Linguagem e do Comportamento Faculdade de

Ciências Humanas e Sociais – Universidade Fernando Pessoa, Porto, Portugal3. Escola Universitária Vasco da Gama, Coimbra, Portugal

The role of Listeria monocytogenes as a foodborne pathogen was definitively recog-nizedduring the 1980s.This recognitionwas the consequenceof anumberof epidemichuman outbreaks due to the consumption of contaminated foods, in Canada, the USand Europe. Listeriosis is especially severe in immunocompromised individuals suchas pregnant women. The disease has a low incidence, although this is undeniably in-creasing, and a high fatality rate amongst those infected. In pregnant women listerio-sis may cause miscarriage, foetal death or neonatal morbidity in the form of septi-caemia andmeningitis. Improved education concerning the disease, its transmissionand preventionmeasures for immunocompromised individuals and pregnantwomenhas been identified as a pressingneed.The aimof this studywas to evaluate the aware-ness, in terms of food safety and listeriosis, of pregnantwomen in the last trimester ofpregnancy orwith babies up to 3months old. For this purpose 956womenwere ques-tioned as to their knowledge and practices relating to what foods to avoid, good hy-giene and cooking practices. Also of interest were the sources of information onListe-ria spp. and listeriosis, and from whom and how women would like to receive suchinformation.Twohundred and twentyninewomen said theyhadnot avoided anykindof food, whilst 306 said they had not made any particular changes to their food safetyrelatedhabitswhen theyprepared and cookedmeals. Fourhundred and seventy sevenwomen considered that they had received enough information about nutrition duringpregnancy, and that this information had been acquired from their doctor, who wasalso the person considered the most competent to give this information. A hundredand seventeenwomen said they had heard about listeriosis, although only 58 of thesehad been able to describe listeriosis symptoms. Women showed plenty of interest innew information about listeriosis, and they would like doctors to provide it, as well asby brochures and in the pregnancy advice book provided by the StateMedical Service.It appears that plan is required, to raise awareness amongst health professionals forthe need for food safety education for pregnantwomen.

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AREA //E // Communication, risk perception and consumer practices – Social sciences in Listeria controlORAL PRESENTATIONS //O / 47–49

The development and progress of a risk-based strategyfor the control of Listeria monocytogenes in New ZealandCastle, M. and Crerar, S.New Zealand Food Safety Authority

The New Zealand Food Safety Authority (NZFSA) has developed a risk-based strat-egy for the control of Listeria monocytogenes. One of the objectives of this strategyis ‘no increase in the reported incidence of foodborne listeriosis over the five yearperiod to 2013’. The strategy proposes a consistent and informed approach to man-aging the risk from L. monocytogenes, through: (i) the categorisation of ready-to-eat foods in relation to their potential to support the growth of L. monocytogenes;(ii) the commissioning of scientific studies and the development of tools to supportindustry and NZFSA make risk based decision; (iii) the application of manage-ment controls for industry using a risk-based approach; (iv) the implementation ofthe principles in the Codex Guidelines on the Application of General Principlesof Food Hygiene to the Control of Listeria monocytogenes; (v) risk communicationfor vulnerable consumers and food businesses. The strategy also recognises thatthere is the potential for the increased incidence of listeriosis cases due to the im-pact of an aging population and the greater availability and range of chilled ready-to-eat foods and the objective will therefore only be achieved by working with theNew Zealand food industry and consumers to enhance the control of L. monocyto-genes. The L. monocytogenes strategy was launched in 2008 and the initial responseto the risk management tools developed will be explored including uptake and re-sponse from industry.

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Compartmentalization of IFN-gamma and IL-6 receptors signallingin Listeria monocytogenes phagosomes: immune vesiclesRamos-Vivas, J., Carrasco-Marin, E., Madrazo-Toca, F., Fernandez-Prieto, L.,Rodriguez-Del Rio, E., Carranza-Cereceda, C. and Alvarez-Dominguez, C.Hospital Santa Cruz de Liencres and FMV-IFIMAV, Spain

Cytokine activated macrophages kill Listeria monocytogenes within the phago-somes. Here, we describe a novel listericidal Stat1 mediated signalling pathway in-volving IFN-γ and IL-6. This pathway is compartmentalized into IFN-γ/IL-6phago-receptosomes, innate immunity vesicles that link LM destruction with spe-cific immunity and IL-6 regulation. The Stat1-mediated signal triggered withinthis compartment, links Rab5a with the activation of the lysosomal enzyme effec-tor, cathepsin-D, and with the induction of oxidative mechanisms such as the phoxregulators, p67phox/Rac2-GTP, and the iNOS. Three downstream events link theselistericidal IFN-γ/IL-6 phago-receptosomes with specific immunity: (i) a Stat1-independent transformation into MIIC-like vesicles regulated by Rab5a-GTP thatallows the acquisition of αβ SDS-stable MHC-II dimers, cathepsin-D, Lamp-2 andLimp-2. (ii) A Stat1-dependent degradation of the Listeria immune-dominant anti-gen, listeriolysin O by cathepsin-D. (iii) A downstream Stat1 signal to regulate IL-6 production and drive a Th-1 cell-mediated immunity.

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Antimicrobial susceptibility among Listeria monocytogenes isolatesfrom non human sources in France over a ten year periodGranier, S. A.1, Moubarek, C.2, Colaneri, C.1, Roussel, S.1, Courvalin, P.2 and Brisabois, A.1

1. AFSSA, France2. Institut Pasteur, France

Listeriosis is public health concern since 1980’s. This food-borne infectious diseasehas very high rates of hospitalization and fatality. It usually requires antimicrobialtreatment. Recommendations are: Penicillin G or ampicillin combined or not withaminoglycosides. Antimicrobial resistance has increased among Gram positive bac-teria during the last past years but very few data are available for Listeria monocyto-genes, especially from non-human sources. In order to determine the frequency ofresistance within the species L. monocytogenes, a collection of 205 food and envi-ronmental isolates, randomly selected among a collection of 10 year period(1996–2006) was tested for antimicrobial susceptibility. This collection of non epi-demiologically related isolates included major serotypes (1/2a,1/2b,1/2c and 4bstrains) encountered, each of them presenting a unique PFGE profile. The pheno-type was determined using the microdilution method as recommended by CLSI.Most of the strains presented a wild type phenotype: susceptible to penicillin G,ampicillin, tetracycline, erythromycin, gentamicin, sulfonamides, trimethoprim,vancomycin and resistant to 3rd generation cephalosporines, quinolones. Only fourstrains displayed an original phenotype: two isolates (serotype 4b) were resistant toerythromycin, two isolates (serotype 1/2a) were resistant to tetracycline, one of thetwo last isolate was additionally resistant to trimethoprim. PCR analysis detectedthe presence of erm(B) gene in the 2 erythromycin resistant strains, tet(M) in thetetracycline resistant strains and dfr(D) was identified in the unique trimethoprimresistant strain. Interestingly, int gene, involved in the mobility of conjugativetransposons, was found in the strains harboring tet(M) gene. The present results donot indicate high frequency of resistance, nor large dissemination of resistance.These data should now be compared to the one obtained for human isolates. Never-theless, those findings need to be regularly updated to make sure that the resist-ance situation in L. monocytogenes doesn’t shift.

Five homologous small RNAs are involved in the responseof Listeria monocytogenes to cell wall acting antibioticsKiil Nielsen, P. and Kallipolitis, B.Department of Biochemistry and Molecular Biology, Denmark

Small RNAs (sRNAs) are important regulatory elements involved in a variety ofcellular responses, including stress tolerance and virulence. In Listeria monocy-togenes five homologous sRNAs, named LhrC1-5, were identified recently by co-immunoprecipitation with the RNA chaperone protein Hfq. The objective of thepresent study is to improve our understanding of the role(s) of LhrC1-5 in L. mono-cytogenes. Our study involves the identification of factors that influence the ex-pression of LhrC1-5 and investigations of genes affected by LhrC1-5. A range ofdifferent environmental conditions and regulatory mutant strains were employedto identify factors that affect the expression of LhrC1-5. The presence and stabilityof the RNA transcripts were determined by Northern blotting. Furthermore, ex-pression of lhrC1-5 was followed by construction of promoter-lacZ fusions for allfive sRNA-encoding genes. These studies revealed a differentiated transcription ofLhrC1-5 in response to the presence of cell wall acting antimicrobial agents, suchas ethanol and β-lactam antibiotics. Cell wall acting agents have previously beenshown to activate the two-component systems LisRK and CesRK in L. monocyto-genes. Our experiments revealed that transcription of lhrC1-5 is completely de-pendent on the presence of a functional LisRK system, whereas the CesRK sys-tem appears to be important for a differentiated transcription of lhrC1-5 inresponse to the above-mentioned cell wall acting agents. Finally, a mutant strainlacking lhrC1-5 was constructed and characterized, and genes regulated by LhrC1-5 were investigated by comparing the global gene expression of a wild type strainvs. a ∆lhrC1-5 mutant strain. In summary, this study shows that L. monocytogenesresponds to cell wall acting agents through a complex gene regulatory networkwhich includes five small non-coding RNAs.

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Phenotypic analysis of selected listerial secretion mutantsHalbedel, S.*, Galander, S. and Flieger, A.Robert Koch Institute, Germany

To be secreted into any extracellular compartment, proteins need to be translo-cated across the cytoplasmic membrane first. Protein translocation is an energydriven process and mediated by different sophisticated nano-machines that linkenergy consumption to substrate translocation. Bulk protein translocation in theFirmicutes is mediated by the Sec translocon together with the YidC proteins thathave their function in the lateral release of translocation substrates into the phos-pholipid bilayer. Listeria monocytogenes YidC homologues are encoded by the spoI-IIJ (lmo2854) and yqjG (lmo1379) genes. In addition, several minor secretion sys-tems have been described that transport certain subsets of translocationsubstrates only, such as the Tat system, or that are supposed to be involved in pro-tein secretion since they seem to form proteinaceous membrane pores. Amongthe latter is the Bacillus subtilis SpoIIIAH protein that recently was shown to bethe mother cell-specific part of a channel connecting the mother cell with the pre-spore compartment in sporulating B. subtilis cells. A homologue of this gene can befound in L. monocytogenes as well (lmo0791), whereas the prespore-specific com-ponent (spoIIQ) and the other seven genes of the B. subtilis spoIIIA operon seemto be absent from the listerial chromosome. L. monocytogenes SpoIIIAH consists ofan N-terminal transmembrane helix and a putative extracellular domain at the C-terminus that shares similarity with the ring forming proteins of the YscJ/FliFfamily associated with type III protein secretion. We therefore hypothesized thatSpoIIIAH might be involved in secretion of proteins or other biomolecules intothe extracellular space in L. monocytogenes. To test this hypothesis and to describethe impact of YidC homologues on protein secretion we generated strains withdeletions of the spoIIIAH, yidC or yqjG genes. Here, we will present data describ-ing the phenotypic and proteomic analysis of these mutants.


Distribution of serotypes and pulsotypes of Listeria monocytogenesin pig farms (France 2008)Boscher, E.AFSSA, France

This work was undertaken to estimate the prevalence of L. monocytogenes and, dis-tribution of serotypes and genotypes in French farrow-to-finish pig farms in 2008,at the breeding pig level. A total of 730 faeces (10 per farm) were sampled fromsows in 73 pig farms localized in Brittany, France. Detection of L. monocytogeneswas carried out according to the ISO 11290-1/A1 method. Isolates were serotypedand typed by PFGE. We found that 46.6% of the farms (34/73) had at least one pos-itive sample and 10.7% of the samples (78/730) were positive for L. monocytogenes.A total of 125 strains were collected. Isolates belonged to four serotypes (1/2a, 1/2b,4b and 1/2c) with a percentage of 41.6%, 36.0%, 20.8% and 1.6% respectively. Out ofthe 34 positive farms, 19, 12 and 3 had respectively 1, 2, and 3 serotypes. Moreover,21, 17, 11 and 1 positive farms had respectively the serotype 1/2a, 1/2b, 4b and 1/2c.The genetic diversity of the isolates collected in this study was very important(DI=0.986). In total, 50 genotypes were obtained; 25 for 1/2a, 15 for 1/2b, 9 for 4band 1 for 1/2c. Moreover 17, 6, 4, 3, 3, and 1 positive farms had respectively 1, 2, 3, 4,5 and 6 genotypes. Furthermore, strains showing similar genotypes occurred inseveral farms; as example, genotypes G18, G12 and G10 were found in 4, 5 and 5farms respectively. This study provided recent valuable information on the occur-rence of L. monocytogenes in French pig farms. This microorganism is prevalent inthe sows in farrow-to-finish pig farms (46.6%). Our work highlighted for somefarms several serotypes and genotypes suggesting several sources of contamina-tion. Furthermore, the serotypes found in this study are identical to those usuallyinvolved in human listeriosis in Europe (Goulet et al. 2008).

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Comparative phylogenomics of Listeria monocytogenes revealsan adaptation profileSilveira Nalério, E.1, Padilha Silva, W.1, Stabler, R.2 and Wren, B. W.2

1. Universidade Federal de Pelotas, Brazil2. London School of Hygiene and Tropical Medicine, UK

Listeria monocytogenes is the causative agent of listeriosis which may cause a rangeof diseases from gastroenteritis to death. In fact, disease outcome can be related tostrain serotype/lineage thus molecular analyses has demonstrated that L. mono-cytogenes is a highly diverse species which can be grouped into three lineages.Whole-genome microarray can be employed to study phylogenetic relationshipsamong Listeria strains either species or serotype level, in addition to demonstratedifferences on their virulence potential and/or environmental adaptation. Theaim of this study was the whole genome comparison of L. monocytogenes strainsfrom different origins. Ninety-nine L. monocytogenes strains from different geo-graphical origins (Brazil, Denmark, Austria, Ireland, USA and unknown), includingclinical strains (humans and animals), food and food industries strains were ana-lyzed. DNA from all strains were competitively hybridized on a L. monocytogenesDNA microarray based on the whole-genome sequence L. monocytogenes EGD-e.DNA labeling and hybridization protocol were followed according to Dorrell et al.(2001). Data acquisition, processing and comparative phylogenomics were per-formed as previously described by Stabler et al. (2006). Comparative phyloge-nomics clustered the L. monocytogenes strains into two central clades which isrepresentative of the two main lineages of this species. In addition each of theseclades was divided into two further subclades. Clade formation was independent ofthe geographical origin of strains with the exception of the clade containing per-sistent strains (strains that persist in food-processing environment), where noneof the Brazilian strains were present. It was found 18 specific genes for lineage Istrains (1/2a and 1/2c serotypes). These genes are related to carbohydrate metab-olism, two component regulatory system, ABC transporter complex and bvrB andbvrC genes. Significantly all persistent strains clustered together in the same lin-eage I clade. We achieved a set of unique genes belonging exclusively to L. mono-cytogenes persistent strains pointing to be responsible for its adaptation profile.The genes are involved in stress resistance and are related to carbohydrate trans-port and metabolism, environmental information processing, signal transductionmechanisms, cell surface protein, amino acid transport and metabolism, nu-cleotide transport and metabolism, translation, cell wall biogenesis, replication, re-combination and repair, transport of small molecules similar to ABC transporter,metabolism of lipids and unknown function. Interestingly from 14 virulence listedgenes most of them were present in all studied L. monocytogenes strains with ex-ception of inlE and inlG genes. These findings indicate that genetic variability of L.monocytogenes strains point to niche adaptation instead virulence differentiationdespite of different origins. Persistent strains clustered suggesting genetic origin tosurvival in this environment.

Serotyping and PFGE patterns of Listeria monocytogenes isolatedfrom poultry meatVasantrao Kurkure, N.1, Kalorey, D. R.1, Rodrigues, J.2, Gunjal, P.1 and Barbuddhe, S. B.2

1. Nagpur Veterinary College, Maharashtra Animal & Fishery Sciences University, India2. ICAR Research Complex for Goa, India

Listeria monocytogenes is a gram positive, facultative, intracellular bacteria which isubiquitous in nature. Poultry meat and products frequently have been implicated inoutbreaks of food borne illness. In India there is practice to sell freshly slaughteredpoultry meat. A total of 119 poultry meat samples were collected from poultry meatshops. These samples were processed for isolation of Listeria spp. by adopting stan-dard protocol. Out of 47 Listeria spp. recovered 17 isolates were confirmed as L.monocytogenes by biochemical and in vitro pathogenicity test. The L. monocytogensisolates were subjected to multiplex PCR based serotyping and PFGE analysis. Allthe isolates were grouped as 4b, 4d and 4e serotypes. By PFGE analysis four Asc Iprofiles were observed. Thirteen isolates were observed to be of similar profile in-dicating homogenecity among the isolates recovered from poultry meat. Presentfindings indicated the need of improvement in hygienic practices while slaughter-ing and sell of poultry meat as L. monocytogenes is of zoonotic pathogen.

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Genotypic characterization of Listeria monocytogenes isolatedfrom fresh leafy vegetablesWarke, S.1, Kalorey, D. R.1, Umap, S.1, Sonegaonkar, A.1, Patil, V.1, Kurkure, N V.1

and Barbuddhe, S. B.2

1. Nagpur Veterinary College, India2. ICAR Research Complex for Goa, India

Listeria monocytogenes is a food borne pathogen responsible for listeriosis, an ill-ness characterized by meningitis, encephalitis, abortion and septicemia in humanbeings. A total of 417 samples comprising eleven different types of fresh leafy veg-etables were collected from supermarkets and local markets in and around Nagpur,Central India to detect L.monocytogenes. The vegetables included spinach (78),Fenugreek (82), Green Sorrel (22), Coriander (53), radish (57), amaranthus (30),sour greens (12), lettuce (33), Mint Leaves (43) and dill leaves (07). All sampleswere processed for isolation of L. monocytogenes by two step enrichment followedby plating on selective media. Confirmation of the isolates was on basis of bio-chemical characters, haemolysis on blood agar and Christie, Atkins, Munch Pe-tersen test (CAMP). The isolates were also tested for the virulence associatedgenes namely, hlyA, plcA, actA, iap multiplex and prfA by PCR. A total of 31 (7.43%)isolates of L. monocytogenes were recovered from spinach (11), fenugreek (9), co-riander (3), radish (4), amaranthus (2) and mint leaves (2). All the genes were de-tected in seven strains, four genes (hlyA, actA iap and prfA) in three strains. ThehlyA, actA and iap in six strains. Three genes (hlyA, iap and prfA) were detected infive strains. The hlyA and iap genes were detected in ten strains. Fourteen isolateswere subjected to multiplex PCR serotyping and all belong to 4b, 4d and 4eserogroup. Contamination of vegetables with L.monocytogenes is a cause of con-cern as most of the vegetables are consumed raw as salad. Thus, there is increasedrisk for transmission of listeriosis. Therefore proper washing and cleaning of freshleafy vegetables is recommended at harvesting and consumption.

Comprehensive appraisal of the exoproteome of Listeria monocytogenesby genomic and proteomic analysesDesvaux, M., Dumas, E., Chafsey, I., Chambon, C. and Hébraud, M.INRA, France

Secreted proteins are the main tools used by bacteria to interact with their envi-ronment and are relevant to the bacterial lifestyle. By definition, secreted pro-teins are proteins actively transported via secretion systems but they can haveradically different final destinations. In monoderm (Gram-positive) bacteria, suchsecreted proteins can (i) anchor to the cytoplasmic membrane, (ii) associate withthe cell wall, or (iii) be released into the extracellular milieu and are the called ex-oproteins. The subset of proteins localized in the extracellular milieu constitutesthe extracellular proteome, also called exoproteome. Listeria monocytogenes is abacterial species both pathogenic and saprophytic, which exhibits a highly vari-able level of virulence from one strain to another and such biodiversity might ex-press within the exoproteome. The present investigation aimed at developping ofa generic genomic strategy to predict exoproteins in monoderm bacteria as well asdetermining those differentially expressed within a panel of representative strains.Genomic analysis was performed following a novel rational bioinformatic ap-proach to predict exoproteins, which expression was determined by proteomicanalyses following 2-DE and identification by MALDI-TOF MS. The rational bioin-formatic approach integrates the structural properties of the proteins potentiallysecreted as well as the respective secretion systems involved. Comparison of the-oretical and experimental 2-DE maps provided a comprehensive picture of the ex-oproteome. Exoproteins were further discriminated between those found in all oronly in a subset of L. monocytogenes strains. While the core exoproteome includedproteins related to virulence and cell wall biogenesis, variation in the proteinmembers of these categories constituted the variant exoproteome. The novel andrational in silico strategy here developed to predict exoproteins is adaptable andfully applicable to other monoderm bacteria. This proteomic analysis resulted inthe first definition of the core and variant exoproteomes of this species and pro-vides auxiliary insight in bacterial physiology.

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Investigating differences in lineages of Listeria monocytogenesusing comparative genomicsMcIlwham, S., Farber, J. and Pagotto, F.Bureau of Microbial Hazards, Canada

Listeria monocytogenes causes foodborne listeriosis and is ubiquitous in the envi-ronment. Strains are often classified into lineages I (food); II (human illness); andIII (food-production). It is not clearly understood how lineages develop a tropismfor a particular niche. Using comparative genomics, we attempted to identifyunique genetic attributes within lineages that contribute to the tropism of thepathogen. A mixed genome array, created from a genomic library of 15 L. monocy-togenes isolates, was used to compare the genomes of different strains. An isogenicmutant for a glycoside hydrolase family 65 (GH65) was created based on microar-ray screening. Forty-five strains from different sources were studied in Mueller-Hinton broth with ampicillin, vancomycin or acriflavine. Additionally, the growthrates of eleven strains in Listeria Enrichment Broth (LEB) were compared. AGH65 gene was found to be present in lineage II isolates but absent in lineage I iso-lates. A GH65 isogenic deletion mutant and other lineage I isolates showed a re-duced ability to proliferate in the presence of vancomycin and ampicillin. Whengrowth rates were compared in LEB, the GH65 deletion mutant behaved similarlyto lineage I strains. The GH65 gene, present in lineage II strains, may provide acompetitive advantage in the host environment. The difference in growth rate ofthe lineage I and II strains in LEB may provide insight as to why certain serotypesare more readily isolated from food commodities.

Autolysis in Listeria monocytogenes – a proteomic approachPinto, E.1, Marques, N.2, Andrew, P. W.3 and Faleiro, M. L.1

1. Instituto de Biotecnologia e Bioengenharia, Centro de Biomedecina Molecular e Estrutural,Universidade do Algarve, Portugal

2. BioFig, Universidade do Algarve, Portugal3. Department of Infection, Immunity and Inflammation, University of Leicester, UK

Autolysins play a role in many cellular processes including cell growth and divi-sion, cell wall turnover, motility and also pathogenicity. Several growth condi-tions may interfere with cell wall synthesis, leading to unrestricted activity of theautolysins which can degraded the cell wall to levels that compromise the cell in-tegrity. During our work differences between Listeria monocytogenes strains inthe tendency to undergo autolysis was found. The objective of the present studywas to test the hypothesis that as cells enter stationary phase the pattern of pro-tein expression in autolytic and non-autolytic strains is different. To achieve this,two L. monocytogenes strains (an autolytic [C897] and a non-autolytic [EGD])were grown in a defined medium at favourable (30 °C) and non-favourable (20 °C)autolysis growth conditions. The bacterial cells and the supernatant samples werecollected immediately before autolysis. The samples were subjected to a proteomeanalysis. Remarkably at favourable autolysis condition the autolytic strain ex-pressed a series of stress proteins and was depleted of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase an essential component of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Injuries in this pathway may cause anegative impact on cell wall synthesis. In both proteomes of the non-autolyticstrain EGD and the autolytic strain C897 in non favourable autolyis conditions re-vealed a higher expression of proteins involved in the energy generation, aminoacid and fatty acid metabolism. In the secretome of C897 the p60 protein wasover expressed at 30 °C but no significant differences were found between thestrain C897 and EGD. However p60 levels were higher in the autolytic strain in-tracellular proteome when grown at 20 °C. The levels of the autolysin p45 werehigher in the secretome of C897 at 30 °C and significant differences between thetwo strains were found.

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Role of flhA, cheR and motA in growth of Listeria monocytogenesat low temperatureMattila, M., Lindström, M., Somervuo, P. and Korkeala, H.University of Helsinki, Finland

Temperature-dependent induction of flagella is a well-characterized phenomenonin L. monocytogenes. At 37°C L. monocytogenes is virtually non-motile and the motil-ity genes are down-regulated whereas at 30°C and below the strains are highly flag-ellated and motile. However, the essentiality of increased flagellum production dur-ing growth at low temperatures is still unclear. To study this relationship, weprepared knock-out mutants of three motility and chemotaxis genes, flhA, cheR, andmotA on L. monocytogenes strain EGD-e. We compared the expression levels of thesegenes in cultures grown at 3, 25 and 37°C by using qRT-PCR. The role of these genesin the cold adaptation of L. monocytogenes was also studied with growth curve analy-sis, motility assays and electron microscopy. The relative expression of flhA, cheRand motA in EGD-e cells grown at 3°C was significantly (p<0.01) increased com-pared to 37°C. At 3°C the level of flhA and cheR transcripts was also significantly(p<0.05) higher than at 25°C. The growth curve analysis showed that in culturesgrown at 3°C both the growth rates and maximum optical densities at 600 nm weresignificantly (p<0.001) lower for the mutant strains ∆flha, ∆cheR and ∆motA com-pared to the wild-type strain. At 37 and 25°C we did not detect any significant differ-ences between the wild-type strain and the mutants. The motility assays showedthat ∆flhA and ∆motA strains were completely non-motile at all three temperatureswhereas ∆cheR showed similar motility pattern to the wild-type at both 3 and 25°C.At 37°C the motility of ∆cheR mutant and the wild-type strain was negligible. Theresults suggest that flhA, cheR, and motA have a role in the cold tolerance of L. mono-cytogenes strain EGD-e, and that flagellum production and chemotaxis are linked tocold adaptation of L. monocytogenes.

The effect of acetic acid (at pH 5.5) or benzoic acid (at neutral pH)on lipid composition and fluidity of Listeria monocytogenes membraneIoannis, D.1, Anita, B.1, Eleni, S.2 and Mastronicolis, S.1

1. Food Chemistry Laboratory of Chemistry Dept, University of Athens, Panepistimiopolis Zografou,Athens, Greece

2. National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Athens,Greece

Listeria monocytogenes is able to alter its membrane fatty acids (FA) composition orthe composition of the polar group of the lipid molecules after a reduction in pH(due to Acid Tolerance Response, ATR). The lipid changes might play a protectiverole upon (acidic) stress decreasing the membrane fluidity and thus obstructing theentrance of the stressful compounds. The aim of our work was to investigate themodification of L. monocytogenes membrane lipids molecules (neutral, NL or polar,PL lipids) and also their FA composition in response to low (acetic acid) or neutralpH (benzoic acid) and their consequences on membrane fluidity. L. monocytogenes(DP-L1044, D. Portnoy, University of Pennsylvania) total lipids,TL extracted fromstationary phase cells cultured (1L BHI broth, 30°C) with low initial pH (5.5) byacetic acid, LmAA (optical density OD600=0.210±0.031, 72h, n=12) or with neutralpH by benzoic acid (1g/L), LmBA (OD600=0.682±0.013, 10h, n=9) and compared withthose of optimal conditions cells, Lmcontrol (OD600=0.816±0.032, 10h, n=8). The re-sults revealed: i) Only acetic acid enhances the antimicrobial activity. ii) The sumsof straight saturated chain FA(16:0 and 18:0) and unsaturated FA(16:1 and 18:1)were similarly increased, at the expense of branched chain FA(a-15:0 and a-17:0) ofeach TL acidic adapted culture (increase of high-melting FA). iii) Singularly aceticacid adaptation in L. monocytogenes was correlated with a higher content (49.3%among TL) of neutral lipids class than those of Lmcontrol. Moreover, we correlatedthe lipid changes with thermodynamic analysis (Differential Scanning Calorimetry,DSC) of lipids in order to estimate experimentally the initial hypothesis, that bothadaptation mechanisms (the high-melting point FA increase and the NL accumula-tion) would promote the decrease of membrane fluidity. The results revealed thatin each culture LmBA and LmAA TL was increased the phase transition tempera-ture,Tc (5.3 °C and 3 °C) and enthalpy difference, ∆Η (10.2% and 53.8% respec-tively) than Lmcontrol. The thermodynamic findings confirmed the above hypothe-sis of decreasing fluidity. The high ∆Η value of LmAA (high energy amount tochange its lipids) is interpreted by NL accumulation.

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Elucidation of the responses to weak acids in the human pathogenListeria monocytogenes using gene microarraysO’Byrne, C.1, Heavin, S.1 and Morrissey, J.2

1. NUI Galway, Ireland2. University College Cork, Ireland

Contamination of processed food with Listeria monocytogenes is a major humanhealth risk. Food preservation regimes often include the use of weak organic acidsto reduce microbial growth. Although the response of L. monocytogenes to strongacids has been investigated extensively, there little is known about how L. mono-cytogenes responds to weak acids, particularly at the molecular level. The objectiveof this study was to investigate the molecular response of L. monocytogenes to fivefood-grade organic acids, acetic, lactic, sorbic, citric and benzoic acid. Carefulgrowth experiments were carried out to determine the inhibitory concentrationsof each of these acids on the growth of L. monocytogenes. Microarray and quanti-tative RT-PCR experiments were then performed to elucidate the molecular re-sponses of the organism to weak acids. In total 222 genes were found to be differ-entially expressed in response to at least one of the weak acids. The temporalresponse of 9 of these genes to sudden exposure to lactic acid or citric acid wasstudied using RT-PCR. These data highlighted the distinct responses observed tothe 2 weak acids and identified genes that showed strong unique transcriptionalresponses (both up-regulation and down-regulation) to each. At a global level therewas surprisingly little overlap between genes that responded to each acid. For ex-ample, the 7 genes differentially expressed in the presence of acetic acid were notinfluenced by any of the other acids. Together the data suggest that each organicacid influences the cell in a very specific way, a finding that suggests that each acidhas a different inhibitory mode of action.

The immunogenic surface protein IspC acts as anN-Acetylglucosaminidase in Listeria monocytogenes serotype 4bRonholm, J.*University of Ottawa, Canada

IspC is a surface protein isolated from Listeria monocytogenes serotype 4b whichacts as an autolysin and virulence factor. Several autolysins including P60, Amiand Auto have been isolated from various L. monocytogenes serotypes but the rolefor their apparent functional overlap remains unknown. Further characterizingthese autolysins may help to gain insights into their roles in Listeria biology. Thenovel autolysin IspC remains understudied. A panel of 15 monoclonal antibodies(mAbs) directed against the surface of L. monocytogenes serotype 4b were gener-ated, characterized as recognizing IspC and will facilitate further study of this pro-tein. Based on serological assays with these mAbs we have determined that IspCexpression is limited to certain L. monocytogenes serotypes including 4a, 4ab, 4b,and 4e, although, other serotypes have genes highly homologous to IspC. Sequencehomology to other autolysins predicted IspC functioned as an N-acetylmuramoyl-L-alanine amidase. However, mass spectrometry analysis of peptidoglycan (PG)hydrolysis with IspC has shown that IspC acts as an N-acetylglucosaminidase.Based on the site of PG cleavage, it was hypothesized that the amount of PG acety-lation would affect IspC mediated PG hydrolysis. PgdA, the only one of three PGdeacetylases predicted to localize to the cell surface, is required for de-acetyla-tion of PG, which is assembled fully acetylated. To determine how acetylation af-fects IspC PG hydrolysis, a pgdA deletion mutant was created. The mutant PG(fully acetylated) was much more susceptible to hydrolysis by IspC than PG fromthe wild-type, as assessed by re-naturing SDS-PAGE. Currently we are making apgdA complement to restore PG deacetylation in the deletion mutant. Further in-vestigation is being done on IspC hydrolysis of PG from the deletion mutant usingmass spectrometry. This will provide insight into the effects of the pgdA deletionon PG structure and IspC PG hydrolase activity.

* Participation Supported by IUFoST.

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Listeria monocytogenes EGD chitinolytic activity is regulated bycarbohydrates but also by the virulence regulatory gene, PrfAHalberg Larsen, M., Leisner, J. J. and Ingmer, H.Faculty of Life Sciences, University of Copenhagen, Denmark

Chitin, a highly insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), is a majorcomponent of fungal cell walls and insect and crustacean exoskeletons and is one ofthe most abundant polymers in marine and terrestrial environments. Chitin hydrol-ysis by Listeria monocytogenes depends on two chitinase encoding genes chiA(lmo1883) and chiB (lmo105) and the aim of this study was to investigate their regu-lation. The production of the chitinases is substrate regulated and subjected tocatabolite control. Thus, chitin induces expression of both chitinases however chiAbut not chiB is furthermore induced by the monomer GlcNAc. In growth mediumsupplemented with chitin and glucose the expression of both chitinases is repressed.Expression of chiA and chiB is growth phase dependent with higher expression inlate exponential growth phase compared to early exponential phase. Chitinases ex-pressed by bacterial pathogens have proven to be important not only for nutrientacquisition and environmental survival but also for infecting humans and animals.Interestingly, we found that the central L. monocytogenes virulence gene regulator,PrfA, is required for the chitinolytic phenotype as chitinase activity was significantlyreduced in ∆prfA mutant cells compared to the wild type cells. In agreement withthis result northern blot analysis showed that the amounts of chiA and chiB tran-scripts were significantly lower upon induction by chitin in the ∆prfA mutant com-pared with the wild type. Furthermore, in contrast to the wild type, no chiA tran-script could be detected in the mutant lacking PrfA during growth in mediumsupplemented with GlcNAc. Regulation of chitinolytic activity in L. monocytogenesis complex and the results obtained in this and other studies indicate that the bio-logical role of this activity may not be limited to the external environment.

Infectious dose curves for guinea pigs challenged with a Listeriamonocytogenes epidemic clone strain and a strain carrying a naturally-occurring virulence-attenuating mutation in inlA show a significantshift in median infectious doseNightingale, K.1, Van Stelten, A.1, Simpson, J. M.1, Chen, Y.2, Scott, V. N.3,Ross, W. H.4, Whiting, R. C.5 and Wiedmann, M.6

1. Colorado State University, USA2. Grocery Manufacturers Association, USA3. U.S. Food and Drug Administration, USA4. Health Canada5. Exponent, USA6. Cornell University, USA

Listeria monocytogenes contains two subpopulations, including (i) epidemic clone(EC) strains, which have been linked to the majority of listeriosis outbreaks world-wide and are overrepresented among sporadic cases in some countries along with(ii) strains carrying mutations leading to a premature stop codon (PMSC) in inlA,which are commonly isolated from ready-to-eat foods (approx. 45%) but rarely as-sociated with human disease (approx. 5%). The virulence factor Internalin-A (InlA;encoded by inlA) binds certain isoforms of the cellular receptor E-cadherin to facili-tate crossing of the intestinal barrier by L. monocytogenes. The currentFDA/FSIS/CDC L. monocytogenes risk assessment was developed with dose re-sponse data from murine challenge experiments, a model that fails to probe InlAmediated virulence due to the inability of InlA to bind the murine isoform of E-cad-herin. Guinea pigs, which express the human isoform of E-cadherin that binds InlA,were intragastrically challenged with (i) a fully-invasive EC strain associated with alisteriosis outbreak or (ii) a strain carrying the most common inlA PMSC mutation.Dose-response curves for tissue infectivity were constructed with either a log-logis-tic, beta poisson, or exponential (for spleen data) fit to the raw individual and com-bined organ data. The log logistic and beta poisson models based on combined organdata showed an approx. 1.3 log10 CFU increase in the median infectious dose for thestrain carrying a PMSC in inlA relative to the EC strain. Inclusion of strain effectsignificantly improved the ability of the model to explain the observed data, sup-porting a significant difference in tissue infectivity between EC and PMSC strains. L.monocytogenes strains thus show notable differences in infectious dose required toestablish an infection. Results from this work support the dose-response relation-ship for L. monocytogenes is strain-specific and will provide critical data for en-hancement of existing and development of future risk assessments.

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MudPIT based proteomic analysis of alkaline adapted, environmentallypersistent Listeria monocytogenes strainsNilsson, R. E., Ross, T. and Bowman, J. P.University of Tasmania, Australia

Soluble protein expression profiles of environmentally persistent and sporadicListeria monocytogenes strains in mid-exponential and stationary growth statesat pH 7.3 and adapted to pH 8.5 were compared using multidimensional proteinidentification technology (MudPIT). Searches employed the X!Tandem algorithmand an in house data validation criteria was applied using the Trans ProteomicPipeline (Version 3.4) and comparison against a decoy database. Qualitative andrelative abundances were compared through functional ontology (TIGR) and de-termination of spectral abundances. A total of 1661 proteins were identified fol-lowing integration analysis. Significant differences in protein expression were ob-served for both growth state and culture condition. Increased presence andabundance of proteins associated with the cell wall, transport and binding, aminoacid / cofactor biosynthesis and protein stabilization were identified. The studyfindings suggest alkaline adapted L. monocytogenes strains may use cytoplasmicacidification by surrogate proton sources (both endogenous and exogenous) as ameans of persisting in high pH environments. Furthermore, proteins associatedwith stabilization of cellular processes and cell wall modification may be aidingin minimizing cellular damage under these conditions. This trend was identified inboth growth states; however it was more pronounced in exponential phase. Nosignificant difference was observed between the L. monocytogenes strains studied.The results of this work support the notion that exposure to conditions favouringalkaline homeostasis may be an important contributor to the development of per-sistent L. monocytogenes, independent of strain.

RpoN, the alternative sigma factor, is associated with the growth phasetransition and pathogenesis in Listeria monocytogenesOkada, Y., Suzuki, H., Monden, S., Igimi, S. and Okada, N.Ministry of Health, Labour and Welfare, Japan

Listeria monocytogenes has 5 types of sigma factor. RpoN, an alternative sigma fac-tor, is involved in the bacteriocin-resistance and the osmotolerance in this bac-terium, however, its’ function remains almost unknown. We found that the rpoNdeletion mutant showed higher growth than its parental strain at the stationaryphase. To identify the RpoN regulated genes which are associated with the growthphase transition, DNA microarray analysis was performed. From L. monocytogenesstrain EGD and its rpoN in-frame deletion mutant, total RNA samples were iso-lated at early exponential and stationary phase. The gene expression profiles inboth strains at each growth phase were compared, and in the results, especially, 27genes associated with oxidoreductase showed the different patterns of expressionbetween two strains. So we examined the survival of these strains after exposure tothe sub-lethal concentration of hydrogen peroxide. Both strains showed the simi-lar levels of tolerance against 100mM hydrogen peroxide at stationary phase. How-ever, the mutant was significantly tolerant to hydrogen peroxide than EGD at themid exponential phase. From the result of electrophoresis, the chromosomal DNAwas degraded after exposure to hydrogen peroxide only in EGD, but not in the mu-tant. Next, we examined the pathogenicity of the rpoN mutant. The mutantshowed the higher growth in J774 cells and the peritoneal adherent cells fromBALB/c mice. In spleen and liver of mouse, the mutant was able to grow at thesimilar level of EGD, however, the growth was slowly. These results suggest that therpoN mutant is able to grow higher than the parental strain at the stationary phasebecause of the increased DNA stability against the active oxygen accumulated inthe bacterial cells as a result of the respiration during the exponential growth, andRpoN is associated with the pathogenicity of L. monocytogenes.

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Cellular lipid fatty acid pattern differences between reference andice-cream isolate of Listeria monocytogenes as response to cold stressAnita, B., Ioannis, D. and Mastronicolis, S.Food Chemistry Laboratory, Department of Chemistry, National University of Athens, Greece

One of the salient features of L. monocytogenes as a food-borne pathogen is itsability to grow at low temperatures, allowing refrigeration at 5 °C to act as an ef-fective enrichment for the organism. There is little information about its ability towithstand in frozen foods. L.monocytogenes is characterized by >85% branched-chain fatty acids (FA), anteiso-15:0 (a-15:0) and anteiso-17:0 (a-17:0). Cells grown inthe cold (5 °C) contain significantly less a-17:0 than those grown at higher tem-peratures. The purpose was to investigate the effect of ice-cream storage inL.monocytogenes FA profile, after inoculation of pilot ice-cream with referencestrain DP-L1040 (D. Portnoy, University of Pennsylvania). The inoculation wasperformed with reference strain Lmref, (BHI, 30 °C, 8h, OD600=0.8) in order toyield 6.30 log cfu/g ice-cream. Cells of L. monocytogenes isolated from inoculatedice-cream, (Lmice), after 6 month of storage and cells of Lmref were grown in BHIbroth, until late exponential phase at two different temperatures, at 30 °C (10h,OD600=0.8) and at 5 °C (8d, OD600=0.6). Each culture (Lmice30°C, Lmice5°C,Lmref30°C and Lmref5°C) total lipids were extracted and FA methyl esters were an-alyzed. Our results show that significant differences exist between the FA profile ofLmref30°C and Lmice30°C. The Lmice30°C was characterized by a-15:0 and a-17:0 FA.Additionally, increase of straight chain 18:0 FA, (from 0.7 to 3.9%) was observed incells of ice-cream isolate. There was also an increase of unsaturated FA. TheLmice5°C showed an a-15:0/a-17:0 ratio of 11.2, while Lmref5°C was characterizedby significantly higher ratio of 19.3. Furthermore, there was a decrease (3.2 fold) of∑branched/∑straight chain FA ratio in ice-cream isolate compared to referencestrain. These results showed that L. monocytogenes strain origin needs to be con-sidered when physiological studies are performed with this bacterium. The foodmatrix and environmental conditions can influence the FA pattern and L. mono-cytogenes survival. A greater understanding of cold adaptation mechanism mayoffer methods for controlling L. monocytogenes in chilled and frozen foods.

Role of the dihydroxyacetone metabolism in the resistance of Listeriainnocua to pediocinMilohanic, E.INRA, France

Listeria monocytogenes is a Gram-positive bacterium, the causative agent of liste-riosis, which affects humans and animals. This bacterium which is a recurring con-taminant of the food chain represents a major problem for the food industry. Wewere interested in the dha system of Listeria innocua that involves the general PTSenzymes, the main transport and phosphorylation system of carbohydrates, whichalso occurs in L. monocytogenes. It catalyses the intracellular PEP-dependent phos-phorylation of dihydroxyacetone (Dha). In silico studies allowed us to hypothesizethat the last gene of the dha system, pedB, could encode resistance to pediocin, abacteriocin produced by the lactic acid bacterium Pediococcus acidilactici. Wesought to understand why this gene is associated with the dha system. We first con-firmed the operon organization of this system. Quantitative PCR experiments re-vealed that the expression of the dha system seems to be partially constitutive. El-evated dha expression was observed in a dhaR mutant, which lacks the repressor ofthis operon. We also showed that the wild-type strain of L. innocua is sensitive topediocin while the dhaR mutant is resistant. The pedB mutant is under investiga-tion. In addition, we purified the five proteins involved in Dha phosphorylationand could reconstitute in vitro these metabolic steps. Interestingly the dha operonencodes many enzymes involved in the pentose phosphate pathway especially theTpi-like protein. These results would suggest a link between Dha metabolism, pen-tose phosphate pathway and virulence in L. monocytogenes.

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Glucose transport system in Listeria monocytogenes and their impacton virulence gene expressionMoussan Ake, F.INRA, France

In Listeria monocytogenes at least two phosphoenolpyruvate (PEP): carbohydratephosphotransferase systems (PTS) of the mannose class and also two non-PTSpermeases can transport glucose. Mutants lacking EIIAB (Lmo0096) or EIIC(Lmo0097) of the glucose/mannose PTS grew slightly slower on minimal mediumcontaining up to 10 mM glucose and consumed glucose 3-times slower than thewild-type strain. A lmo0784 mutant (EIIA, mannose type PTS) exhibited an ex-tended lag phase before it started growing on glucose and glucose consumptionwas slowed 3.5-fold. All PTS transporters are inactive in a ptsI mutant, which nev-ertheless grew at high glucose concentrations (> 15mM) after an extended lagphase. Two GlcU-like non-PTS glucose transporters (Lmo0169 and Lmo0176)probably catalyze glucose uptake in the DptsI mutant. Expression of either one ofthem in an Escherichia coli DptsHIcrr mutant restored glucose utilization. The ef-ficient utilization of glucose affects the PrfA-dependent expression of L. monocy-togenes virulence genes, including hly. Nevertheless, inactivation of the glcU genesin a strain carrying a Phly-gus fusion had no effect on PrfA activity. However, dele-tion of the gene for EIIABGlc/Man caused 3- to 5-fold elevated gus expression,whereas deletion of EIICGlc/Man had only a slight effect. b-Glucuronidase activityin the EIIABGlc/Man mutant was also elevated when it was grown on solid mini-mal medium containing 5 or 10mM glucose. A similar behaviour was observed forthe lmo0784 mutant, which in addition exhibits very low man operon expression.The highest gus expression was observed in the DptsI mutant grown at glucoseconcentrations between 25 and 50mM.

Antimicrobial susceptibilities of Listeria monocytogenes isolatedin JapanMonden, S., Okutani, A., Suzuki, H., Asakura, H., Nakama, A., Igimi, S., Okada, Y.and Maruyama, T.Japan

Antibiotics, such as ampicillin and co-trimoxazole, are usually used as the firstchoice for treatment of listeriosis. However, the antimicrobial resistant strains ofListeria monocytogenes are often isolated in many countries. In this study, in vitroantimicrobial susceptibility of 232 L. monocytogenes strains (101 isolates from pa-tients of listeriosis and 131 isolates from food and food-related environment) ob-tained in Japan from 1974 to 2008 were examined their susceptibilities against 6kinds of antibiotics by plate dilution method. According to the breakpoint from theguideline of National Comittee for Chemical Laboratory Standard, all strains testedhere were susceptible to gentamicin and kanamycin, and all clinical isolatesshowed susceptibility to ampicillin and erythromycin. One strain from beef was re-sistant to ampicillin, and the another one from pork showed resistance to 3 kinds ofdrug, chloramphenicol, erythromycin and enrofloxacin. 56.4% of clinical isolatesand 37.8% of food and environmental isolates were resistant to enrofloxacin, andthe rest of isolates showed intermediate phenotype to this antibiotics. The rates ofenrofloxacin-resistance were very high in isolates belonging to serotype 1/2a(63.3%) and 1/2c (75%) compared with those of 1/2b (41.8%) and 4b (38%). In thisstudy, most of clinical isolates were susceptible to antibiotics except enrofloxacin.On the other hand, some strains from food and environment showed resistance orintermediate to ampicillin, chloramphenicol or erythromycin. Furthermore, allstrains were more than intermediate to enrofloxacin, a kind of quinolone that usedfor treatment of animal infection in Japan. These results suggest that the origin oflisterial isolates in involved in their antibiotic susceptibility.

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Influence of sub-lethal concentrations of disinfectantson Listeria monocytogenes adhesion and invasion in Caco-2 cellsGaedt Kastbjerg, V.1, Halberg Larsen, M.2, Ingmer, H.2 and Gram, L.1

1. National Institute of Food Research, Technical University of Denmark2. Faculty of Life Sciences, University of Copenhagen, Denmark

Listeria monocytogenes is frequently detected in the food processing environment,where it, ideally, must be exposed to disinfectants daily. However, L. monocyto-genes is not always eliminated by the disinfection process. One reason could bethat the bacteria are only exposed to sub-lethal concentrations of the disinfectant.We have recently shown that sub-lethal concentrations of disinfectants used inthe food industry affect virulence gene expression on transcript level in L. mono-cytogenes, and the effect depend on the active components of the disinfectants.The aim of the present study was to determine if disinfectants used routinely inthe food industry affect the virulence of this pathogen studied in a cell-model.Two different disinfectants were used. Compound 1 contains peracetic acid andhydrogen peroxide as the active ingredients and reduces the expression of viru-lence genes in L. monocytogenes whereas Compound 2 contains quaternary am-monium compounds (QAC) and induces virulence gene expression. L. monocyto-genes EGD was exposed to the two disinfectants for one hour in a non-inhibitingand a sub-lethal concentration, and subsequently the bacterial cells were addedto a monolayer of Caco-2 cells. Bacterial adhesion and invasion were monitored.Exposure of L. monocytogenes to the sub-lethal concentration (0.0031%) of theQAC disinfectant significantly increased adhesion to Caco-2 cells as compared tobacteria exposed to water (control) and resulted in a slightly higher invasion. Noeffect was observed of the non-inhibiting concentration of the QAC disinfectant(0.0016%). In contrast, the adhesion was unaffected and the invasion slightly de-creased after exposure to the non-inhibiting concentration of 0.125% of the per-acetic disinfectant as compared to bacteria exposed to water. On-going studieswill evaluate how long term exposure to disinfectants will affect adhesion and in-vasion to Caco-2 cells and these data will also be discussed.

The SOS response in Listeria monocytogenes – a stresssurvival mechanismKiil Nielsen, P., Zahle Andersen, A. and Haahr Kallipolitis, B.University of Southern Denmark

The SOS response is a functionally conserved DNA repair system found in a varietyof bacteria. The SOS response involves two central regulatory components, RecAand LexA, which coordinate the expression of target genes leading to arrest of celldivision, DNA repair and mutagenesis. Historically, the SOS response is linked tothe presence of single stranded DNA and stalled replication forks induced by UV ra-diation. However several other stress conditions, which are not expected to causemassive DNA damage, have been shown to induce SOS response e.g. mild heattreatment, antibiotics, ethanol and salt stress. The SOS response of the food bornepathogen Listeria monocytogenes is presently not well understood. We find it likelythat the SOS response is important for the growth and survival of this pathogen incontaminated foods and during infection. It is therefore of great interest to investi-gate this response in L. monocytogenes into more details. To this end, we focusedour studies on the auto regulated repressor protein LexA, the genes targeted by thisregulator, and the environmental signals leading to activation of the SOS responsein L. monocytogenes. LexA regulated gene expression is analyzed by comparing ex-pression profiles of WT L. monocytogenes with ∆lexA and lexA* strains expressing anon functional and a constitutive active LexA, respectively. In order to further elu-cidate the regulatory mechanisms and timing of the SOS response, we constructpromoter-reporter gene fusions, which report the size and duration of activation ofLexA controlled protein expression. As reporter genes we employ both lacZ anddegradation tagged gfp. We thus expect to be able to monitor time resolved changesin SOS gene expression and thus come closer to the apparently versatile role of thisstress response mechanism in Listeria monocytogenes.

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Acid shock triggers heavy metal detoxificationin Listeria monocytogenesMüller, S., Neuhaus, K. and Scherer, S.Technische Universität München, Germany

L. monocytogenes is a food borne pathogen ubiquitous in the environment. Thebacterium is exposed to low pH in various ecological niches, such as the stomach,macrophages or acidic soil. Acidic conditions are also found in ecological niches as-sociated with food production. In this work, the acid shock response at pH 5 for15min, 30min, 45min, 60min and 120min and the acid adaptation at pH 5.2(growth to mid log-phase) was studied at the transcriptional level using microar-rays. Besides transcriptional induction of well characterized genes which are in-volved in acid tolerance, an up regulation was also found for genes whose prod-ucts are potentially participating in heavy metal detoxification, such as ion exportproteins (heavy metal exporting ATPase: lmo0641, lmo1852-1854; cation effluxprotein: lmo2231, lmo2231) or heavy metal reduction proteins (arsenate reduc-tase: lmo2230). Furthermore, it was demonstrated that, after induction of the acidtolerance response (60 min, pH 6) L. monocytogenes exhibits a higher resistanceagainst various heavy metal ions, e.g. against CdII, HgII, and NiII; and especiallyagainst CuII and AsV. Acidic pH appears to serve as a trigger not only for the in-duction of genes involved in acid tolerance itself, but also for the induction ofgenes involved in heavy metal resistance. Since metals become more bioaccessibleat low pH, especially in soil, acid solubilized heavy metals might reach noxiousdoses in certain environments and, therefore, detoxification genes may be up reg-ulated. Ongoing analysis using deletion mutants will help to elucidate the role ofdetoxification under acidic growth conditions.

Listeria monocytogenes mutants defective in growth at 5 °Cand in high salt environmentBurall, L., Laksanalamai, P. and Datta, A.US Food and Drug Administration, USA

Listeria monocytogenes can survive and grow in refrigerated temperature and inthe presence of high salt. In an effort to better understand the associated mecha-nisms, a library of 5280 transposon mutants of LS411, an isolate from the Jaliscocheese outbreak, were screened for their ability to grow in brain heart infusion(BHI) broth at 5 °C or in the presence of 7% NaCl. Two mutants with alteredgrowth profiles have been identified. 63-C8 showed a significant reduction ingrowth in BHI at 5 °C. Additionally, the mutant had reduced growth in the pres-ence of 9% but not 7% NaCl. Sequencing located the transposon between two di-vergently transcribed genes, iap and one encoding a putative SecA subunit. qPCRrevealed a substantial reduction in the expression of iap in 63-C8 relative to LS411,though a slight increase in expression was observed for the putative SecA subunitgene. 36-F11 showed no growth reduction at 5 °C but had attenuated growth in thepresence of 7% NaCl. The salt attenuation was exacerbated at 9% NaCl, which re-sulted in reduced colony counts despite apparent increases in biomass. Prelimi-nary microscopy work showed that the majority of cells in these cultures are “live”,indicating the possible presence of either a viable but non-culturable state or in-creased avidity in the bacterial associations within the clumps. Sequencing of theinsertion region of 36-F11 identified the transposon between two divergently tran-scribed genes. The insertion site is 36bp upstream of a gene encoding a glycinebetaine/L-proline ABC transporter, which is down-regulated in the mutant whilethe divergently transcribed putative mechanosensitive ion channel appearedslightly upregulated. Further studies are being conducted to identify the mecha-nisms underlying these phenotypes.

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Revival of 5,000 Listeria strains from Seeliger’s historical collectionwith a semi-automated microbiological pipelineHaase, J.1, Hof, H.2 and Achtman, M.1

1. Science Foundation Ireland2. University of Heidelberg, Mannheim, Germany

Prof. Seeliger from the University of Würzburg, Germany, collected more than7,000 Listeria strains, globally, between the 1920s and the 1980s from humans, an-imals, food and the environment. Within this project, this collection, the “SpecialListeria Culture Collection” (SLCC), has been recultured and frozen at -80 °C.Strain information has been transcribed from the original notebooks and is nowstored in an SQL-based database (Bionumerics, Applied Maths) and the physicalstrain locations are stored in a LIM system (ItemTracker). A large collection suchas this required the set up of a comprehensive “strain pipeline”. This ensures cor-rect cataloguing, efficient storage and retrieval of cultures. This was achieved viacustom scripts (Python) that allow communication and archiving of data obtainedfrom the initial culturing to isolation of DNA and downstream phylogenetic analy-ses. The collection now provides an exceptional opportunity to investigate thepopulation structure and the evolutionary history of L. monocytogenes.

Two point mutations are responsible for the lack of glycosidic substitionin cell wall teichoic acids in Listeria monocytogenes serovar “7”Eugster, M. R.1, Huwiler, S.2, Morax, L.1 and Loessner, M. J.1

1. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland2. Institute of Microbiology, ETH Zurich, Switzerland

The currently defined six Listeria species are further differentiated into at least 15distinct serovars based on serological reactions with specific antisera directedagainst O and, to a lesser extent, H antigens. Some L. monocytogenes strains (suchas WSLC 1034) have previously been assigned to serovar “7”. These cells containribitol phosphate wall teichoic acids (WTA) similar to serovars 1/2 and 3, but with-out glycosyl substition. Purified WTA polymers and associated sugars were ana-lyzed by a novel technique using electrospray ionization tandem mass spectrome-try (ESI-MS/MS). In addition, WTA carbohydrates were identified usingfluorescently labeled N-acetylglucosamine (GlcNAc) binding proteins (CBD-P35)and Listeria phage A118 which requires rhamnose-substituted WTAs for adsorp-tion. We found that the absence of rhamnose and GlcNAc in WTAs of strain 1034 isbased on two single point mutations only. The loss of GlcNac is due to a mutation(P300L) in the protein of gene lmo2550, which is involved in wall teichoic aciddecoration with GlcNAc. The second mutation is a single base deletion and pro-duces a frameshift at position 165 in gene lmo1083, whose product is involved inrhamnose biosynthesis, resulting in a truncated enzyme and lack of rhamnose inWTA. Deletion mutants constructed in a serovar 1/2a background (EGDeDlmo2550 Dlmo1083) resulted in loss of the rhamnose and GlcNAc WTA compo-nents. Complementation with wt lmo2550 and lmo1083 in the EGDe mutant, inserovar “7” strain 1034, and in other mutants featuring a lack of sugar substitu-tion not only restored the rhamnose and GlcNAc content in EGDe, but also pro-duced a serovar 1/2 phenotype in the “serovar 7” strain and other mutants lackingGlcNAs from their WTA. These findings suggest that Listeria serovar “7” strains (atleast WSLC 1034) are likely mutants originating from a serotype 1/2 background,and that Listeria monocytogenes serovar “7” may not exist.

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High-throughput genome sequencing of two Listeria monocytogenesclinical isolates during a large foodborne outbreakGilmour, M.1, Graham, M.1, Van Domselaar, G.1, Tyler, S.1, Kent, H.1, Trout-Yakel, K.1,Larios, O.1, Allen, V.2, Lee, B.3, Nadon, C.1 and Kearney, A.1

1. Public Health Agency of Canada2. Ontario Agency for Health Protection and Promotion, Canada3. Canadian Food Inspection Agency, Canada

A large, multi-province outbreak of listeriosis associated with ready-to-eat meatproducts contaminated with Listeria monocytogenes serotype 1/2a occurred inCanada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gelelectrophoresis (PFGE) revealed two similar but distinct AscI PFGE patterns.Whole genome sequencing of two L. monocytogenes isolates was completed usingthe Roche GS FLX™ platform to rapidly determine the genome sequence of theprimary outbreak strain and to investigate the genetic diversity associated with achange of a single restriction enzyme fragment observed by PFGE. The chromo-somes were collinear, but differences included 28 single nucleotide polymor-phisms (SNPs) and three indels, including a 33kbp prophage that accounted forthe difference in PFGE pattern. The distribution of these traits was assessedwithin clinical, environmental and food isolates associated with the outbreak, andthis comparison indicated that three distinct, but highly related strains may havebeen involved in this nationwide outbreak. Notably, these two isolates were foundto harbor a 50 kbp putative mobile genomic island encoding translocation and ef-flux functions that has not been observed in other Listeria genomes. High-throughput genome sequencing therefore provided a more detailed real-time as-sessment of genetic traits characteristic of the outbreak strains than could beachieved with routine subtyping methods. These data allowed us to determineevolutionary lineages and unequivocally define the full breadth of genetic variationbetween two subtype variants identified by the internationally standardizedPulseNet PFGE typing method. This study confirms that the latest generation ofDNA sequencing technologies can be applied during high priority public healthevents, and laboratories need to prepare for this inevitability and assess how toproperly analyze and interpret whole genome sequences in the context of molec-ular epidemiology.

Expression of antimicrobial activity in food and clinicalListeria monocytogenes isolatesBarbosa, J., Ferreira, V., Borges, S., Azevedo, I., Magalhães, R., Santos, I, Almeida, G.and Teixeira, P.Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Portugal

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen capa-ble of causing fatal infections in risk groups, such as pregnant women, elderly andimmunocompromised individuals. Little information is available on the antimicro-bial susceptibility of L. monocytogenes, particularly of strains isolated from foodand production environments. Minimum inhibitory concentrations (MIC’s) ofampicillin, penicillin G, chloramphenicol, erythromycin, tetracycline and van-comycin were assessed for 370 and 118 food and clinical L. monocytogenes isolates,respectively. All the clinical isolates were susceptible to all the antibiotics tested.Amongst food isolates, 0.5% were resistant to tetracycline and 4.1% and 2.7% wereintermediary and resistant, respectively, to erythromycin. Only one isolate demon-strated a resistance profile to tetracycline, an antibiotic largely used in veterinarypractice, but the resistance of nine isolates to erythromycin is a reason for concern,since erythromycin is used to treat pregnant women diagnosed with listeriosis or asa drug of second choice in cases of ampicillin allergy. Although in vitro antibioticsusceptibility testing does not always reflect the in vivo situation, results demon-strated that some of the isolates investigated are resistant to antibiotics of clinicalimportance commonly used to treat listeriosis. Listeriosis is usually an easily treat-able disease and no reports have been presented on antibiotic resistant isolatesfrom disease cases. However, it is an important area to keep under surveillance.Whilst the present study does not in a scientific sense present de novo science, itcontains very valuable and important data. Only by keeping an on-going track ofthe resistance patterns can these be monitored for unexpected changes.

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The Listeria monocytogenes sigma B and sigma H regulons overlap, butonly sigma B appears to be important for survival of acid, alkaline andoxidative stressChaturongakul, S.1, Raengpradub, S.2, Wiedmann, M.3 and Boor, K. J.3

1. Mahidol University, Thailand2. Silliker, Inc., USA3. Cornell University, USA

Previous transcriptomic analyses in Listeria monocytogenes using whole genome mi-croarrays and Hidden Markov Model (HMM) based promoter searches identifiedmany genes that were controlled by more than one transcriptional regulator (e.g.,σB,σH, σL, PrfA, HrcA or CtsR). The regulons of the two stationary phase sigma factors,σB and σH shared the largest number of genes. To further investigate the regulatoryfeatures shared between σB and σH, whole genome microarrays were performed andtranscript profiles for∆sigB,∆sigH, and∆sigB∆sigH strainswere compared to that ofthe parent strain 10403S. In the present study, using cut-off criteria of a 1.5 fold dif-ference at p<0.05, we identified 301 σB-dependent genes, 82 sH-dependent genes,and a total of 61 genes with expression profiles that suggest regulatory interactionsbetween σB and σH. The 61 co-regulated genes grouped into 4 different qualitythreshold (QT) clusters: Cluster 1 is comprised of 19 genes that are positively regu-lated by both σB and σH (e.g., mRNA levels for cspD, encoding a cold shock proteinand for rpoD, encoding an RNA polymerase sigma factor, were significantly lower inthe ∆sigB∆sigH strain than in the other strains), Cluster 2 contains 17 genes that arenegatively regulated by bothσB andσH (e.g., mRNA levels for genes encoding riboso-mal proteins (rpsR and rplJ), RNApolymerase beta subunit (rpoB), and a sigma factor(sigC) were significantly higher in the ∆sigB∆sigH strain than in the other strains),Cluster 3 contains 11 genes with reduced expression in both the ∆sigB and ∆sigHstrains, but with transcript levels that are the same in both the ∆sigB∆sigH and wildtype strain (e.g., ATP-dependent Clp protease clpP), and Cluster 4 has 14 genes thatare positively regulated in the ∆sigB strain but that are negatively regulated in the∆sigB∆sigH strain or vice versa (e.g., mreD, which contributes to cell shape and rpsDand rplK, which encode ribosomal proteins). In addition to transcript profiling,10403S and the 3 otherwise isogenicmutant strains were also phenotypically charac-terized. After exposure to acid (1 h at final pH of 2.5), alkaline (1 h at final pH of 12), oroxidative stresses (15min at 13mM cumene hydroperoxide final concentration), thephenotype of the∆sigB∆sigH strain was identical to that of the∆sigB strain, i.e., withcell numbers that were significantly reduced (CFU/ml) when compared to those ofthe parent strain. This latter finding further verifies the central role ofσB in stress re-sponse, and suggests thatσH is less important for survival of these stresses.

Virulence gene expression in Listeria monocytogenes strains isolatedfrom different sourcesAlessandria, V., Rantsiou, K. and Cocolin, L.University of Turin, Italy

Listeria monocytogenes is an important food-borne pathogen that has the capacity tocause severe infections. Ubiquitous microorganism, it is commonly isolated fromfoods of animal origin,mainlymeat andmilk products.However, due to its capacity todevelop at refrigeration temperatures, human listeriosis outbreaks aremost often as-sociated with ready-to-eat food products that are consumed without prior cooking.The incidence of the disease depends ondifferent factors including the infective doseand the immunity conditions of the host. The presentwork focuses on the expressionanalysis of four virulence genes (sigB, plcA, hly and iap) in 11 different strains of L.monocytogenes and, more in detail, 3 collection strains (EGDe, NCTC10527, SCOTTA), 7 isolated from food matrices (4 of which isolated from meat products and threefrom diary products) and 1 isolated from humans. In the first step, the expressionanalysis was performed in vitro, culturing each strain in BHI (Brain Heart Infusion)medium, in order to identify possible differences in the expression level for the dif-ferent strains. The combineduse of reverse transcription andquantitativePCR (qRT-PCR) was used to evaluate gene expression. As a second step, we wanted to evaluatethe trend of expression of the genes in food. Analyses were performed inoculating L.monocytogenes in milk at different conditions and in particular at two temperatures(4 and 12°C) for different times (24 and 48 hours). Significant expression differencesemerged for the different genes,without showing any significant association betweenthe expression of the genes and the origin of the different strains.

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Differentiation of Listeria monocytogenes, Listeria innocua andListeria marthii, a novel Listeria species isolated from the naturalenvironment, Finger Lakes National ForestGraves, L. M.1, Helsel, L. O.1, Steigerwalt, A. G.1, Morey, R. E.1, Daneshvar, M. I.1,Roof, S. E., 2 Orsi, R. H.2, Fortes, E. D.2, Milillo, S. R.2, den Bakker, H. C.2, Wiedmann, M.2,Swaminathan, B.1 and Sauders, B. D.3

1. Centers for Disease Control and Prevention, USA2. Cornell University, USA3. New York State Department of Agriculture and Markets Food Laboratory Division, USA

Four isolates (FSL S4-120T, FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-sporeforming bacilli that were phe-notypically similar to Listeria spp. were isolated from soil, standing water, and flow-ing water samples obtained from the natural environment in the Finger LakesNational Forest, New York, USA. The four isolates were closely related to one an-other and were determined the same species by whole genome DNA-DNA hy-bridization studies, but sufficiently different from L. monocytogenes and L. innocuaby DNA-DNA hybridization to form a separate species. 16S ribosomal RNA se-quence analysis confirmed their close phylogenetic relatedness to L. monocytogenesand L. innocua and more distant relatedness to L. welshimeri, L. seeligeri, L. ivanovii,and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prsshowed that these isolates form a well-supported sistergroup to L. monocytogenes.The four isolates showed >82% relatedness at 55°C and >76% relatedness at 70°Cwith 0.0–0.5% divergence. Labeled DNA from the type strain (FSL S4-120T)showed an average of 73% relatedness to three L. monocytogenes strains and one L.innocua strain (range, 69–75%) in reactions at 55°C, the divergence in related DNAsequences was between 7.5% and 9.5%. In reactions at 70°C, FSL S4-120T DNAshowed an average of 45% relatedness to the three L. monocytogenes strains andone L. innocua strain (range, 30–53%). The isolates yielded positive reactions inthe AccuProbe® test that is purported specific for L. monocytogenes, did not fer-ment L-rhamnose, were non-hemolytic on blood agar media, and did not contain ahomologue of the L. monocytogenes virulence gene island. A new species L. marthiiis described which phenotypically and genotypically is distinct from all other Lis-teria species including its closest neighbors L. monocytogenes and L. innocua.

Differed roles of L,D-carboxypeptidases encoded by lmo0028and lmo1638 genes.Yurov, D.*, Varfolomeev, A., Kaminskaya, A. and Ermolaeva, S.Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia

L,D-carboxypeptidase hydrolyses a bound between m-A2pm and C-terminal D-alanine in murein. Listeria monocytogenes carried two genes, lmo0028 andlmo1638 that encode L,D-carboxypeptidases. The lmo0028 promoter has a recog-nition site for the virulence regulator PrfA. Mutant L. monocytogenes strainsGIM0028 and GIM1638 were obtained by site-specific insertions into the chro-mosome of the wild type EGDe strain. Morphology was characterized with lightmicroscopy. Septation was studied with fluorescent vancomycin. Genome copynumbers were determined with quantitative PCR. L. monocytogenes virulence wasstudied on BALB/c mice and human colon carcinoma HT29 cells. The mutationin lmo0028 caused dispersion in cell lengths: the length/width ratio ranged be-tween 1.5 and 15 for GIM0028 and 1 and 4.4 for EGDe. The insertion in lmo1638caused shortening in cells. The average length/width ratios were 3.75, 1.2 and 3.0for GIM0028, GIM1638 and EGDe, respectively. The longish GIM0028 cells weredefected in septum formation, GIM1638 were not. GIM0028 demonstrated twofold decreasing in invasion efficiency. Doubling times in HT29 cells determined byplating were 51 minutes and 70 minutes for EGDe and GIM0028, respectively.However, evaluation of intracellular bacterial genome numbers with qPCR did notreveal a difference between the strains. GIM0028 bacteria increased their lengthupon intracellular growth: average length/width ratio was about 4.8 for invadingbacteria and 9.2 after 6 h of intracellular growth while for the wild type bacteria itwas 3.5 (p<0.005). GIM0028 demonstrated reduced virulence for BALB/c micewith LD50 of 1x106 and 1.5x104 CFU/mouse for GIM008 and EGDe strains, re-spectively. The Lmo0028 L,D-carboxypeptidase is involved in septum formationwhile Lmo1638 seems to be involved in cell elongation. A mutation in lmo0028decreases L. monocytogenes virulence.


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Antimicrobial susceptibilities of Listeria monocytogenes isolatedfrom retail beef, pork and poultry in JapanIda, M., Shimojima, Y., Kaneko, S., Higuchi, Y., Nakama, A. and Kai, A.Tokyo Metropolitan Institute of Public Health, Japan

Meat and meat products are the major source of food-borne infections and themost important link between food-producing animals and humans. The aim ofthis study is evaluate antimicrobial susceptibilities of Listeria monocytogenes iso-lated from meat in Japan. A total of 125 strains isolated from beef (n= 10), pork(n=49) and poultry (n=66) between 2001 and 2009 in Tokyo area were examined.Serotypes of tested strains were 1/2a (n=46), 1/2b (n=26), 1/2c (n=15), 4b (n=26)and others (n = 12, including untypable). Ampicillin, penicillin G, gentamicin,kanamycin, erythromycin, oxytetracycline, norfloxacin and chloramphenicol wereincluded in the study. The minimum inhibitory concentrations of antimicrobialagents were determined by the agar dilution method according to the guidelines ofClinical Laboratory Standards Institutes (CLSI). CLSI breakpoint guidelines for L.monocytogenes and other Gram-positive bacteria were used for data analysis. En-terococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922 were used asquality control strains. All isolates were susceptible to antimicrobials except foroxytetracycline. Three strains, isolated from domestic pork, domestic poultry andbeef imported from Australia were resistant (>32µg/ml). All of them were isolatedin 2001 and belonging to serotype 1/2c. This study showed that antimicrobial re-sistance is not highly prevalent in L. monocytogenes in meat in Japan.

Genetic basis of two low pathogenic L. monocytogenes strainswith apparent phospholipase C activityJiang, L., Bai, F., Chen, J., and Fang, W.Zhejiang University Institute of Preventive Veterinary Medicine, Hangzhou, China

Two Listeria monocytogenes isolates showing apparent in vitro phospholipase Cactivity were characterized for their genetic basis and pathogenecity. Sequencinganalysis found two major mutations in plcB at position 1 [from A (ATG) to G(GTG)] and position -26 [from C (ACG) to T(ATG)], resulting in 9-aa extension ofthe enzyme at the N-terminus. The strains were of serovar 4a, had low patho-genecity with LD50 at log10CFU 8.21-8.35 in BALB/c mice, and presented no mi-croscopically visible plaques on the L929 cell monolayers. Site-directed mutationof these sites to the plcB version of strain 10403S abolished the enzyme activity, in-creased virulence by one log (from 8.12 to 7.07), but did not have significant effecton plaquing. This abolishment did not seem to be related to presence of G145S inthe central regulator prfA, which was found to induce overexpression of virulencegenes independent of environmental conditions in other strains. However, thisdoes not mean that prfA was not involved in regulation of its expression becausetransposon mutagenesis identified two mutant strains lacking apparent phospho-lipase activity due to disruption of the prfA by insertions at nt364 and nt828. Thereare two additional changes of T165A and K197N in prfA, as compared with other L.monocytogenes serovar 1/2a and 4b strains. Further work is under way to examineif apparent activity of the novel phospholipase in these strains results from easyaccess to zinc metalloprotease encoded by mpl for maturation, or if amino acidsubstitutions of T165A and K197N other than G145S in prfA also contribute to reg-ulation of downstream virulence genes. Using one of the strains as a model organ-ism, we are testing how phospholipase C and listerolysin interact in determiningits pathogenecity and if there are other genes involved in their low pathogenecityby genome wide scanning using Solexa sequencing technology.

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Molecular genotyping and antimicrobial resistanceof Listeria monocytogenes from foods and the environmentParisi, A.1, Miccolupo, A.1, Fraccalvieri, R.1, Latorre, L.1, Normanno, G.2 and Santagada, G.1

1. Experimental Zooprophylactic Institute Apulia and Basilicata, Italy2. University of Bari, Faculty of Veterinary Medicine, Italy

Listeria monocytogenes is generally susceptible to a wide range of antibiotics, how-ever an increasing number of strains resistant to one or more antibiotics have beenreported. Objectives of the present study were to evaluate molecular genotypingand antimicrobial susceptibility in L. monocytogenes isolates from foods and theenvironment. Over all a total of 100 L. monocytogenes isolates from foods (n=74)and the environment (n=26) were subjected to molecular genotyping using Multi-Locus Sequence Typing as previously described, moreover the susceptibility to 21antimicrobials was performed using Gram-positive GPN4F kit (TREK DiagnosticSystem). All the isolates resulted sensitive to ampicillin, chloramphenicol, gen-tamycin, penicillin G, rifampicin, streptomycin, sulfamethoxazole trimethoprim.Antimicrobial resistance was recorded for oxacyllin (24%), and tetracycline (1%)whereas some isolates resulted moderately sensitive toward clindamycin (19%),quinupristin/dalfopristin (2%) and ciprofloxacin (1%). The isolates belonged tosix different serotypes: 1/2a (43%), 1/2c (24%), 1/2b (15%), 4b/4e (12%), 3a (4%)and 3b (2%). MLST identified 50 different Sequence Types (ST) most of whichrepresented by a single isolate. ST9 (21%), ST121 (12%), ST3 (5%) and ST199 (5%)were the most prevalent STs. Moreover MLST allowed a good discrimination ofthe two main genetic lineage of L. monocytogenes. Our results confirm the lowprevalence of food and environmental isolates resistant to antimicrobials. Inter-estingly, a good correlation between resistance to oxacillin and genetic lineage Iwas observed (Yates corrected =35,46; p<0.005); this is in agreement with theclonal organization of L. monocytogenes population. The severity of L. monocyto-genes infections and the reported increasing of antimicrobial resistance make nec-essary the continuous monitoring of this trend although the recorded data do notappear to be alarming. The comparison of phenotypic and genetic characters allowto validate the evolutionary models speculated for this microbial species.

Virulence transcriptome analysis of Listeria monocytogenesby application of microarrays in vitro and in situRantsiou, K., Alessandria, V. and Cocolin, L.University of Turin, Italy

With the complete genome sequence available for Listeria monocytogenes as wellas other Listeria spp., it is nowadays possible to use tools that allow global analysisof the biology of this pathogenic microorganism. Microarrays give the possibility tostudy concurrently a large number of genes and deduce information regardingtheir expression. This approach accelerates significantly the rhythm by which newinformation is generated. In this way, the effect of different conditions on the ex-pression of a series of genes and as a consequence on the physiology of a microor-ganism can be investigated. Within the 6th FP project Pathogen Combat, it wasdeveloped a microarray including 72 genes of L. monocytogenes, encoding for vir-ulence, adhesion and stress response genes. In this study we sought to apply thisarray to study the effect of different environmental conditions on the expression ofvirulence genes. First, different strains of L. monocytogenes were grown in vitro, atdifferent temperatures, pH and salt concentrations. Then, L. monocytogenes wasartificially inoculated in different food matrices, to simulate the conditions of ‘vir-ulence’ of this microorganism at time of consumption. Global analysis of the ex-pression of virulence genes in different L. monocytogenes strains will lead to a bet-ter undestanding of the physiology of this microorganism and eventually to theprediction of its potential to cause disease to humans.

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Effects of growth conditions on surface properties of Listeria;a proposed role for AI-2Wong, H. T. L., Nwaiwu, O.* and Rees, C. E. D.School of Biosciences, University of Nottingham, UK

Listeria monocytogenes is recognized as a particular problem in factory environ-ments due to its ability to colonize surfaces and then become persistent by adapt-ing to low nutrient and low temperature conditions. We have been investigatingthe effect of low nutrient conditions on the ability of Listeria to attach to surfaces.Cultures were prepared using a minimal medium (D10), a rich medium (BHI) andalso the D10 medium supplemented with duck meat extract. Growth in the BHImedium was fastest, but significant increases in growth were seen when D10 wassupplemented with 10% duck meat extract. One feature of cells that become per-sistent in factory environments is their ability to form biofilms that are resistant tocleaning and disinfection. We have found that the surface hydrophobicity (mea-sured using Microbial Attachment to Solvents (MATS) assays) alters significantlywhen the organism is grown under low nutrient conditions, with cells becomingmore hydrophobic. Also these cells have a greater tendency to flocculate in liquidculture. This affect is seen when cells are grown above 30°C and therefore is notrelated to the synthesis of flagellae. The ability to form biofilms in Listeria isknown to be affected by the synthesis of a functional autoinducer 2 (AI-2)-likesignal, and that an intact luxS gene is associated with inhibition of attachment andbiofilm formation. Using a Vibrio harveyi reporter strain we have found that whencells are grown in minimal media, the amount of detectable AI-2 is reduced. AsAI-2 has a role in the down-regulation of components required for attachmentand biofilm formation, this result is consistent with our observation of changesin the surface properties of Listeria under low nutrient conditions that are morelikely to promote surface attachment.


Stress behaviour of a Listeria monocytogenes 568 Lmo1634transposon mutantTruelstrup-Hansen, L.1, Holman, D. B.1 and Ells, T. C.2

1. Dalhousie University, Canada2. Agriculture and Agri-Food Canada

Listeria monocytogenes is an important psychrotrophic foodborne pathogenic bac-terium with tendency to persist in food processing plants. Heat treatment is rou-tinely used as a means to control pathogenic bacteria in food products. Therefore,the thermotolerance of L. monocytogenes and mechanisms responsible are of inter-est. Previous research using transposon mutagenesis yielded several heat resistantmutants, including one, named 1B4, that was mapped to a putative alcohol/ac-etaldehyde dehydrogenase gene, lmo1634 or adh, also identified as Listeria adhe-sion protein. The objective of this study was to investigate the function of this genein relation to a variety of environment stresses and in biofilm formation. The trans-poson insertional mutant 1B4 demonstrated significantly greater tolerance to acid(pH 2.7), 20% NaCl, and heat treatment of 52°C than the wild-type (WT) Lm568strain. Attachment of 1B4 (7.06 log10 CFU/cm2) to stainless steel coupons was notsignificantly (p>0.05) different from the WT Lm568 (6.91 log10 CFU/cm2). Neither1B4 nor Lm568 exhibited greater survival after desiccation (43% RH) at 20°C for 7days. The alcohol dehydrogenase activity of Lm568 (0.197U/mg) was found to besignificantly (p>0.05) greater than in 1B4 (0.0458U/mg). 1B4 was complementedin trans with the WT lmo1634 gene and the successful transcription of lmo1634 inthe complemented strain was confirmed by RT-PCR. In addition, no polar effect onthe genes immediately downstream of lmo1634 in 1B4, i.e., lmo1635, lmo1636, orlmo1637, was observed. The trans complementation of 1B4, however, did not com-pletely restore the WT phenotype. Attempts to create a deletion mutant in lmo1634ultimately proved unsuccessful. Despite these technical issues, it is clear that a mu-tation in lmo1634 yields a multi-stress resistant phenotype.

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Investigation of the conditions that trigger the activation of the alter-native sigma factor σB in Listeria monocytogenesUtratna, M., Shaw, I. and O’Byrne, C.National University of Ireland, Galway, Ireland

Listeria monocytogenes has evolved multiple strategies to overcome diverse stressconditions and it is able to occupy extreme environmental niches. As a facultativeintracellular foodborne pathogen L. monocytogenes successfully invades host cells,surviving in gastric tract before the final colonization of the target organs. The al-ternative sigma factor (σB) plays an essential role in the stress tolerance and viru-lence of L. monocytogenes. Many components of the σB regulon have been identi-fied but the mechanisms that regulate σB are still unknown. The current modelfor σB regulation, based on work done in Bacillus subtilis, suggests that the activ-ity of σB is independent of the levels of σB in the cell. The expression of OpuCA hasbeen shown to be controlled by σB. Therefore the levels of this protein can act as anindirect reporter of the activity of σB in L. monocytogenes. Polyclonal antibodiesagainst OpuCA were developed in chickens and used in Western blotting. Basedon this reporter system it was shown that σB is more active in stationary phasewhen compared to exponential phase (OD600=0.6). σB activity under conditionsencountered in the human gut was also determined. Levels of OpuCA increasegradually in the range of salt up to 0.9M NaCl and when the pH is reduced from pH7.2 to pH 5.0. Higher σB activity was also detected under limited oxygen condi-tions and at 4 °C. However, no change in the levels of OpuCA was observed in thepresence of low concentrations of bile salts up to 5mM. Additionally an attemptwas made to develop a transcriptional reporter fusion system by cloning the σB

promoter from strongly σB-dependent gene, lmo2230, into the reporter vectorpTCV-lac allowing the measurement of β-galasctosidase activity under variousconditions. This genetic system will also be a useful tool in monitoring the activityof σB in L. monocytogenes.

Characterization of Listeria monocytogenes 1/2a, 1/2b, 1/2c and 4bby Amplified Fragment Length Polymorphism and evaluation of theirgeographical distributions in PortugalMaia, C. H.1, Goulão, M. M.2, Santos, M. I.1, Ferreira, M. A. S. S.3 and Pintado, C. M. B. S.2

1. Instituto Nacional de Saúde Doutor Ricardo Jorge, Portugal2. Escola Superior Agrária, Instituto Politécnico de Castelo Branco, Portugal3. Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Portugal

Listeriosis is a bacterial infection caused by the ingestion of foods contaminatedwith Listeria monocytogenes, that can affect humans, especially the group that in-cludes pregnant women, newborn infants, the elderly and immunocompromisedindividuals. AFLP molecular subtyping, using EcoRI restriction enzyme, was usedto do the differentiation between strains isolated from different Denomination ofProtected Origin regions of cheese production in Portugal and as well from clinicalspecimens. A set of 159 strains belonging to serotypes 1/2a, 1/2b, 1/2c and 4b iso-lated from samples of Serra da Estrela ewe’s cheese, São Jorge cow´s cheese, Serpaewe´s cheese, Tolosa ewe´s cheese and Castelo Branco ewe´s cheese were analyzed.Evaluation of similarities between strains was obtained by analyses with BioNu-merics™ software by Applied Maths, Belgium. With AFLP molecular subtyping,nearly all the isolates were divided according to their serovar. Therefore it waspossible to predict the serovar from the AFLP pattern found in most of the iso-lates. In the opposite direction, each one of the serovars was associated to severalAFLP molecular types, which demonstrates the grater discriminatory capacity ofthe AFLP subtyping. Similarities between profiles, based on band positions, werederived from the Dice correlation coefficient (SD) with a maximum position tol-erance of 1%, 3% or 4%. Listeria monocytogenes strains were clustered by the tech-nique of the UPGMA and a dendrogram was constructed to reflect the genetic dis-tance between them.

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Global analysis of the Listeria monocytogenes surface proteinsof the LPXTG familyBotello-Morte, L.1, Calvo, E.2, Mariscotti, J.1, D’Orazio, V.1, García-del Portillo, F.1

and Pucciarelli, M. G.1,3

1. Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superiorde Investigaciones Científicas (CSIC). Darwin 3, Madrid, Spain

2. Unidad de Proteómica, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain3. Departamento de Biología Molecular, Universidad Autónoma de Madrid. Campus de Cantoblanco,

Madrid, Spain

Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen thatcauses serious systemic diseases in humans and animals. Comparative genomicsrevealed the presence in the Listeria genus of a large number of genes encodingsurface proteins containing an LPXTG sorting motif. This signature makes theseproteins recognized by sortases, the enzymatic machinery that anchors proteinscovalently to the peptidoglycan. In the case of the L. monocytogenes EGD-e strain,a total of 41 genes encoding LPXTG proteins were discovered. Despite the rele-vance of surface proteins for communication with the environment and the host,the biological role of most members of this family remains unknown in L. mono-cytogenes. In a collaborative effort with other European groups that contributed togenerate mutants defective in specific LPXTG proteins, we accomplished a com-prehensive proteomic analysis in peptidoglycan material purified from each ofthese mutants. A total of 30 LPXTG mutants have been analysed to date. Pro-teomic analysis was also performed in peptidoglycan purified from intracellularwild-type bacteria upon infection of epithelial cells. A major finding of this globalapproach was the cross-regulation existing among certain LPXTG proteins. Thisphenomenon involved in most cases the down-regulation of concrete LPXTG pro-teins in response to the lack of a specific LPXTG protein. Interestingly, the inversephenomenon, the up-regulation of a LPXTG protein in response to a deficiencyin another LPXTG protein, was also observed. These proteomic data were con-firmed with specific antibodies. Proteins subjected to these regulatory processes,currently investigated in detail by our group, include Lmo0320 (Vip), Lmo0514,Lmo0610, Lmo0880, InlE, InlG, InlH, and Lmo2085.

Antimicrobial susceptibility of Listeria monocytogenes strainsderived from food and food-processing bakery plantEusébio, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G., Silva, J. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

The susceptibility of 167 strains of Listeria monocytogenes isolated from a bakeryindustry, from food and food-processing environment, to 11 antibiotics was deter-mined by the standard agar dilution methodology. The tested antibiotics were:ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, nitrofu-rantoin, penicillin, rifampicin, streptomycin, tetracycline and vancomycin; mini-mal inhibitory concentrations values were used to classify the strains into sensi-tive, moderately resistance and resistant. All the tested isolates were found to besusceptible to ampicillin, chloramphenicol, gentamicin, nitrofurantoin, penicillin,rifampicin, tetracycline streptomycin and vancomycin. In the case of erythromy-cin, 54 isolates (32%) were susceptible, 68 (41%) displayed moderately resistanceand 45 (27%) were resistance. Concerning to ciprofloxacin, a moderate resistancewas observed in 12 strains (7%) against 155 strains (93%) that were susceptible.Generally, this study showed that L. monocytogenes strains are susceptible to theantibiotics commonly used in the treatment of listeriosis. Concerning that antibi-otic resistance in some L. monocytogenes strains has already been described, a con-tinued study of emerging antimicrobial resistance is important to guarantee aneffective treatment of human listeriosis.

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A physiological study to purpose a new formal methodto obtain L. monocytogenes cells adapted to BACSaá Ibusquiza, P., Cabo, M. L., Herrera, J. J. R., Vázquez, D., Carrera, S. and Eiriz, E.Instituto de Investigaciones Marinas (Marine Research Institute), CSIC, Vigo, Galicia, España

Maximum adaptation potential of Listeria monocytogenes (L. monocytogenes) cellsto benzalkonium chloride (BAC) was classically checked by successive expositionof stationary cells to increasing sublethal BAC concentrations. However, thismethod is time consuming and do not ensure we reach the maximum level of adap-tation of the strain. So, in the present work we develop a new method to obtainBAC-adapted cells of L. monocytogenes that is based in only one exposition ofL. monocytogenes exponential-phase cells to sublethal concentrations of BAC.Moreover, in order to predict the optimal conditions for achieving the maximumlevel of adaptation, a factorial design to formalize the effects of the initial concen-tration of cells and the concentration of BAC during the exposition was carriedout. Obtained empirical model demonstrated a positive effect of inoculum and apositive interaction between both variables. However, a negative first order effectfor the BAC indicated the convenience of using moderately high concentrations ofit during the expositions. As a consequence of applying this procedure, adaptationof L. monocytogenes 5873 to BAC was increased aproximately 4 times respecting tothe wild type strain in only 36h. Finally, comparison between the expression pro-files in the wild and the highest BAC-adapted variant was also carried out.

Characterization of a RNA-helicase in the human pathogenListeria monocytogenesNetterling, S. and Johansson, J.Department of Molecular Biology, Umeå University, Umeå, Sweden

The intracellular pathogen Listeria monocytogenes grows in a wide range of mi-lieus and shows a great acceptance to different environmental conditions, such asbeing able to grow in a span from -1 to 45°C. Listeriosis can cause severe symptomssuch as meningitis, with high lethality rate. The ability to grow at low tempera-tures is one of the most interesting aspects of this pathogen. We have looked atRNA helicases; proteins that are able to unwind short dsRNA structures in an ATP-dependent manner. Most mRNAs develop complex secondary and tertiary struc-tures during the maturation process, these structures needs to be unwound be-fore translation initiation. The stability of these complex structures is influencedby temperature, often becoming more rigid at lower temperatures thereby hin-dering e.g. translation, resulting in undesired cold-stabilized RNA structures. RNAhelicases may function by relaxing local RNA-RNA interactions or by dissociat-ing RNA from proteins. In L. monocytogenes RNA helicases have earlier beenshown to be up-regulated during growth in a cold environment. RNA-helicaseshave been suggested, but not proven, to participate in small regulatory RNA events.We have looked at a mutant lacking one DExH-box RNA helicase in L. monocyto-genes. This DExH-box RNA helicase deletion strain displayed an inability to growat 4°C and had an impaired growth at temperatures below 37°C. Motility was alsoaffected in the deletion strain implying that it cannot spread in food sources aswell as the wild-type strain. All phenotypes were rescued in a complementedstrain. Our results indicate that RNA helicases is of high importance to the bac-terium for growth at lower temperatures, and provides a link between RNA heli-cases and motility.

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AREA //B // Listeria monocytogenes as a human and animal pathogenPOSTER PRESENTATIONS // P / 47–81, 182

A Listeria monocytogenes strain is still virulent despite non-functionalmajor virulence genes: Optical Mapping shows a potential mechanismRoche, S. M.1, Grépinet, O.1, Corde, Y.1, Teixeira, A. P.1, Kerouanton, A.2,Témoin, S.1, Mereghetti, L.3, Brisabois, A.2 and Velge, P.1

1. INRA, UR 1282 Infectiologie Animale et Santé Publique, F-37380 Nouzilly and IFR 136, Agentstransmissibles et Infectiologie, France

2. AFSSA LERQAP, Unité Caractérisation et Epidémiologie Bactérienne, F-94706 Maisons-Alfort,France

3. Université François Rabelais de Tours, EA3854 “Bactéries et risque materno-foetal”, Tours, Franceand CHRU, Tours, France

L. monocytogenes is a pathogenic species, but some strains exhibit low virulence. Inprevious studies, 24 naturally occurring low-virulence L. monocytogenes strainswere identified using a method combining a plaque-forming (PF) assay with sub-cutaneous (SC) injection into the left hind footpad of mice. Based on their phe-notypic characteristics, these low-virulence strains have been assigned by clusteranalysis to one of four groups. The 11 strains belonging to Group I exhibit a mu-tated PrfA whereas five out of the six strains belonging to Group III have causalmutations in plcA, inlA and inlB genes. New strains exhibiting the same PFGE spe-cific profile than the low-virulence Group III strains have been identified and char-acterized. All were low-virulence strains exhibiting the same mutations charac-teristic of the Group III strains, beside the A23 strain which has beencharacterized as a virulent strain in the mouse model. Interestingly, this strainexhibited the same mutations than the low-virulence Group III strains in the inlA,inlB, plcA genes and another one in the mpl gene. This led to expression of inactiveInlB, PI-PLC and PC-PLC proteins and to a lack of InlA expression which areknown to be important for virulence. Despite these mutations in these major vir-ulence genes, the A23 L. monocytogenes strain was still virulent in vitro and in vivo.Analysis by Optical Mapping (Phylogene-France, OpGen-USA) of the A23 and twoGroup III strains in comparison with the EGDe genome showed specific fragmentsinserted in the genome of the Group III strains. Identification of the genes modi-fied by the insertion could explain the virulence of the A23 strain compared to theGroup III strains. Phylogenetic analysis of these strains could be fruitful to un-derstand the evolution of virulence trait as well as plasticity of virulence genes.

Protein expression of lineage I, II, and III Listeria monocytogenesstrains in murine macrophagesDonaldson, J. R., Nanduri, B., Pittman, J. R., Burgess, S. C. and Lawrence, M. L.Mississippi State University, USA

In the current study, we compared the ability of Listeria monocytogenes strains fromgenetic lineages I, II, and III (serovar 1/2a strain EGD, serovar 4b strain F2365, andserovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1.We found that the lineage III strain HCC23 was able to initiate an infection butcould not establish prolonged infection within the macrophages. By contrast, strainsEGD and F2365 proliferated within macrophages for at least 7 hr. We further char-acterized this interesting phenotypic difference by determining the protein expres-sion profiles of these strains at 0 hr, 3 hr, and 5 hr post-infection using two dimen-sional liquid chromatography coupled with electrospray ionization tandem massspectrometry. We found that metabolic and cell wall associated proteins were ex-pressed by all three strains at 3 hr post-infection. However, increased expression ofstress response and DNA repair proteins was detected in the lineage I and II strainsat 5 hr post-infection. These proteins were not significantly increased in the lineageIII strain at this time point. By comparing the protein expression patterns of thesethree L. monocytogenes strains during intracellular growth in macrophages, we wereable to detect biological differences that may determine the ability of L. monocyto-genes to survive and persist in macrophages.

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Prevalence of L. monocytogenes in bovine mastitic milk samples:Possible source of food borne infectionKalorey, D. R.1, Warke, S.1, Kurkure, N. V.1 and Barbuddhe, S. B.2

1. Nagpur Veterinary College, India2. ICAR Research Complex for Goa, India

Listeria monocytogenes is a food borne pathogen responsible for listeriosis, dis-ease characterized by meningitis, encephalitis, reproductive disorders and sep-ticemia in human beings. One of the major source of Listeria is milk. A study wasconducted to know the prevalence of Listeria monocytogenes in mastitic milk fromcentral India. Quarter milk samples (n=1221) were collected from dairy cows andscreened for sub clinical mastitis by California mastitis test (CMT). CMT positivesamples were examined for the presence of L. monocytogenes following two stepenrichment and plating on selective agar. Confirmation of isolates was based onbiochemical tests, haemolysis on blood agar and CAMP test. The L. monocytogenesconfirmed isolates were subjected to PCR assay for detection of virulence markergenes (hlyA, actA, iap and prf A). A total of 71 (5.81%) milk samples harbored L.monocytogenes. The hlyA gene was detected in all the isolates. The hlyA and prfAgenes were detected in 33 strains. The hlyA and actA were in seven strains. Elevenstrains harbored hlyA, actA and iap genes. All isolated strains were subjected toserotyping by PCR and revealed all to be of 4b, 4d and 4e serogroup. Excretion of L.monocytogenes in milk may contribute to food borne infection in human. Detectionof 4b serogroup is of highly significance owing to its association with food bornelisteriosis outbreaks.

Agr-dependent peptide sensing in L. monocytogenes – Effects on biofilmformation, virulence and global gene expressionWaidmann, M. S.1*, Monk, I. R.2, Auchter, M.1, Preising, N. P.1, Hill, C.3 and Riedel, C. U.1

1. Institute of Microbiology and Biotechnology, University of Ulm, Germany2. Trinity College Dublin, Ireland3. University College Cork, Ireland

Autoinducing peptides are signalling molecules used in population-dependent generegulation of Gram-positive microorganisms. The best studied example of bacterialpeptide sensing is the agr system of staphylococci. An operon with high structuraland sequence similarity to the staphylococcal arg system was recently identified inthe genome of L. monocytogenes EGDe. The listerial agr system is composed of a fourgene operon with agrB involved in the proteolytic processing/export of the geneproduct of agrD, the posttranscriptionally modified signalling peptide, andagrA/agrC encoding a typical two-component system with histidine kinase (AgrC)and response regulator (AgrA). Here, we present our recent advances in identifyingand characterizing the native agr peptide of L. monocytogenens from spent cell cul-ture supernatant and the effects of agr peptide sensing on biofilm formation, viru-lence and global gene expression. Furthermore, the mechanisms of agr-dependentgene regulation in L. monocytogenes were investigated. A ∆argD mutant showed asignificant defect in biofilm formation. Invasion of EGDe ∆argD into Caco-2 andHep-2 cells was significantly reduced compared to EGDe wt. Moreover, the ∆argDmutant showed an unusual subcellular distribution after primary invasion. In linewith these observations promoter activities of important virulence genes were re-duced in the ∆argD mutant. Using bioluminescence in vivo imaging, a significant at-tenuation of EGDe ∆argD in a murine model of listeriosis could be shown. More-over, microarray analysis revealed that expression of a large number of genesbelonging to all functional categories was affected in EGDe ∆argD both in exponen-tial and stationary growth phase indicating a global impact of agr-dependent peptidesensing on all aspects of physiology in L. monocytogenes.


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agrD-deletion affects InlA- and InlB-regulation viaa temperature-dependent and -independent mechanismWaidmann, M. S.1*, Monk, I. R.2 and Riedel, C. U.1

1. Institute of Microbiology and Biotechnology, University of Ulm, Germany2. University College Cork, Ireland

Quorum sensing via the secretion of autoinducing peptides is a commonly usedmechanism for gene regulation among Gram-positive bacteria. A well known ex-ample is the accessory gene regulator (agr) in Staphylococcus aureus, which isknown to affect virulence. Following the sequencing of Listeria monocytogenesEGDe genome and the identification of a homologous gene locus, suggestions of aregulatory role in virulence arose. The first step of listerial pathogenic lifecycle isthe entry into a host cell, a process mediated by members of the Internalin family.Therefore, Internalin A and B are described as the main players for uptake by ep-ithelial and endothelial cells. To testify the role of agr in regulation of these viru-lence factors, we evaluated the invasive capability of an agrD deletion mutant,which lacks the putative autoinducing peptide, into Caco-2 and HEp-2 cells. Theseassays showed that the Internalin A-dependent invasion of the mutant was sig-nificantly decreased in comparison to the wild type. The temperature chosen forgrowing pre-cultures did not influence this defect. By contrast, the effect of theagrD deletion for Internalin B-mediated invasion into HEp-2 cells was only im-paired in pre-cultures grown at 37 °C. Furthermore, the total numbers of invadedbacteria were much higher as compared to 30°C. These results indicate a temper-ature-independent role of agr in the regulation of Internalin A and a temperature-dependent mechanism in case of Internalin B. This influence could occur via af-fecting either the expression itself or the activation of the identified regulatorsPrfA and σB. According to these results agr seems to be an additional player for lis-terial virulence.


Bovine cranial nerve Schwann cells express E-cadherin, a candidatekey-player in the brainstem invasion of Listeria monocytogenes in cattleMadarame, H.1, Seuberlich, T.2, Vandevelde, M.2, Zurbriggen, A.2, and Oevermann, A.2

1. Veterinary Teaching Hospital, Azabu University, Japan2. Neurocenter, Vetsuisse Faculty Bern, Switzerland

The interaction of internalin A, a major surface ligand of Listeria monocytogenes(LM), and its host cell-receptor E-cadherin is required for the entry of LM intocells and for the crossing of the intestinal and placental barrier during infection. Inruminants, listeriosis occurs most commonly as rhombencephalitis, specificallytargeting the brainstem, and it is believed that LM enters the brain via cranialnerves. However, the host cell receptors involved in brain invasion are not known.The aim of this study was to investigate the expression of E-cadherin in cattlebrain, trigeminal nerve (TN) and its ganglion (TG), and thus its putative role inbrain invasion. To this end, brains, TN and TG of cattle were examined by im-munohistochemistry (IHC, n=6) and Western blot (n=2). In the brain, strongmembranous E-cadherin expression was observed in choroid plexus epithelialcells by IHC, whilst no other brain structures were labeled. TN and TG exhibitedstrong E-cadherin expression in Schwann cells and satellite cells (which are spe-cialized Schwann cells), respectively. The labeling was particularly evident at theintercellular boundary of satellite cells and at the Schmidt-Lantermann incisures.Confirming the IHC results, E-cadherin specific bands of molecular masses of ap-proximately 37kDa and 130kD were evident in the WB of the TG, but were absentin the WB of the brainstem. These results indicate that E-cadherin expressingSchwann cells of cranial nerves might be the initial port of entry for LM in cattlerhombencephalitis. From there, LM might gain access to the brain by cell-to-cellspread via axons. However, this hypothesis needs to be confirmed by complemen-tary in vitro investigations of the molecular host-pathogen interactions.

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Pathogenic potential of Listeria monocytogenes isolatesfrom New Zealand seafood premises: implications for controlDurante Cruz, C. and Fletcher, G.The New Zealand Institute for Plant and Food Research

Listeria monocytogenes is likely to persist in food premises, leading to contaminationof products processed therein. It is therefore important to assess the virulence po-tential of these isolates in order to evaluate the risk they can pose to consumers’heath. In this work, we assessed the invasion capacity of L. monocytogenes isolatesobtained from New Zealand (NZ) seafood premises and correlated this with theirpersistence and genetic profile. From 120 isolates obtained from four seafood prem-ises, 30 (28 from environmental and 2 from product samples) belonging to differentserotypes and pulsotypes were compared in this study. These were compared withfive human isolates obtained from the NZ Reference Culture Collection, two iso-lates (human and food) from a NZ seafood-related outbreak of listeriosis and onemeat isolate shown to be resistant to high pressure conditions. ScottA was used as areference strain and assigned an invasion index of 100%. In vitro studies were con-ducted using Caco-2 cells (100:1 Listeria:Caco-2) followed by 40 min incubation at37°C. Three independent experiments were performed on each isolate in duplicate.In Greenshell™ mussel premises, isolates from Plant I showed higher invasivenessthan those from Plant II. All isolates were serotype 1/2a, except for one 1/2b fromPlant I. The persistent and predominant pulsotype from Plant II had a low invasionindex. Serotype 1/2b and 4b isolates from the two fish premises showed higher in-vasiveness than the serotype 1/2a isolates from mussel plants. Product (serotypes1/2a and 1/2b) and one human (serotype 1/2a) isolates showed higher invasion in-dices than environmental ones. The seafood outbreak strains had an intermediateinvasion index. This study improves our understanding of L. monocytogenes pres-ent in NZ seafood premises and highlights the need to improve control. Althoughsome environmental factory isolates were potentially pathogenic, persistent strainswere generally less invasive in Caco-2 cells.

Copper homeostasis and virulence in Listeria monocytogenesDavid, C.1, Schuler, S.1, Glenn, S.2, Jen, C.1, Andrew, P.2 and Roberts, I. S.1

1. University of Manchester, UK2. University of Leicester, UK

Copper is essential for many bacteria being an important prosthetic group for cer-tain enzymes. However at high levels copper is toxic in part due to its ability toredox cycle and catalyse the formation of oxygen derived free radicals. As suchbacteria need to be able to maintain copper homeostasis. In L. monocytogenes theproblem of copper homeostasis is particularly acute. It inhabits a range of envi-ronments including soil, effluents and food where it will be exposed to fluctuatingcopper levels. The use of copper as an anti-microbial in animal feeds, as a disin-fectant in factory-based farming and as a biocide in fruit production means L.monocytogenes will have to combat potentially toxic levels of copper during foodproduction and processing. Adapting to fluctuations in the level of copper is im-portant during infections. L. monocytogenes replicates in a number of host tissuesthat will offer different challenges with regard to copper availability. Early in in-fection L. monocytogenes grows in the gall bladder, where there are high levels ofcopper, whereas in the liver and spleen it replicates in an environment in whichthere may be little free copper. Adapting to these different niches within the hostrequires effective copper homeostasis. In this paper we describe and analyse theoperon that encodes for the only P1 type-ATPase copper exporter in L. monocyto-genes and describe the molecular basis by which transcription of this operon isregulated by environmental copper and identify the role played by this operon ininfection of L. monocytogenes.

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Pattern of cytokine production during murine listeriosisDussurget, O.Institut Pasteur, France

Listeria monocytogenes causes listeriosis, an infection whose manifestations ex-tend from gastroenteritis to septicemia, meningitis, encephalitis, abortions andperinatal infections. Cytokines are important mediators of host defense againstpathogenic microorganisms. They control innate immune responses and con-tribute to shape adaptive immune responses. Several studies have previously ana-lyzed the production of one or several cytokines in response to Listeria monocyto-genes in vitro and also in vivo. In this work, we used a murine model of primarylisteriosis to systematically follow in the same animal the protein levels of 20 cy-tokines in the blood and in the liver and spleen, two important organs in which L.monocytogenes replicate. Infection was shown to trigger the secretion of the majoractivator of macrophage antimicrobial activity, interferon (IFN) g, in blood, liverand spleen 24h after intravenous inoculation. The early response to infection wasalso characterized by a significant increase in proinflammatory cytokines tumornecrosis factor (TNF) a, interleukin (IL) 1a, IL1b and IL6, chemokines KC, IP10,MCP1, MIP1a and MIG, and growth factors GM-CSF and V-EGF. Secretion of cy-tokines important for the T cell response, e.g. IL2, and more specifically for Thelper 1 (Th1) cell response, IL12 and IL10, was induced after infection. Surpris-ingly, the level of IL4 and IL13, two cytokines involved in Th2 cell response, alsoincreased upon infection. The level of most cytokines increased in correlation withthe number of bacteria in each organ during the first three days of listeriosis. Incontrast, a strain of L. monocytogenes which does not produce the major virulencefactor listeriolysin O (LLO), and is cleared very early from infected mice, failed tostimulate cytokine production. Together, this first multiplex analysis of cytokineproduction in vivo represents an important basis to identify virulence determi-nants involved in the modulation of host immune response.

Analysis of the post-translocation chaperone PrsA2 and its unique rolein facilitating Listeria monocytogenes pathogenesisAlonzo, F. and Freitag, N.University Of Illinois at Chicago, USA

Listeria monocytogenes is a Gram-positive bacterial pathogen whose ability tocause disease is dependent upon the coordinated expression and activity of se-creted products that promote virulence. We have shown that the post-transloca-tion chaperone PrsA2 is critical for L. monocytogenes pathogenesis and that prsA2mutants exhibit a substantial cell-to-cell spread defect in tissue culture. We there-fore hypothesized that a loss of PrsA2 results in perturbation and/or altered ac-tivity of factors responsible for intracellular survival. PrsA2 was found to promotethe activity and stability of two secreted virulence factors: listeriolysin O (LLO)and the broad range phospholipase PC-PLC. Culture supernatants from strainscontaining deletions of prsA2 exhibited reduced hemolytic activity as measuredby the lysis of red blood cells in vitro, and this reduced activity appeared to be theresult of altered stability and increased proteolytic cleavage of LLO. prsA2 mu-tants were also defective for processing of PC-PLC from its pro-form to its enzy-matically active mature form, resulting in decreased phospholipase activity in vitroas well as within infected host cells. PrsA2’s role in bacterial virulence was found tobe unique from the related L. monocytogenes chaperone PrsA1, a protein thatshares a high degree of sequence similarity with PrsA2 but makes no discernablecontribution to pathogenesis. Proteomic analysis of L. monocytogenes secretedproteins suggests that PrsA2 is required for the correct localization of additionalbacterial gene products. Taken together, these data indicate that PrsA2 has beenfunctionally adapted to promote the secretion and activity of multiple L. monocy-togenes factors that play important roles in bacterial virulence.

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CtaP is a multifunctional cysteine-transport associated proteinrequired for Listeria monocytogenes pathogenesisXayarath, B.University of Illinois at Chicago, USA

The bacterial pathogen Listeria monocytogenes survives under a myriad of condi-tions in the outside environment and also within the human host where infectionscan result in severe disease. Bacterial life within the host requires the expressionof genes with roles in nutrient acquisition as well as the biosynthesis of bacterialproducts required to support intracellular growth. A gene product identified asthe substrate-binding component of a novel oligopeptide transport system (en-coded by lmo0135) was recently shown to be required for L. monocytogenes viru-lence. We report here that the gene product encoded by lmo0135 makes multiplecontributions to L. monocytogenes growth and survival in environments both in-side and outside of host cells. The lmo0135 gene product was required for bacter-ial growth in the presence of low concentrations of cysteine in vitro, suggestinglmo0135 and its associated transport system function in high affinity cysteinetransport. We have therefore designated the lmo0135 gene products as CtaP forcysteine-transport associated protein. Interestingly, CtaP was not required forbacterial replication within the host cytosol, but was required for bacterial adhe-sion implicating a role for this protein in bacterial attachment to host cells. CtaPappears to function directly as an adhesin as latex beads coupled to CtaP werefound to specifically bind epithelial cell monolayers in tissue culture assays. Inaddition, loss of CtaP increased membrane permeability and acid sensitivity, re-sulted in altered bacterial surface hydrophobicity, and severely attenuated viru-lence following both intragastric and intravenous inoculation of mice. Taken to-gether, the data presented indicates that CtaP is part of a unique ABC transportsystem that contributes to multiple facets of L. monocytogenes physiology, growth,and survival both inside and outside of animal cells.

Enzymatic activity of the metalloprotease of Listeria is regulated by pHForster, B. M.*, Pavinski Bitar, A., Slepkov, E. R. and Marquis, H.Cornell University, USA

The broad-range phospholipase C of Listeria monocytogenes, PC-PLC, contributesto escape from vacuoles. During infection, PC-PLC accumulates at the membranecell wall interface until a decrease in vacuolar pH triggers its proteolytic maturationand release. PC-PLC maturation is mediated by the metalloprotease of Listeria(Mpl), which is produced as a zymogen and matures via intramolecular autocatal-ysis. We tested the hypothesis that Mpl activity is regulated by pH. Radiolabeledinfected cells were perfused to manipulate host cell cytosolic pH, and secreted Mplwas immunoprecipitated. Mature Mpl was detected in samples treated at pH6.5,but not at pH7.3. To distinguish whether pH regulates autocatalysis or cell walltranslocation of bacterium-associated Mpl, we inserted a Flag tag at the N-terminusof the pro or catalytic domain of Mpl, and used an antibody that recognizes onlyN-terminal Flag to detect Mpl. Results from fluorescence microscopy indicatedthat the zymogen is bacterium-associated at physiological pH, but not followingacidification of the host cell cytosol. Mature Mpl was not detected bacterium-as-sociated at either pH. Lastly, using purified mature Mpl and the proform of PC-PLC, we determined that processing of the PC-PLC propeptide by mature Mpl oc-curs only at acidic pH. Together, these results indicated that Mpl enzymatic activityis regulated by pH, whether it undergoes autocatalysis or mediates the maturationof PC-PLC. Future studies will aim at assessing the possibility that specific aminoacid residues act as pH sensors in the regulation of Mpl autocatalysis.


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Identification of propeptide residues regulating thecompartmentalization, maturation, and activity of the broad-rangephospholipase C of Listeria monocytogenesSlepkov, E. R., Pavinski Bitar, A. and Marquis, H.Cornell University, USA

The broad-range phospholipase C of Listeria monocytogenes, PC-PLC, is made asan inactive proenzyme whose activation is regulated by pH and by the metallo-protease of Listeria (Mpl). The propeptide of PC-PLC is comprised of 24 aminoacid residues (C28-S51) and regulates PC-PLC compartmentalization and activity.In this study, we generated a series of propeptide mutants to determine the mini-mal requirement to prevent PC-PLC activity, and to identify amino acid residuesregulating compartmentalization and maturation. To determine how manyresidues were required to prevent PC-PLC activity, nested deletions were gener-ated in the PC-PLC propeptide of an Mpl-minus strain and phospholipase activitywas assessed on egg yolk plates. We found that a single amino acid residue at posi-tion P1 (S51) of the cleavage site is sufficient to prevent activity, which is consistentwith P1’ (W52) being located within the active site pocket. The requirements toretain the proform of PC-PLC bacterium-associated were assessed by immuno-precipitating secreted PC-PLC from radiolabeled infected cells maintained atphysiological pH, whereas the influence of the propeptide on the ability of Mpl tomediate PC-PLC maturation was assessed by immunoprecipitating secreted PC-PLC from radiolabeled infected cells perfused with buffer at acidic pH. We ob-served that the triple mutant E31A Y32A L33A is translocated across the cell wallmore effectively than wild-type at physiological pH, and that Y32A and L33A sin-gle point mutants are less effectively processed by Mpl at acidic pH. These resultsindicated that the N-terminus of the propeptide regulates PC-PLC compartmen-talization and the ability of Mpl to process PC-PLC. Considering that Y32 and L33are located 19 and 18 residues upstream of the propeptide cleavage site, we suggestthat these two residues initiate the interaction with mature Mpl leading to pro-cessing of the propeptide at S51.

A mouse model of fetoplacental Listeria monocytogenes infectionand abortionPoulsen, K. P., Faith, N., Laura Knoll, L. and Czuprynski, C.School of Veterinary Medicine, University of Wisconsin Madison, USA

Foodborne outbreaks of Listeria monocytogenes, particularly with serotype 4b,continueto occur. Pregnant women are over represented in listeriosis outbreaks(17-fold increasein incidence of disease). Infection of the fetus and placenta re-sults in abortion, stillbirth,or premature parturition. The latter carries with it ahigh risk for neonatal sepsis andmeningitis. Alterations in cellular and molecularcomponents of the immune systemoccur during pregnancy to prevent rejectionof the maternal allograft (i.e. fetus). How these pregnancy associated changes af-fect vertical transmission of L. monocytogenes to the fetus, is poorly understood.We have developed a mouse model for infection with aserotype 4b strain of L.monocytogenes isolated from a foodborne listeriosis outbreak thatresulted in abor-tion and fetal death. Intragastric infection of pregnant mice resulted insevere in-fection of the maternal and fetal tissues, in both C57BL/6J and A/J mousestrains.Use of a luciferase tagged L. monocytogenes strain and bioluminescent technologyallowed us to visualize infection of maternal and fetal tissues and shedding of L.monocytogenes cells in feces and vaginal secretions of aborting mice. These datasupport the use of pregnant mice as a model for maternal and fetal L. monocyto-genes infection. This model will prove useful in future investigations of the ma-ternal and fetal immune response to listeriosis during pregnancy.

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Listeria infection of the insect model system Galleria mellonellaJoyce, S. A. and Gahan, C. G.APC and department of Microbiology UCC, Ireland

With the exception of mice, alternative model systems are constrained by an uppertemperature limit for their survival. This temperature varies from 25°C for Acan-thamoeba and Danio rerio to 28°C for C. elegans and for Drosophila species. Thislimitation questions the relevance of these models for use with human pathogenswhere virulence gene expression is temperature dependent. Galleria mellonella,the Greater Wax Moth Larvae, is well documented as an infection model amenableto elevated temperature of 37 °C. Here, we examine and establish Galleria mel-lonella as a relevant model for infection by Listeria species. Mutants attenuated forvirulence in mice are similarly attenuated in this model system. We demonstratethat haemolysin production is the dominant factor in the pathogenic process of L.monocytogenes to G. mellonella. Real time visualization of infection reveals that thesame sub-sets of virulence genes required for L. monocytogenes infection of bothhumans and mice are expressed (differentially) and required for insect infections.L. monocytogenes replication occurs in G. mellonella. On examining the host re-sponse we find that L. monocytogenes is mainly internalized within one hour of in-fection. In the presence of hemolysin, hemocyte viability decreased eight- fold andin the absence of PrfA a four-fold increase in viability was evident relative to WTstrain. Bacterial load in hemocytes was significantly increased on infection with amutant in hemolysin (DhlyA) production and a mutant in stress factor production(DsigB) compared to wt infection. We found that the Phenyloxidase (PO) systemmounts a response to the presence of Listeria species within 4 hour of insect in-fection and that infection by L. monocytogenes results in the production of anti mi-crobial peptides (AMPs). Taken together, we propose that G. mellonella is a rele-vant model for infection by L. monocytogenes at 37°C.

Construction of a murinised Listeria monocytogenes H7858 (4b) strainfor improved murine infectionCummins, J. and Gahan, C.Science Foundation Ireland

Listeria monocytogenes is Gram-positive food borne pathogen that is capable ofcausing listerosis culminating in various diseases in humans such as gastroenteri-tis, meningitis, encephalitis and spontaneous abortions. L. monocytogenes is ca-pable of internalisation into non-phagocytic cells and crossing the intestinal, theplacental and the blood-brain barriers. Internalisation into non-professionalphagocytic cells is mediated in particular by the surface protein, InlA. InlA pro-motes listerial uptake into enterocytes by targeting the N-terminal domain of thehuman E-cadherin. However, this interaction is species specific with its interac-tion completely impaired in the rat and mouse models due to the absence of a pro-line at position 16 of the N-terminal E-cadherin repeat. A landmark result was thedevelopment of a transgenic mouse line expressing human E-cadherin by intes-tinal enterocytes. However, an alternative approach to overcome the lack of ap-propriate animal models has been the creation of a murinised strain of L. monocy-togenes EGDe (Wollert et al., 2007 Cell. 129(5):891-902). By the substitution oftwo amino acids within the InlA protein we have created a murnised strain in theH7858 4b background. Our results have demonstrated that this mutant has an in-creased ability to infect mice by the oral route and does not increase invasionwithin human cells. The benefits of this approach will be discussed.

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The role of a phosphoinositide phosphatase in the intracellular survivalof Listeria monocytogenesWang, J., Corbett, D. and Roberts, I. S.Faculty of Life Sciences, University of Manchester, UK

L. monocytogenes is capable of invading and growing in a number of host cells. Fol-lowing uptake it escapes from the phagosome and grows in the cytosol recruitingactin filaments to move and spread from cell to cell. Once inside the cell it sub-verts the host cell physiology to promote its own growth and survival. Phospho-inositides are phospholipids present in host cell membranes that play key regula-tory roles in orchestrating cell physiology. They control a broad range of processesincluding organelle identity, signal transduction and cell migration. The basis ofthis control is their relative state of phosphorylation that is controlled throughthe action of host kinases and phosphatases. The central role played by phospho-inositides inside cells make them a prime target for subversion by intracellularpathogens wishing to hijack the cell’s physiology. Recently we discovered that L.monocytogenes expresses a phosphoinositide phosphatase, capable of removingphosphate groups from a number of important phosphoinositides. Importantlywe demonstrated that this enzyme was essential for the growth of L. monocyto-genes inside infected cells and probably is important in escaping the phagosome.This is the first identification of a phosphoinositide phosphatase enzyme playinga key role in the intracellular survival of L. monocytogenes.

Virulence gene expression in Listeria monocytogenes strains isolatedfrom different sourcesAlessandria, V.University of Turin, Italy

Listeria monocytogenes is an important food-borne pathogen that has the capacityto cause severe infections. Ubiquitous microorganism, it is commonly isolatedfrom foods of animal origin, mainly meat and milk products. However, due to itscapacity to develop at refrigeration temperatures, human listeriosis outbreaks aremost often associated with ready-to-eat food products that are consumed with-out prior cooking. The incidence of the disease depends on different factors in-cluding the infective dose and the immunity conditions of the host. The presentwork focuses on the expression analysis of four virulence genes (sigB, plcA, hlyand iap) in 11 different strains of L. monocytogenes and, more in detail, 3 collectionstrains (EGDe, NCTC10527, SCOTT A), 7 isolated from food matrices (4 of whichisolated from meat products and three from diary products) and 1 isolated fromhumans. In the first step, the expression analysis was performed in vitro, culturingeach strain in BHI (Brain Heart Infusion) medium, in order to identify possibledifferences in the expression level for the different strains. The combined use ofreverse transcription and quantitative PCR (qRT-PCR) was used to evaluate geneexpression. As a second step, we wanted to evaluate the trend of expression of thegenes in food. Analyses were performed inoculating L. monocytogenes in milk atdifferent conditions and in particular at two temperatures (4 and 12 °C) for dif-ferent times (24 and 48 hours). Significant expression differences emerged for thedifferent genes, without showing any significant association between the expres-sion of the genes and the origin of the different strains.

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Targeted Signature-tagged mutagenesis for phenotype screeningof Listeria monocytogenes mutantsHenriques, A., Carvalho, F. and Cabanes, D.IBMC, Portugal

Listeria monocytogenes is a foodborne facultative intracellular human pathogenpossessing a wide range of virulence factors tailored for pathogenesis. Commonstrategies used to identify new virulence factors are based on mutagenesis and invivo phenotype analysis, which requires testing of generated mutants versus wildtype strain in separate mice cohorts. To streamline this analysis, a targeted signa-ture-tagged mutagenesis (T-STM) method was developed. Based on the concept ofSTM, where random mutants identifiable by specific tagging are negatively se-lected, we developed a method that enables specific gene targeting in L. monocy-togenes genome for the creation of insertional mutants and their simultaneousphenotypic analysis in mice. Tagged “wild type” and mutants for prfA and two un-known LPXTG genes were constructed by inserting differentially tagged suicidevectors. T-STM uses defined tags that allow the discrimination between tagged-strains in a pool by PCR. The four tagged-strains were pooled in equivalentamounts into one single inoculum (Input) used for oral and intravenous infectionof mice. Three days after infection, intestine, spleen, and liver of mice were col-lected, homogenised and plated on rich medium. Resultant bacterial growth (Out-put) was used as template for PCR screening and evaluation of the relative pro-portion of each tagged-strain. Mutants were considered less virulent whenundetectable (or less detectable) by PCR in the output. This technique allowed usto clearly distinguish the severely attenuated prfA mutant, validating the method.PCR products for the two LPXTG protein-encoding mutants appeared in equalproportion to the wild type strain in the output, suggesting that these proteins arenot involved in Listeria virulence in this infectious model. This study shows thefeasibility and advantage of the developed T-STM method for simultaneous viru-lence analysis of pools of Listeria tagged mutants. This approach should be nowused for larger-scale phenotypic analysis of specific L. monocytogenes mutants.

Listeria monocytogenes cellular infection triggers tyrosine-phosphorylation of Myosin IIA, a new protein involved in invasionAlmeida, M. T., Cabanes, D. and Sousa, S.IBMC, Portugal

Listeria monocytogenes is a human food borne pathogen that may lead, in particu-lar in immunocompromised individuals, to a severe disease characterized by sep-ticemias, meningitis, meningo-encephalitis and abortions. The study of the cellbiology of the Listeria infectious process provided insights in the way bacteria ma-nipulate the host and revealed unsuspected functions of cellular proteins. To causeinfection pathogens interfere with crucial host intracellular pathways, and differ-ent pathogens often hijack the same signaling pathways. In particular, host phos-phorylation cascades are preferential targets of infecting bacteria. In this study,using L. monocytogenes as a pathogen model, we showed that eukaryotic cells pres-ent a variable protein phosphorylation pattern upon infection. We addressed inparticular the tyrosine-phosphorylated protein profile triggered by Listeria infec-tion and identified the motor protein, Myosin IIA (MyoIIA), as differentially ty-rosine-phosphorylated in response to Listeria uptake. We demonstrated that My-oIIA is not only tyrosine-phosphorylated over the time of infection, but is alsorecruited with actin at the bacteria entry site. In addition, we were able to showthat the inhibition of MyoIIA activity affected Listeria entry into non-phagocyticcells. The reduction of MyoIIA expression using RNAi techniques resulted in anincreased Listeria uptake. Together these data point to the role of a novel myosinclass in the internalization of Listeria, correlating for the first time, myosin post-translational modifications and Listeria infection.

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Invasion profile of Listeria monocytogenes strains involvedin invasive and gastroenteritis listeriosis outbreaksLaksanalamai, P., Sahu, S. and Datta, A.Food and Drug Administration, USA

Listeria monocytogenes (Lm), the causative agent of foodborne human listeriosis, isa serious public health concern. The disease is characterized by meningitis, sep-ticemia, abortion and death in immuno-compromised population. Growing num-bers of evidence have revealed that Lm can also cause self-limited febrile gas-troenteritis in healthy individuals. To understand the mechanisms underlying twodifferent disease outcomes several efforts have been made to differentiate gas-troenteritis and invasive strains of Lm. Since the difference between these twotypes of outbreaks is Listerial ability to invade, in this study we have examinedthe expression of two internalin genes, inlA and inlB which encode virulence pro-teins required for the internalization process. The inlA and inlB gene expressionprofiles were determined by quantitative RT-PCR from several strains represent-ing two groups of Lm that cause either invasive listeriosis or febrile gastroenteri-tis. Analysis of the inlA and inlB gene expression from food and patient isolatesshowed similar levels of expression from isolates originated from the same out-breaks. Surprisingly, the inlA and inlB gene expression from Lm isolated from in-vasive listeriosis outbreaks appears to have lower expression that those thatcaused gastroenteritis listeriosis. In contrast the expression of iap, a gene involvedin host cell invasion, was higher in invasive strains. To understand the correlationbetween the inlA and inlB gene expression and invasion of Lm, we also investi-gated the efficiency of invasion of these strains by in vitro invasion assay using eu-karyotic cell lines, Caco2 and HepG2. The results appeared to correlate with theinlA and inlB expression in that the invasion efficiency is higher in the gastroen-teritis strains. Global gene expression profiles of these strains are currently beinginvestigated using a DNA microarray.

Molecular characterization of the Vip-Gp96 interactionMartins, M., Cabanes, D. and Sousa, S.IBMC, Portugal

The study of mechanisms exploited by Listeria monocytogenes to cause infectionprovided new insights in the way bacteria manipulate the host cell machinery. Tosubvert the host cell signalling pathways, Listeria uses a complex set of surfaceproteins called virulence factors that act in concert to allow bacterial infectionand evasion from the host immune system. Recently, we reported that Vip, a L.monocytogenes virulence factor interacts with Gp96, an endoplasmic reticulum(ER) resident chaperone that in cell stress conditions is targeted to cell surface,promoting host cell invasion. The purpose of this study was the identification ofthe Gp96 domain involved in the interaction with Vip. Using protein-protein in-teraction and invasion assays, we demonstrated that the region 22-411 amino acidsof Gp96 (N-terminal) seems to be exposed outside of the cell thus available forVip binding. This interaction is required for Listeria uptake into host cells. Al-though this entire region participates in the interaction, the domain comprisingthe 22-192 amino acids seemed to be the one crucial for the interaction. The exis-tence of cell signalling events downstream Vip-Gp96 interaction was also investi-gated. Signal transducer and activator of transcription 3 (Stat3) activation duringL. monocytogenes invasion was addressed, suggesting that Stat3 was not activatedin response to Listeria entry. In addition, we demonstrated that Vip seems to haveno role in Listeria phagocytosis by bone marrow-derived macrophages (BMMØ)and in bacterial survival within them.

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Investigation of the molecular mechanisms by whichListeria monocytogenes grows in the mammalian gall bladderDowd, G., Joyce, S., Casey, P. G., Hill, C. and Gahan, C. G.University College Cork, Ireland

Listeria monocytogenes is a foodborne pathogen that is a significant cause of mor-talities due to consumption of contaminated foods. Recent work has demonstratedthat the pathogen grows in the mammalian gall bladder and that gall bladder growthforms a significant phase of the infectious process. However, relatively little isknown about the mechanisms that underpin the growth of the pathogen in this mi-lieu. Here we have performed basic experiments which demonstrate that L. mono-cytogenes grows well in bile from the porcine gall bladder and can utilize bile as asource of energy and nutrients. We used bioluminescence-labeled L. monocytogenesto allow visualization of bacterial growth kinetics in porcine gall bladders infectedex vivo. We then performed random mutagenesis of L. monocytogenes and selectedmutants that grow well under laboratory conditions but fail to grow in bile. Se-quencing of the genes mutated in this experiment demonstrated that the pathogenrequires genetic loci that are involved in central metabolism for growth in bile, in-cluding genes encoding proteins required for amino acid biosynthesis and biotinmetabolism. The majority of mutants that were unable to grow in bile were alsoattenuated for virulence in the mouse model of infection. Overall the work is thefirst to demonstrate the requirement for specific gene sets for the growth of L.monocytogenes in the specialized environment of the mammalian gall bladder.

Role of cadmium efflux system in Listeria monocytogenes virulenceCamejo, A. and Cabanes, D.IBMC, Portugal

Listeria monocytogenes is a human intracellular pathogen able to colonize hosttissues after ingestion of contaminated food, causing severe invasive infections.In order to gain a better understanding of the nature of host–pathogen interac-tions, we studied the L. monocytogenes genome expression during mouse infec-tion. We found that the shift of the Listeria genome expression during infection ischaracterized by the activation of genes involved in virulence, stress, subversion ofthe host immune system, and by the adaptation of the bacterial metabolism tohost conditions. Mutagenesis of genes highly induced in vivo allowed the identifi-cation of novel L. monocytogenes virulence factors, including cadC. Cadmium re-sistance in Listeria and other gram positive bacteria is an energy-dependent cad-mium efflux system, involving two proteins, CadA and CadC. CadA has been shownto be a cadmium efflux P-type ATPase; cadC encodes a transcriptional regulatorthat is a member of the ArsR metalloregulatory proteins. The strong in vivo acti-vation of cadC and the significant impaired virulence of the cadC mutant suggestthat this heavy metal resistance system constitutes an advantage for in vivo Liste-ria survival. In addition, this system has never been previously implicated in thevirulence of other pathogens. The detailed regulation of the CadAC system role aswell as its role in cadmium resistance and in the Listeria infectious process will bepresented and discussed.

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Investigation of chitinase as a potential virulence factorin Listeria monocytogenesChaudhuri, S.University of Illinois in Chicago, USA

Listeria monocytogenes is an environmental pathogen that causes disease in hu-mans primarily following the consumption of contaminated food products. Whileconsiderable attention has been given to the identification of bacterial virulencefactors specifically expressed within host cells, relatively little is known regard-ing gene products that may contribute to bacterial life in the outside environmentwhile also potentially serving a role for virulence within the host. L. monocyto-genes has been shown to produce and secrete two chitinases, ChiA and ChiB, aswell as a chitin binding protein encoded by lmo2467. ChiA and ChiB are chitin de-grading enzymes that are presumed to facilitate L. monocytogenes life in the out-side environment where chitin is plentiful. Recently however, chitinases and chitinbinding proteins have been reported to contribute to the pathogenicity of two un-related environmental bacterial pathogens, Legionella pneumophila and Vibriocholerae. The chitinases produced by these bacteria appear to enhance bacterialcolonization of mouse lung (L. pneumophila) and intestine (V. cholerae). To deter-mine if chitinases or chitin binding proteins contribute to L. monocytogenes patho-genecity, in-frame deletion mutants were constructed in chiA, chiB, and lmo2467,and a triple gene deletion mutant was also constructed. All mutants were found togrow similarly to wild type L. monocytogenes in BHI broth culture and within in-fected tissue culture cells. However, following intravenous infection of mice, two ofthe single mutants, ∆chiA and ∆chiB, as well as the triple mutant, ∆chiA ∆chiB∆lmo2467 were found to be defective for bacterial growth in the livers and spleensof infected animals. As mammals do not produce chitin, these results suggest thatL. monocytogenes has adapted its chitinases to exploit recognition of host sub-strates, potentially carbohydrate-linked molecules, to enhance bacterial survivalwithin infected animals.

Sub-lethal concentrations of common disinfectants do not influencesurvival and growth of Listeriamonocytogenes in whole bloodHolch, A., Gaedt Kastbjerg, V. and Gram, L.Technical University of Denmark

Listeria monocytogenes is a serious food borne bacterial pathogen that can colonizefoodprocessing environment. Virulence gene expression is influencedby several en-vironmental factors e.g. temperature andoxygen-concentration. It has recently beenshown that sub-lethal concentrations of disinfectants used in the food industry af-fect the expressionof virulence genes in L. monocytogenes (Kastbjerg et al.2009). Theexpression is either enhanced or reduced depending on the active compound in thedisinfectant.The purpose of the study was to determine if these changes in virulencegene expression influence the virulencepotential of L. monocytogenes in amore com-plex biological assay. We chose to study the ability of L. monocytogenes to grow andsurvive in whole blood as several of the virulence genes are important for survivalandgrowth inblood.Twostrainsof L. monocytogenes (EGDandamaternofetal strain)where grown to exponential phase and exposed to two different disinfectants in anon-inhibitory and a sub-lethal concentration for one hour. The disinfectants con-tained either peroxide or quaternary ammonium compound (QAC) as their activecompound.These cause enhancedor reducedvirulence geneexpression, respectively.The exposed bacteria were mixed with whole blood and the survival and growth wasfollowed for 50 hours by plate counting. L. monocytogenes was able to grow in wholeblood after exposure to the two different disinfectants in two different concentra-tions. However, pre-exposure to peroxide or QAC did not cause any difference ingrowth for both strains of L. monocytogenes as compared to exposure to water (con-trol). In all experiments, the maternofetal strain grew slightly better than the EGDstrain. Hence, the effect of sub-lethal concentrations of disinfectants on virulencegene expressiondidnot affect the survival and growth in a complex biologicalmodel.

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Internalin LRR domain variability in Listeria monocytogenes isolatedfrom different hostsZaytseva, E. A.1,2, Ermolaeva, S. A.2 and Somov, G. P.1

1. Research Institute for Epidemiology and Microbiology, RAMS Siberian Division (Vladivostok)2. N.F. Gamaleya Research Institute for Epidemiology and Microbiology, RAMS (Moscow)

Development of certain clinic manifestations of listeriosis has been recently sug-gested to depend on several factors of L. monocytogenes pathogenicity. A numberof surface and secreted proteins from internalin family, of which A and B inter-nalin proteins are most thoroughly investigated, are involved in the process of Lis-teria invasion into eukaryotic cells. One of the specific features of this protein fam-ily is presence of so called LLR domains involved in the direct interaction witheukaryotic receptors. The purpose of this research is to analyze distribution ofvarious internalin gene alleles among L. monocytogenes isolated from differentsources. Eighty-six L. monocytogenes cultures were used belonging to serovariants1/2a, 1/2 b, 4b, isolated from various sources in the Far East and European part ofRussia in the period from 1952 to 2005. Primers, gene restricting fragment andcoding LRR domain were selected with the help of Oligo38 application. Gene pres-ence in L. monocytogenes was determined with the help of PCR. DNA fragment se-quencing was performed in Genom Center (Moscow). Molecular genetics featuresof Listeria were analyzed in 4 culture groups depending on isolation source: 1)stillborn who died from listerial infection (n=21); 2) wild murine rodents (n=19); 3)marine hydrobionts (n=19); 4) food (control, n=27). Invasion factor genes - inlA,inlB, inlC and inlE - were found in all of L. monocytogenes isolates. Sequence ofLRR domains of genes inlA, inlB, inlC and inlE was determined for all the isolates.L. monocytogenes isolates demonstrated specific distribution of the examined genealleles depending from isolation source. Among clinic isolates (group 1) low vari-ability was recorded for inlA and inlC (p < 0.01). L. monocytogenes isolates obtainedfrom wild rodent organs showed low variability with for inlB (p < 0.01). Specificalleles of inlA, inlC and inlE were found in Listeria cultures isolated from marinehydrobionts. Alleles of inlE were uniform in all the compared groups. In controlgroup all gene alleles were distributed uniformly too. The obtained results con-firm internalin role in L. monocytogenes interaction with a certain host. A microbewith specific gene allele may be more virulent and invasive for a certain type ofhost even at low infection doses.

Reduced virulence of an adenylosuccinate lyase transposon mutantof a serotype 4b strain of Listeria monocytogenesFaith, N. G.1,2, Kim, J.-W.3, Kathariou, S.3, Sahaghian, R.1 and Luchansky, J. B.4

1. School of Veterinary Medicine, Univ. Wisconsin-Madison Madison, WI2. Food Research Institute, Univ. Wisconsin-Madison Madison, WI3. Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State Univ., Raleigh, NC4. Eastern Regional Research Laboratory, USDA-ARS, Wyndmoor, PA

A severe outbreak of listeriosis occurred in 1998–99 as a result of contamination ofhot dogs with serotype 4b Listeria monocytogenes. We compared several character-istics of strain H7550, implicated in the 1998–99 outbreak of listeriosis, and a plas-mid-free derivative (H7550cds) of that strain lacking the cadmium resistance plas-mid pLM80, with that of selected transposon mutant derivatives of those strains.We assessed virulence in a mouse model of intragastric (i.g.) inoculation of anes-thetized A/J mice with approximately 106 CFU of the individual strains. A trans-poson mutant of strain H7550 that lacks adenylosuccinate lyase activity (strainJ22F), and a non-hemolytic (LLO-) transposon mutant of strain H7550cds (J29H),were avirulent in our mouse model. We did not recover viable cells from the spleen,liver, blood gallbladder, or ceca of mice inoculated with either strain J22F or J29H,whereas the respective parent strains H7550 and its cadmium sensitive derivativeH7550cds were equally virulent for mice. We observed no significant difference inthe resistance of stationary phase cells of the plasmid harboring versus plasmid-free strains to synthetic gastric fluid at pH 4.5. All four strains formed biofilms onplastic surfaces within 24 hr in vitro. Strain J22F was better able to form biofilms invitro than its parent strain H7550, whereas the LLO-negative mutant strain J29Hwas not significantly different from its parent strain H7550cds in biofilm formation.These results provide the first evidence that adenylosuccinate lyase activity is re-quired for virulence of L. monocytogenes in mice. They also demonstrate that LLOis not required for biofilm formation in vitro.

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Manifestations and outcome of listeriosis in adult patientsFernández Guerrero, M L.1, Mancebo Plaza, B.2, Torres, R.2, Górgolas, M.1 and Jusdado, J. J.2

1. Fundación Jiménez Díaz. Universidad Autónoma de Madrid, Spain2. Hospital Severo Ochoa, Madrid, Spain

Infections caused by Listeria monocytogenes in adult patients are rarely reporteddespite the increasing prevalence of these infections in western countries. Hence,the manifestations, optimal treatment, prognosis and risk factors for mortalityare not entirely known. We herein report a review of the most relevant clinical as-pects, treatment and outcome of a large series of patients with listeriosis studied intwo hospitals in Madrid, over a period of 15 years. Patient with isolation of L. mono-cytogenes from blood, CSF or other sterile fluids were included in the analysis.Sixty-two cases were seen. The mean age was 58.5 years (from 20 to 85 years) with-out differences in distribution by genders. Seventy percent of cases had comor-bidities: cirrhosis of the liver, hematologic neoplasms, immunosupressive disor-ders and corticosteroid therapy were the most common. Primary bacteremia(48%) and meningoencephalitis (37%) were the most common presentations. Tenpatients (16%) presented with focal infections such as bacterial peritonitis (4),endocarditis (3) and brain abscesses (3). Ampicillin alone or in combination withgentamicin and cotrimoxazol were the antimicrobial treatments most frequentlyused. We did not find differences in the mortality rate among patients treated withmonotherapy in comparison with those that received combined therapy. Cotri-moxazol was successfully used in 10 out of 13 patients (77%) treated. Twenty-threepatients (37%) died within 30-days of diagnosis. Mortality increased according tothe severity of the underlying disease and was significantly higher in immuno-compromised patients (66%) than in those with chronic debilitating conditions(20%; p < 0.001). Human listeriosis is a severe disease producing high mortalityparticularly in immunocompromised patients with hematologic neoplasms andcorticosteroid therapy. Despite synergistic activity of combined ampicillin/gen-tamicin therapy, the survival benefits of this combination remain theoretical. Cot-rimoxazol may be a useful alternative treatment for human listeriosis.

Model for human Listeriosis: in vivo monitoring of orally infected miceusing bioluminescent Listeria monocytogenesBergmann, S., Lengeling, A., Pasche, B. and Schughart, K.Helmholtz-Centre for Infectious Research, Germany

The ubiquitous Gram-positive bacterium Listeria monocytogenes is the causativeagent of listeriosis. After ingestion of contaminated food, this pathogen is able tocross the intestinal, blood-brain and placental barrier and leads to gastroenteritis,meningitis and maternofetal infections which may result in abortion and sponta-neous stillbirth. We have generated a bioluminescent Listeria monocytogenes strainwhich carries two amino acid substitutions in the bacterial invasion protein in-ternalin A (InlAS192NY369S) and enables the bacterium to bind to the murine E-cadherin receptor with increased affinity as compared to wildtype Listeria. Thegenetic modification allows the bacteria to invade intestinal epithelial cells andto cross the murine intestinal barrier with high efficiency. The new bioluminescentListeria strain was used to monitor bacterial dissemination in orally infected mice.For visualization of bioluminescent bacteria during infection we have used theXenogen IVIS system, a highly sensitive, low light system optimized for in vivo im-aging. Here, we present first results of different mouse inbred strains (BALB/cByJ,C57BL/6J, CD1, 129P, C3HeB/FeJ) that behave different in the intensity of infec-tion and show a time variation regarding bacterial spreading, the crisis of infectionand bacterial clearance. We found CD1 and C3HeB/FeJ mice to be resistant toorally transmitted listeriosis, they showed reduced bacterial dissemination in theearly phase of infection (during first 72 hrs), and a fast clearance of the pathogenfrom day 5 p.i. on. In contrast BALB/cByJ and C57BL/6J mice we found to be moresusceptible after oral infection. This is reflected by slower bacterial clearance anda reduced survival.We further give an outlook how we will use this new approach to obtain a detailedinsight in the process of crossing the feto-placental barrier in pregnant mice aswell as the blood-brain barrier after oral infection with our modified Listeriamonocytogenes strain.

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Galleria mellonella as model system to study Listeria species-specificand Listeria monocytogenes serotype-specific pathogenesisMraheil, M. A., Krishnendu, M., Hain, T. and Chakraborty, T.Germany

Essential aspects of the innate immune response to microbial infection are con-served between insects and mammals. This has generated interest in using insectsas model organisms to study host-microbe interactions. We used the greater waxmoth Galleria mellonella, which can be reared at 37 °C, as a model host for exam-ining virulence potential of Listeria spp. Here we report that Galleria is an excel-lent surrogate model of listerial septic infection, capable of clearly distinguishingbetween pathogenic and non-pathogenic Listeria and even between virulent andattenuated Listeria monocytogenes strains and seotypes. Virulence required liste-rial genes hitherto implicated in the mouse infection model, and was linked tostrong antimicrobial activities in both hemolymph and hemocytes of infected lar-vae. We conclude that severity of septic infection of L. monocytogenes is primarilymodulated by innate immune responses and suggest the use of Galleria as a rela-tively simple, non-mammalian model system that can be used to assess the viru-lence of strains of Listeria spp. isolated from a wide variety of settings from boththe clinic and the environment.

Listeria monocytogenes ActA is a key playerin evading autophagic recognitionPillich, H., Loose, M., Hain, T. and Chakraborty, T.Germany

Autophagy is a pivotal bulk degradation system that eliminates undesirable mole-cules, damaged organelles, and misfolded protein aggregates in response to di-verse stimuli, including infection. Autophagy acts to limit intracellular microbialgrowth but intracellular pathogens have evolved strategies to subvert host au-tophagic responses for their survival. We found that Listeria monocytogenes ActA,a surface protein required for actin polymerization and actin-based bacterialmotility, plays a pivotal role in evading autophagy, but in a manner independent ofbacterial motility. We show that L. monocytogenes exploits the biomimetic prop-erty of ActA to camouflage itself with host proteins comprised of Ena/VASP andthe Arp2/3 complex, thereby escaping recognition by autophagy.

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Non-haemolytic and hypovirulent Listeria monocytogenes becamehaemolytic and virulent after passage through miceSecic, I., Lindbäck, T. and Rørvik, L. M.Norwegian School of Veterinary Science

Two different isolates (from a fish processing plant and a broiler abattoir) showingtypical appearance for L. monocytogenes on OCLA and ALOA, did not expresshaemolysis on blood agar with blood from different sources (sheep, horse, bovine,human) and appeared as L. innocua on RLM. However, biochemical reactions and16S RNA analysis identified them as L. monocytogenes. High levels (109) of eachisolate were injected i.p. into five mice. Three of the mice died after 1-3 days forboth strains. Cultivation from livers and spleens revealed a mixture of haemolyticand non-haemolytic colonies. Non-haemolytic isolates showed typical appearancefor L. monocytogenes on OCLA and ALOA, but were identified as L. innocua onRLM. Haemolytic and non-haemolytic isolates from each mouse, as well as the re-spective mother strains were identical pulsotypes. In contrast to passage throughmice, fifty passages of the mother strains on RLM did not change the phenotype,and screening of 107 colonies did not reveal any haemolytic subpopulation. HT-29 cell assay showed that while the non-haemolytic isolates were all hypovirulent,their haemolytic twins were virulent. Part of the LIPI 1 virulence island of non-haemolytic and haemolytic isolates was sequenced. The non-haemolytic isolatehad a seven base insertion in the prfA gene which introduced a stop codon, result-ing in a truncated PrfA protein. The seven base pair insertion was deleted duringinfection in mice, resulting in a normal PrfA protein in the haemolytic version.This study shows that during passage through mice hypovirulent L. monocytogenescan change to a virulent phenotype. It is not known if this phenomenon may occurin humans. These strains would not have been recognized as L. monocytogenes un-less OCLA or ALOA had been used as isolation agar.

Mutants of Listeria monocytogenes (Lm) resistant to the polycationicpeptide protamine appear to be attenuated for virulenceSchlech, W.Dalhousie University, Canada

Previously, we reported the isolation of Lm mutants resistant to protamine, a rela-tively inexpensive polycationic peptide derived from salmon and herring milt.Here, we report the final characterization of two protamine-resistant mutants, em-phasizing their virulence defects, which have led us to believe that protamine-resis-tant mutants are attenuated. We also report that p60 mutants, isolated independ-ently from the protamine-resistance phenotype and previously reported to showvirulence defects, are also resistant to protamine. Protamine-resistant (PtmR) mu-tants from the Canadian Maritimes outbreak strain 15U and from the Massachu-setts outbreak strain Scott A, were picked as isolated colonies from TSA plates con-taining 1 mg/ml of protamine. Electron microscopy and SDS-PAGE wereextensively applied to the initial characterization of the mutants. Swiss-Webstermice were infected by direct gastric inoculation with different Lm doses and spreadto the spleen and liver monitored at 3 days after inoculation. Survival of Lm in dif-fusion chambers surgically implanted in the peritoneal cavity of rats for 3 days, wasalso evaluated. By electron microscopy and SDS-PAGE, the PtmR mutants dis-played significant differences in their surface proteins and structure. However,both showed a reduced ability to infect mice (particularly the liver) and a reducedsurvival in the intraperitoneal diffusion chambers. p60 mutants, which are unableto efficiently infect cells in culture, turned out to also be PtmR. It seems that PtmRmutants, in spite of their different origin and surface characteristics, are attenuatedfor virulence. This possibly represents an example of an inverse fitness-virulencerelationship that could be exploited to select against highly virulent Lm strains infood products treated with inhibitory concentrations of polycationic peptides.

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The role of plasmacytoid dendritic cells in the course of Listeria mono-cytogenes infectionSolodova, E., Lienenklaus, S., Jablonska, J. and Weiss, S.Laboratory of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany

Type I interferons (IFNs) play a key role in linking the innate and adaptive arms ofthe immune system. There are more that 13 IFN-α and a single IFN-β, all using acommon receptor – IFNAR, which is expressed on wide variety of cell types. Pro-duction of type I IFNs in response to infection with viruses is essential for clearanceof the pathogen from the host. But the impact of these cytokines during bacterial in-fection is less defined. Plasmacytoid dendritic cells (pDCs) are known to be majornatural type I IFN producing cells during viral infections, but not much is knownabout their impact in the course of bacterial infections. Listeria monocytogenes isone of the bacteria known to induce type I IFN synthesis. In contrast to viral in-fections these cytokines were shown to have detrimental effects for the host duringinfection with this bacterium. In our study we used tissue specific conditional re-porter and knock-out mice showing that LysMcre-expressing cells but not pDCsare responsible for type I IFN production during Listeria monocytogenes infection.We have also shown that depletion of pDCs revealed a phenotype showing the abil-ity of pDCs to protect mice infected with Listeria monocytogenes. The aim of mypresent work is to investigate this phenomenon und to understand how pDCs areable to provide protection to mice during Listeria monocytogenes infection.

Oxygen restriction increases the infection potentialof Listeria monocytogenes – a transcriptional analysisAndersen, J. B., Bergstrøm, A., Knudsen, G., Bak Christensen, B., Ebersbach, T.,Boye, M. and Rask Licht, T.Technical University of Denmark, National Food Institute, DTU, Division of Microbiology and RiskAssessment, Denmark

Listeria monocytogenes has been implicated in several food borne outbreaks aswell as sporadic cases of disease during the last two decades. Increased under-standing of the biology of this organism is important in the prevention of foodborne listeriosis. This is highly relevant for safety assessment of this organism infood. We have previously shown (Andersen et al., BMC Microbiology; 2007, 7:55)that the environmental conditions to which L. monocytogenes is exposed prior toingestion are decisive for its in vivo infective potential in the gastrointestinal tractafter passage of the gastric barrier. Infection of Caco-2 cells revealed that Listeriacultivated under oxygen-restricted conditions were approximately 100 fold moreinvasive than similar cultures grown without oxygen restriction. This means thatnot only the number of Listeria present in a given food item, but that also the phys-iological condition of these bacteria is important for food safety. The in vitro and invivo data suggest that an oxygen-restricted L. monocytogenes cell represents a sig-nificantly higher risk than a cell grown without oxygen restriction. In order toidentify transcriptional differences contributing to different invasiveness, mi-croarray gene chip technology was applied to cDNA created from RNA isolatedfrom oxygen restricted and non-restricted cultures. The analysis confirmed severalrelevant genes to be differentially transcribed in the two environmental condi-tions e.g. genes related to virulence potential of L. monocytogenes.

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AREA //C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosisPOSTER PRESENTATIONS // P / 82–125, 183–186

Semi-automated repetitive sequenced-based PCR compared to pulsedfield gel electrophoresis for Listeria monocytogenes sub-typingRoussel, S., Félix, B., Vignaud, M.-L., Tam Dao, T., Marault, M. and Brisabois, A.AFSSA, France

Listeriosis is a severe infection which mainly impacts pregnant women, neonatesand immuno-compromised adults. The commercially available, semi-automatedrep-PCR assay system, DiversiLab, has been successfully used for subtyping severalspecies of bacteria. We compared here the DiversiLab System with macrorestric-tion analysis by pulsed field gel electrophoresis (PFGE) which is currently the goldstandard for molecular subtyping of Listeria monocytogenes. We used a panel of116 human and food L. monocytogenes strains for the comparative evaluation.Among these strains, there were 4 pairs of duplicates; 13 strains were epidemio-logically related and the remaining food isolates were epidemiologically unrelated.The strains of different serotypes revealed distinct ApaI-PFGE types, Rep-PCRtypes (RT) and AscI- PFGE types except for one RT and one AscI- PFGE type. Thefour doublets displayed a unique RT, AscI and ApaI PFGE pattern showing thegood reproducibility of the three methods. The epidemiologically related strainswere clustered in the same RT and PFGE types. The Simpson’s index of diversitywas 0.954; 0.988; 0.994; 0.998 for DiversiLab, AscI-PFGE, ApaI-PFGE andAscI/ApaI-PFGE respectively. Thus, PFGE was more discriminating than Diver-siLab. However, for 1/2a serotype strains, six AscI-PFGE and three ApaI-PFGEtypes were divided into different RT. DiversiLab allowed a good discriminationbetween serotype 1/2a strains. This investigation also demonstrated the ability ofthe DiversiLab to differentiate serotypes 4b and 1/2b strains. DiversiLab is lesslabour-intensive than PFGE and provides results in less than 24 hours comparedwith 30 hours to 3 days for PFGE from the time a pure culture of the bacteria is ob-tained. Based on these results, DiversiLab may be useful for tracking the source ofcontamination in food processing facilities and their environments.

Listeriosis: a frequent cause of fatal encephalitis in Francewith high case fatalityMailles, A.1, Vaillant, V., Lecuit, M.2 and Stahl, J.-P.3

1. Institut de Veille Sanitaire, France2. Institut Pasteur, France3. University Hospital of Grenoble, France

In 2007, we carried out a national prospective study in 106 hospital units to as-sess the aetiology, clinical patterns and outcome of infectious encephalitis inFrance. Among 253 case-patients, encephalitis due to L. monocytogenes was iden-tified in 13 people. A case of encephalitis was a patient aged at least 28 days, hos-pitalised in France with an acute onset of illness and at least one abnormality ofthe CSF, and fever and decreased consciousness or seizures or altered mental sta-tus or focal neurological signs. All case-patients had a complete biological investi-gation to assess the etiologic diagnosis. Clinical data were collected using stan-dardised questionnaires. Listeriosis case-patients were compared to othercase-patients enrolled in the study. Thirteen case-patients were identified withencephalitis due to L. monocytogenes among 253 case-patients (5%) and 131 case-patients with an etiological diagnosis (10%). Listeriosis was the fourth most fre-quent cause of encephalitis identified after HSV (n=55), VZV (n=20) and tubercu-losis (n=20). Listeria was isolated in CSF from XX patients, from blood only from1 case. XX cases had a positive PCR on CSF. The last patient was diagnosed on acombination of clinical and epidemiological criteria. Eleven listeriosis case-pa-tients were confirmed cases, 1 was a probable case and 1 was a possible case. Theirmean age was 71 years and 9 were men. Listeriosis case-patients were more likelyto have a current treated cancer (23% vs 5%, p=0.005) and to present with cra-nial nerves impairments (38% vs 17%, p=0.02). Six of 13 (46%) listeriosis case-patients had a fatal outcome vs 20/216 (8.5%) of other patients (p=0.007). Theseresults showed that L. monocytogenes is a frequent cause of encephalitis in Franceand confirmed its severity. Listeriosis should be considered early in the course ofencephalitis, as specific and efficient treatment can be proposed.

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Listeria monocytogenes: identification and subtypingFavretti, M.Istituto Zooprofilattico Sperimentale delle Venezie, Italy

Listeria monocytogenes is a common environmental organism and an importantfood-borne pathogen too. Pregnant women, neonates, elderly and immunocom-promised patients are at greatest risk of acquiring listeriosis. Since the recogni-tion of Listeria monocytogenes as a food-borne pathogen there have been rapid ad-vances in the development of suitable methods for isolation and identification.Although all Listeria monocytogenes strains are considered pathogen, only someserotypes are predominant and frequently involved in outbreaks. So it is very im-portant to obtain viable isolates for typing, elucidating outbreaks and tracing thesource of infection. The differentiation of Listeria monocytogenes isolates withphenotypic or molecular subtyping methods has provided useful information re-lated to the phylogeny, epidemiology and ecology of the organism and also has per-mitted the correlation of isolates from food and from human case in order to un-derline any relationship between them. Although there are several techniques foridentification and typing available at the moment, it is not possible to identify the“gold standard” method yet. Therefore, using in parallel different methods en-sures a correct and complete identification of strains. We present an overview ofthe techniques used to isolate, to identify and to subtype Listeria monocytogenes;the various subtyping systems provide different degrees of discrimination amongisolates. Subtyping methods for Listeria monocytogenes include phenotypic (e.g.serotyping and phage-typing) and different DNA–based subtyping methods (e.g.multilocus enzyme electrophoresis, ribotyping, pulsed-field gel electrophoresis(PFGE), polymerase chain reaction (PCR)).

Virulotyping of Listeria monocytogenes by high resolution melt analysisAmar, C. F.1, Tamburro, M.2, Dear, P.3 and Grant, K.1

1. Health Protection Agency, Centre for Infections, UK2. University of Molise, Campobasso, Italy3. Laboratory of Molecular Biology, Cambridge, UK

L. monocytogenes causes a severe foodborne infection in vulnerable people. Iso-lates are known to vary in their ability to cause infection but current typing meth-ods do not provide information on strain pathogenicity. Six key L. monocytogenesvirulence genes, clustered together on pathogenicity island, LIPI-1 (9Kb), areknown to be essential for intracellular survival, whilst two other virulence genes,internalin A and B, are responsible for host cell invasion. Many food isolates pro-duce truncated internalin proteins due to point mutations and thus have reducedpathogenicity. Detection of these mutations and those in other virulence genesare likely to provide valuable information on strain pathogenicity. High ResolutionMelt (HRM) analysis is a powerful technique capable of identifying sequence vari-ations in PCR amplicons by accurately determining their melting temperature(Tm). This technique was used to generate virulence fingerprints, using 81 markersspanning LIP1 and Internalin A and B genes. Strains of L. monocytogenes fromhuman cases, food and the environment were compared with results generatedusing L. monocytogenes EGDe. Considerable variations in Tm were found acrossmarkers for LIP1 and internalin A and B genes which were confirmed by sequenc-ing. Eight markers had Tms specific to genetic lineage I and 11 to genetic lineage II.Fifteen markers had conserved sequences across both lineages with those span-ning the hlyA gene being the most conserved. In the housekeeping gene, prs,marker variations were only detected in serotype 4b strains. Tm variations weredetected in markers for actA, intA and intB genes in which point mutations areassociated with the expression of truncated proteins and a concomitant reductionin virulence. HRM is an accurate and rapid technique for detecting sequence di-versity within bacterial genes and not only presents a novel method for strain char-acterisation but also offers a unique potential to inform on strain pathogenicity.

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Risk factors for nonperinatal listeriosis mortalityin Los Angeles County, California, 1992–2004Guevara, R. E.*, Mascola, L. and Sorvillo, F.County of Los Angeles Department of Public Health, USA

Listeriosis is a relatively rare foodborne disease but has significant public healthimplications as it has a case-fatality rate of 20% and it causes 28% of all food-borne disease related deaths. Yet, data on the risk factors for listeriosis mortalityhave been limited. A 13-year retrospective cohort study was performed to describenonperinatal listeriosis mortality in Los Angeles County, California, during1992–2004. A nonperinatal listeriosis case was defined as a non-pregnant person>42 days old with a positive culture of Listeria monocytogenes and residence inLos Angeles County. Causal modeling based on prior knowledge and bivariableanalyses was performed to determine a comprehensive unconditional multivari-able logistic regression model with 29 main effect variables. With 281 nonperina-tal listeriosis cases, multivariable analysis found non-hematological malignancy(odds ratio: 5.92, 95% confidence interval: 1.85-18.9), alcoholism (4.63, 1.36-15.8),age ≥ 70 years (3.44, 1.50-7.87), steroid medication (3.34, 1.38-8.08), and kidneydisease (2.94, 1.18-7.31) to be statistically significant risk factors for mortality.Other listeriosis mortality risk factors with adjusted odds ratios >1.5 includedblood transfusion, asthma, black race-ethnicity, Asian race-ethnicity, antibiotics,hypertension, chemotherapy, and Hispanic race-ethnicity. Cases admitted withsepsis only had the highest mortality (23.7%), while cases with meningitis onlyhad the lowest mortality (3.13%). The findings of this study should highlight theimportance of certain underlying risk factors and their association with nonperi-natal listeriosis-related deaths. (This study was published in Clinical InfectiousDiseases 2009; 48:1507-15 ©2009 by the Infectious Diseases Society of America.http://www.journals.uchicago.edu/loi/cid).


Molecular typing of Listeria monocytogenes isolated from ovine sausageNives, M. R.1, Mele, P.1, Parisi, A.2, Latorre, L.2, Virgilio, S.1 and Tola, S.1

1. Istituto Zooprofilattico Sperimentale of Sardinia, Italy2. Istituto Zooprofilattico Sperimentale of Apulia and Basilicata Italy

Sardinia, an island located in the middle of Mediterranean sea, has about four mil-lion of milking sarda sheep. In recent years in addition to milk and cheese pro-duction other products from sheep meat have been commercialized (ham andsausage). In this survey, between 2007 – 2008, five sampling were performed in-side a factory located in the northern side of Sardinia. We monitored the differentstages of sausage production for detecting the presence of Listeria monocytogenesin food matrices, the equipment and the environment. Isolation and identifica-tion of L. monocytogenes were carried out according to ISO 11290-1/2:1996/98amendment 1-2004. The isolates were characterized using serotyping and threedifferent molecular methods: PFGE, MLST, VNTR according to previous publishedprotocols. Moreover five virulence genes (actA, hlyA, plcB, prfA, iap) were detectedby PCR. L. monocytogenes was isolated in all the phases of the sausage production(raw minced meat, meat mixture after salt and spice addition, sausage after desic-cation time, sausage during different seasoning periods) and from the floor drain.On the whole 21 out of 126 samples resulted contaminated. The L. monocytogenescount resulted in all the samples higher than 100 UFC/g (Regulation EC n°2073/05). All isolates resulted PCR-positive for the investigated virulence genes.PFGE, MLST and VNTR identified two different molecular profiles. Five isolates,marked as type A and belonging to the serotype 1/2a, were isolated just from onebatch samples whereas the type B (n=16), belonging to the serotype 1/2b, was iso-lated from three different food batches and from the floor drain. Our resultsdemonstrate an high occurrence of L. monocytogenes in the studied facility. So thesheep sausage production must be object of a careful and constant vigilance ac-tivity and the isolates should be characterized using molecular methods for timelymonitoring the contaminations and to play out the due prevention actions.

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A novel phage-PCR assay for the rapid detection of viable Listeria mono-cytogenes in food within 24 hElemam, M. M.School of Biosciences, University of Nottingham, UK

Listeria monocytogenes is a pathogen of public health significance causing occa-sional outbreaks and sporadic cases of foodborne illness. This microorganism is ofparticular concern in the food industry due to its widespread distribution in na-ture, its ability to survive in a wide range of environmental conditions, and its abil-ity to grow at refrigeration temperatures. The standard microbiological methodfor L. monocytogenes detection in foods (ISO 11290-1/ A1 - 2004 and NHS-2009) iscostly and time consuming; require at least 6 days obtaining a results. The devel-opment of a rapid and reliable test capable of detection very low numbers of the or-ganism in ready to eat products is of significant importance to the food industry.The Phage Amplification assay has been developed as a rapid method for the de-tection of L. monocytogenes. We have been using the broad host range phage A511to develop such a method for detection of Listeria monocytogenes in both cheeseand stomached food samples. Successful development of the assay requires a viru-cide to achieve differential inactivation of the phage without affecting the viabilityof the target cell to be detected. Several different chemicals were evaluated as po-tential virucides, but tea extracts were found to be the most effective virucidalagent. The efficacy of the new assay was tested using cheese samples and was toshown to be able to detect low numbers of cells, in only 24 h in compared to the 6days to conventional culture methods. Because of the broad host range of thephage the identity of the cell detected must be confirmed using PCR amplifica-tion of signature sequences from the phage plaque and several different PCRstrategies have been evaluated. The combined phage-PCR method would allowrapid screening for food products prior to release from the factory.

Perinatal listeriosis in Los Angeles County, California, 1992 – 2004Guevara, R. E.* and Mascola, L.County of Los Angeles Department of Public Health, USA

In Los Angeles County (LAC), California, physicians and laboratories are requiredto report all listeriosis to the LAC Department of Public Health. From this passivesurveillance, a population-based epidemiologic study was performed to further de-scribe perinatal listeriosis. Perinatal listeriosis was defined when Listeria monocy-togenes was identified from a normally sterile site between a mother-fetus ormother-infant (infant age < 43 days old) pair. During 1992–2004, of 413 listeriosiscases reported, 122 (30%) were perinatal listeriosis cases. Perinatal listeriosis hada higher average annual incidence rate (5.9 cases/100,000 live births/year) andmortality (32.2%) than nonperinatal listeriosis (2.5 cases/million people/year, and18.6%, respectively). Disease onset during pregnancy defined intrapartum cases(n=93, 76%), during 0-6 days after birth defined early onset cases (n=21, 17%), andduring 7-42 days after birth (n=8, 7%) defined late onset cases. For intrapartum,early-onset, and late-onset cases, fetal/infant mortality was 36.6% (n=34), 14.3%(n=3), and 0%, respectively. No maternal deaths occurred. Median gestational age atadmission of intrapartum cases was 28 weeks (range 10-40 weeks) with maternalsymptoms commonly including fever (84%), abdominal pain (35%), chills (29%),back pain (24%), and headache (20%). Intrapartum listeriosis cases had maternalinfections of both sepsis and meningitis (n=2, 2.2%) and sepsis only (n=72, 77.4%),but mothers with symptoms in 19 (20.4%) intrapartum cases were culture-nega-tive or not cultured. Among intrapartum cases with hospital admission for liste-riosis before 38 weeks gestation, 33 (35%) had fetuses continue into pregnancy formore than 7 days (6 fetal demises/stillbirths with median of 13.5 days after onset, 4sick newborns with median 30.5 days after onset, 17 healthy newborns with me-dian 25 days after onset, and 6 unknown birth outcomes). Although annual inci-dence of perinatal listeriosis has been decreasing, opportunities such as culturingsymptomatic mothers for L. monocytogenes exist to further prevent perinatal liste-riosis morbidity and mortality.


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Antimicrobial resistance of Listeria monocytogenes human strainsisolated since 1926 in FranceMorvan, C., Moubareck, A., Leclercq, M., Herve-Bazin, S., Bremont, M., Lecuit, P.and Le Monnier Courvalin, A.Institut Pasteur, France

Amoxicillin plus gentamicin is the treatment of choice for listeriosis, a foodborneinfection caused by Listeria monocytogenes (Lm). Lm is considered universallysusceptible to antibioticsactive against gram-positive bacteria, but resistant strainshave been reported. Lm is considered universally susceptible to antibiotics activeagainst Gram-positive bacteria except 3rd generation cephalosporins, but resistantstrains have been reported. We have studied retrospectively the antimicrobial re-sistance of Lm human strains isolated between 1926, the year of its discovery, and2007. 4 816 human Lm strains were picked randomly in the strain collection of theWHO collaborating for Listeria. Their susceptibility to 23 clinically relevant an-tibiotics was determined by disc diffusion. MICs of fluoroquinolones and peni-cillins were determined by E-test. All resistant strains were typed by PFGE andthose displaying a unique profile were screened for the presence of antibiotic re-sistance genes by PCR. The mechanisms leading to detected resistance was studiedfor each resistant strain. Sixty-one human strains (1.27%) were resistant to oneor more antibiotics. Resistance to the tetracyclines (n=34) and fluoroquinolones(n=20) was more common and emerged since the late 80s and the late 90s, re-spectively. Resistance to fluoroquinolones was attributed to active efflux. Resist-ance to the tetracyclines was encoded by tet(M), presumably carried by a conjuga-tive transposon in the 14 strains which also harboured the int gene. We alsodetected the first human isolate with high-level resistance to trimethroprim, dueto the dfrD gene. Although no penicillin-resistant strain was identified, MICs havesignificantly increased since 1926 with values up to 2 µg/mL. In conclusion, L.monocytogenes has not acquired significant clinically-relevant antibiotic resist-ance. Importantly, no resistance to amoxicillin and gentamicin, the recommendedantibiotic regimen for listeriosis, was found. Yet, significant increase of penicillinMICs and the description of a strain resistant to trimethoprim reinforce the needfor surveillance and are in favour of systematic antibiotic susceptibility testing in-cluding penicillin MICs determination.

Today and tomorrow: The molecular epidemiology of listeriosis in the UKGrant, K., Amar, C., Matos, J., Mook, P., Little, C. and Gillespie, I.Health Protection Agency, UK

Listeriosis is a rare but severe foodborne infection caused by Listeria monocyto-genes: in the UK it is the leading cause of death due to a foodborne pathogen. Since2000 there has been an increase in the number of cases associated with patientsaged > 60 years presenting with bacteraemic infection in the UK and in other Eu-ropean countries and, yet, the reason for this increase remains to be established. Inthe UK, the number of pregnancy related cases of listeriosis has remained rela-tively stable over the past few years, although, more recently there has been a no-ticeable increase in the proportion of cases describing themselves as of ethnic ori-gin. Particularly concerning is that since the beginning of 2009, there has been anincrease in the total number of pregnancy related cases. Because the incubationperiod for listeriosis is long and those at risk of infection are often ill with seriousunderlying conditions the investigation of individual cases and outbreaks is oftendifficult with an increased emphasis on microbiological evidence. Approximately170 human and 900 food Listeria monocytogenes isolates from across England andWales are received annually by the Laboratory of Gastrointestinal Pathogens. Allisolates are identified and characterised using molecular methods including PCR–based serotyping and amplified fragment length polymorphism. This data is usedin conjunction with standardised epidemiological data to confirm and link spo-radic cases; to identify and investigate clusters and outbreaks; to provide infor-mation on routine food testing as well as for national surveillance purposes such asidentifying risk exposures for human infection. This paper will demonstrate, withrecent examples from the UK, the contribution of molecular typing to epidemio-logical investigations and will outline a new direction for typing Listeria monocy-togenes which will not only be discriminatory but provide additional informationon the relatedness of strains and their pathogenic potential.

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Use of Fluorescent Amplified Fragment Length Polymorphismfor improved typing of Listeria monocytogenesMatos, J., Amar, C., Desai, M., Cross, L. and Grant, K.Health Protection Agency, UK

Listeria monocytogenes is a ubiquitous, psychrotrophic, Gram positive foodbornepathogen which has the ability to persist in food processing environments andgrow in a range of ready-to-eat foods. It causes the disease listeriosis which mainlyaffects those with a weakened immune system including pregnant women,neonates, the elderly and immunocompromised individuals. Although a rare dis-ease it has a high mortality rate (20 –30%) and is currently responsible for moredeaths than any other foodborne pathogen in the UK. Typing of L. monocytogenesstrains is critical to investigating both sporadic cases and outbreaks enabling theidentification of the food vehicle and tracing the origin of contamination. The suc-cess of any typing method depends on its discriminatory ability, reproducibility,cost of the procedure and speed to getting results. At present Pulse field Gel Elec-trophoresis is generally accepted as the gold standard typing method for epidemi-ological investigations involving L. monocytogenes. However, it is not without itsdrawbacks including being lengthy and labour intensive to perform, making it un-suitable for real time analysis. PFGE also suffers from a lack of interlaboratoryreproducibility and the analysis of profiles is open to subjective interpretation.This study investigated the use of Fluorescent Amplified Fragment Length Poly-morphism (FluAFLP) for typing 842 isolates of L. monocytogenes from clinicalcases, foods and food processing environments using two restriction enzymes(HindIII and HhaI). All isolates were previously subtyped by multiplex PCR-basedserotyping and a 133 subset had also been previously typed by PFGE using AscI.fAFLP was found to be highly discriminatory (D.I. = 0.987), rapid to perform, re-producible and readily amenable to automation. Cluster analysis of isolatesshowed that fAFLP efficiently separated lineage I isolates from those of lineageII and that there was a high level of interlineage discrimination.

Towards an application for field samples of a multipathogen platformfor molecular detection of raw milk pathogensOmiccioli, E1, Amagliani, G.2, Brandi, G.2, Tonucci, F.3, Foglini, M.3 and Magnani, M.2

1. Diatheva s.r.l., Italy2. Department of Biomolecular Science, University of Urbino, Italy3. Istituto Zooprofilattico Sperimentale Umbria e Marche, Italy

Listeria monocytogenes, together with Salmonella spp. and Escherichia coli O157,are pathogens associated with various food categories, included raw milk, whichcommercialisation for human direct use has been encompassed by the853/2004/EC. Since this product can constitute a risk for human health, the ab-sence of such bacteria should be assessed by sensitive and specific diagnosis. Mo-lecular diagnostic tests in multiplex format proved to be useful for the rapid iden-tification of food contaminations caused by more than one microbial species. Aspart of a European Research project (DIAGNOSIS), we developed an enrichmentmultiplatform Real-Time PCR, including an internal amplification control ensur-ing the reliability of results, for the detection of all three pathogens. The assaycombines an enrichment step in a medium properly formulated for the simulta-neous growth of target pathogens (Multipathogen enrichment medium), a DNAisolation method, and a multiplex Real-Time PCR detection system based either ondual-labelled probes (MultipathogenFLUO), or on melting curve analysis (Multi-pathogenHRM). The entire system enables the detection of as few as 1 CFU of eachpathogen in 125 ml milk in only two working days and it is now available fromDiatheva srl, Fano, Italy. An application study for field samples is currently inprogress. A local dairy farm, licensed to the direct selling of raw milk through au-tomatic distributors, was selected for the collection of raw milk samples and swabsfrom filters located between milking rooms and storage tanks. Samples areanalysed by the official reference ISO methods and also culture-enriched in theMultipathogen enrichment medium. Aliquots from all enriched cultures are sub-jected to DNA column-based extraction and PCR amplified by both the Multi-pathogenHRM and MultipathogenFLUO kits. The comparison of results will allowthe estimation of method equivalence and the compliance of the molecular kitswith law provisions, contributing to promote the adoption of PCR-based methodsalongside the traditional diagnostic procedures.

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Detection and recovery of Listeria species from stainless steelBoone, R., Iugovaz, I, Trottier, Y.-L. and Pagotto, F.Health Canada

The 2008 Canadian listeriosis outbreak in ready to eat meat raised many issuesover the safety and reliability of the national food supply, including the reliabilityof detection methodologies aimed at food-contact surfaces made of stainless steel.Five methods pertaining to the recovery of Listeria species including L. monocy-togenes from stainless steel were assessed. A cocktail of L. monocytogenes (serovars1/2a, 1/2b, 4b), L. innocua, plus a Gram-positive organism (Staphylococcus aureus)was inoculated at different levels onto stainless steel. Inocula were recovered usingMFLP-41 and assessed using different detection methods, including Petrifilm(3M), BAX Genus Listeria (Qualicon), BAX Listeria monocytogenes, and fourVIDAS kits (BioMérieux) (LDUO, LIS, LMO2 and LSX). Direct plating onto selec-tive and chromogenic agars were done simultaneously from each broth and eachkit was compared to the “gold standard”, MFHPB-30. Experiments using skimmilk powder assessing cell viability revealed a consistent 1-log die-off after 24 h.The VIDAS strips DLIS, LIS and LSX had 74 positive samples out of 84 tested fol-lowing 52 hours of incubation in LX Broth while the BAX Genus Listeria had 72positive samples out of the 82 tested. The VIDAS strips DLMO and LMO2 yielded52 and 51 positive samples out of 79 tested, respectively; whereas the BAX L. mono-cytogenes detected 50 of 81 samples tested. Petrifilm modifications to increase in-cubation time from 30 h to 48 h are being suggested due to 27 out of 84 sampleswere negative at 30 h but positive at 48. Differences amongst methods were notedat the single cell inoculum level. None of the methods had false positive results.Through the study of different rapid detection methods, options can be broughtforward to improve and ensure optimal conditions for Listeria detection (and re-covery) from stainless steel environments.

Natural carriage of Listeria in fresh-water fish in RussiaEgorova, I.1, Voronin, M.2, Selyaninov, Y.1 and Kolbasov, D.1

1. National Institute of Veterinary Virology and Microbiology Pokrov Russia2. National Park “Zavidovo”, Russia

To study the extent of Listeria natural carrier stage in fresh-water fish, theIvankovo water storage reservoir was observed. In the period of 2006 to 2008, 642fish specimens belonging to 18 fresh-water fish species were examined. In total, 34listerial cultures of monocytogenes and innocua species were isolated in fish, 35,3%of them belong to L. monocytogenes. Listeria isolation rate was 5,38%. L. monocy-togens were isolated predominantly from plant-feeder fish like bream, cruciancarp and silver bream. L. innocua - in bream, pike, crucian carp, redeye and perchof all age groups. Listeria carriers were found most often in areas where animalfarm buildings and treatment facilities were located. The primary Listeria loca-tions in fish were muscle tissues, gill rakers and parenchymatous organs. In twocases L. innocua were isolated from fresh-water fish intestines. The peak of Liste-ria carrying in fish falls on summer periods (40%). In autumn and winter periodsthe detection rates were 28%. In spring the index dropped to 4%. All the culturesare quite typical specimens of their species. L. monocytogenes cultures producedhemolysins and phospholipases, were agglutinated with specific sera and lysedwith phage L2A, and induced purulent conjunctivitis in guinea pigs. L. innocuacultures were hemo- and phospholipase-negative and were agglutinated with aserum of serogroup II, were lysed with phages L4A and did not induce mice deathdue to intraperitoneal inoculation at a dose of 109 m.b. The experimentally deter-mined diversity of the of Sma I pulse electrotypes suggests there are both associ-ated and independent Listeria reservoirs and there is a circulation of several clonevariants of the pathogen in the fresh-water fish population.

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Prevalence of Listeria in wild fauna in RussiaEgorova, I.1, Fertikov, V.2 and Kolbasov, D.1

1. National Institute of Veterinary Virology and Microbiology Pokrov Russia2. National Park “Zavidovo”, Russia

Monitoring of Listeria in environmental objects and also among ungulate wildlifeinhabiting forest hunting ranges of the Central region of Russia has been carriedout. 662 samples of soil from feeding grounds, decaying plant residues, foragesand wildlife faeces, and also brain, muscle tissue and/or parenchymal organs ofthe axis deer, the maral, the elk and the wild boar, were tested and 68 listerial cul-tures were found. The organism carriage indices for the axis deer, the maral andthe wild boar in the areas of Tver, Moscow and Kaluga regions were at a similarlevel making up 2.0 to 2.7%. In Vladimir region the indices turned out to be a littlehigher gaining 12% for the wild boar and 5.45% for the axis deer, which is mostlikely due to some differences in the forms of economic activities and inadequateobservance of sanitary-and-hygienic requirements. Examinations of elk faeces re-vealed Listeria in none of the cases which is probably explained by specificity ofthe elk nutrition. Examinations of environmental objects showed that all of themwere to a variable extent contaminated by Listeria of both pathogenic and non-pathogenic species. Listeria were mainly isolated in soil samples taken from feed-ing grounds used for seasonal feeding of the wildlife. In samples from wild ani-mals Listeria were found in two cases in axis deer brain and lymph node, and alsoin boar liver and lung. The isolate taken from the axis deer is of a certain researchinterest, as according to all the phenotypic indications it was classified to L. in-nocua, although investigation of its genetic structure showed presence of patho-genicity genes typical for pathogenic Listeria species in its genome. The remainingisolates of Listeria found in the course of the monitoring investigations belongedbasically to two species, namely L. monocytogenes and L. innocua.

Quantitative assessment of the exposure to Listeria monocytogenesfrom soft-ripened cheese consumption in North America: a jointFDA/Health Canada projectGendel, S.1, Pouillot, R.1, Murray, C.1, Farber, J.2, Ross, W.2, Couture, H.2 and Jean, A.2

1. Food and Drug Administration, USA2. Health Canada

The United States and Canada continue to experience sporadic illnesses and out-breaks of listeriosis associated with the consumption of cheese, particularly softand soft-ripened cheese. Therefore, the US Food and Drug Administration andHealth Canada jointly conducted a risk assessment to assess the public health im-pact of Listeria monocytogenes in soft-ripened cheese in the US and Canada, and toevaluate the impact of current and potential mitigation and control strategies. Tosupport this risk assessment, a probabilistic model was developed to assesschanges in L. monocytogenes prevalence and levels from “farm to fork” in multiplescenarios. This model evaluated the impact of contamination of the milk used forcheese making, in-plant environmental contamination, partitioning, mixing, bac-terial growth and bacterial inactivation though the complete product pathway.Model calculations and parameters for a base model were derived from data in thepublished literature and from specific databases from both countries. The generalframework of the model was a second-order simulation that allowed separate eval-uation of variability and data uncertainty. An original back-calculation was used toevaluate the frequency and level of environmental in-plant contamination basedon published data on L. monocytogenes prevalence and levels in soft-ripenedcheeses at retail in the US. This back-calculation model was integrated using theMonte-Carlo method. The results of the back calculation suggest that in-plantcontamination is infrequent and generally leads to a low levels of L monocytogenesper cheese (<25 cfu in each whole 250g cheese). Because data on production lev-els and practices for the cheese making industry are limited, the model was used tocompare exposure estimates obtained using the base model to estimates obtainedusing alternate scenarios. For example, scenario analysis suggested that, for con-taminated cheese, the level of exposure is highly sensitive to storage conditions.

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Human listeriosis in England, 2001-2007: association with neighbour-hood deprivationGillespie, I. A., Mook, P., Little, C. L., Grant, K. and McLauchlin, J.Health Protection Agency, UK

Listeriosis is a rare but severe disease affecting mostly the elderly and the im-munocompromised, and, to a lesser extent, pregnant women, unborn or newborninfants. Despite a high mortality rate, the socioeconomics of listeriosis has notbeen studied in detail, meaning that health inequalities which might exist in rela-tion to this disease are not apparent. Laboratory surveillance data on cases of Lis-teria monocytogenes infection reported in England between 2001 and 2007 werelinked to indices of deprivation and denominator data using patients’ postcode.Incidence relative to increasing quintiles of deprivation was calculated by fittinggeneralized linear models whilst controlling for population. Patient exposure datawere scrutinised and compared with commercial food purchasing denominatordata to further quantify the risk. For all patient groups, listeriosis incidence washighest in the most deprived areas of England when compared to the most affluent,and cases were more likely to purchase foods from convenience stores or fromlocal services (bakers, butchers, fishmongers and greengrocers) than the generalpopulation. Patients’ risk profile also changed with increasing neighbourhood dep-rivation. With increased life expectancy and rising food prices, food poverty couldbecome an increasingly important driver for foodborne disease in the future.Whilst Government policy should continue to focus on small food businesses toensure sufficient levels of food hygiene expertise, tailored and targeted food safetyadvice on the avoidance of listeriosis is required for all vulnerable groups. Failureto do so may enhance health inequality across socioeconomic groups.

Characterisation of antibodies for use in an antibody-based sensor foron-site detection of L. monocytogenesGilmartin, N., Hearty, S. and O’ Kennedy, R.School of Biotechnology and National Centre for Sensor Research, Dublin, Ireland

L. monocytogenes is a facultative anaerobic, non-sporing, Gram-positive, motile,rod-shaped bacterium and is an important food-borne pathogen. It is a majorcausative agent of listeriosis, an infection which can kill vulnerable people suchas the elderly, newborns, pregnant women and people suffering from immuno-compromising diseases. The current methods for sampling and detection of L.monocytogenes can result in extensive treatment times, limited sensitivity andvery low recovery rates of the microorganism (due to the formation of biofilms).Antibody-based sensors permit the rapid and sensitive analysis of a range ofpathogens and associated toxins. They offer many advantages such as improvedsensitivity, real-time analysis and quantification. An anti-L. monocytogenes mon-oclonal antibody (2B3) was isolated from a panel of hybridomas produced using L.monocytogenes serotype 1/2a as the immunogen. This antibody reacted with L.monocytogenes serotypes 1a, 1/2a, 1/2b, 1/2c, 3a, 4a and 4b but did not react with re-lated Listeria spp. and non-Listeria spp. The mAb was further evaluated in bothenzyme-linked-immunosorbent assay (ELISA) and fluorescence-linked-immunosorbent assay (FLISA)-based formats and in a surface plasmon resonancebased biosensor platform. This antibody will be exploited for use in an antibody-based sensor as part of the BIOLISME project which aims to develop a system tomonitor the contamination levels of L. monocytogenes in industrial food-producingplants. Preliminary work on the use of this antibody in the detection of L. mono-cytogenes in biofilms will also be presented.

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Food Investigation of sporadic cases of neuroinvasive listeriosisGoulet, V.1, Leclercq, A.2, Laurent, E., King, L., Dusch, V., Salem, S., Vaillant, V.1,Chenal-Francisque, V.2, de Valk, H.1 and Pihier, N.3

1. Institut de Veille Sanitaire, France2. National Reference Centre and WHO collaborating centre for Listeria, France3. Direction générale de l’Alimentation, France

Listeriosis in France is mandatory notified. For each case, a detailed food history iscollected. For cases with central nervous system involvement who consent, sam-ples are taken from foods and the environment in the kitchen of their residence orof hospital for patients hospitalized before disease onset. We present results ofinvestigations 2003-2008. Samples are taken by officers of inspection services ofthe ministry of agriculture, within 14 days after diagnosis of the case, and sent totheir authorised laboratories. Strains of Listeria monocytognes (Lm) isolated fromthese samples are sent to the National Reference Centre who performs PCRserotype, and PFGE (Apa1 and Sma1) typing of the food and patient isolates. Lmcontaminated food is traced back with further investigations at the productionsites. 140 cases have been investigated. Lm was isolated in 63 (45%) investiga-tions, from cheeses, meat products, and the environment. For 23 investigations,the food or environmental strains had the undistinguishable PFGE profiles of thepatient strain. At 2 occasions, the patient strains were isolated from the kitchenenvironment in their hospital. In 3 other investigations, the patient strain was alsoisolated at the production site of the contaminated food. At one occasion, the earlyinvestigation of a case later shown to be part of a cluster, allowed the prompt in-vestigation in a cheese producing factory and the confirmation of the source ofthe outbreak. Food investigation of listeriosis cases are of interest because theycan lead to control measures in contaminated production sites and limit the size ofoutbreaks of listeriosis by rapidly identifying their sources. However the yield ofthese investigations is limited because numerous unpacked food products in thepatient’s refrigerators can not be easily identified. An important finding is thatLm strains can colonise the environment of hospital kitchens.

Evaluation of the antimicrobial activity of Vaccinium myrtillus, Prunusdomestica and Myrtus communis against Listeria monocytogenesSerio, A., Di Pasquale, F. and Paparella, A.Department of Food Science, University of Teramo, Mosciano Stazione (TE), Italy

Essential oils and plant extracts have been increasingly used as natural foodpreservatives. They are considered consumer-friendly and can be used withoutany specific authorization or labelling change. In this study, three aqueous plantextracts were evaluated for their antimicrobial activity against 45 Listeria mono-cytogenes strains. Among blueberry, plum and myrtle extracts, the latter was themost effective against the strains isolated from smoked salmon and meat prod-ucts, with MIC (Minimal Inhibitory Concentration) values generally ranging from0.5 to 2.0%. Interesting results were also obtained with the plum extract, althoughwith MIC values from 1 to 4%. Blueberry was effective only at high concentrationlevels, even above 10%. Isolates from the smoked salmon industry were generallyless sensitive, suggesting that resistance to plant extracts may somehow be relatedto strain origin. A 3-factor-5-level Central Composite Design was used to evaluatethe extracts activity in combination with compounds commonly present in somefood products, where they can constitute a hurdle for bacterial growth. Myrtle ex-tract activity was boosted by lactic acid and salt, while ATCC7644 strain, used ascontrol, was particularly susceptible to all combinations. These results are in linewith our previous research on volatile compounds, where L. monocytogenes wasinhibited at low extract concentrations, without significantly affecting food fla-vor. Further studies will be carried out to evaluate the potential of myrtle extract infood biopreservation.

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High throughput quantitative detection of Listeria monocytogenesin food by RealTime PCRCammà, C., Ancora, M., Rizzi, V., Sperandii, A., Prencipe, V. and Migliorati, G.Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy

Listeria monocytogenes is one of the most important food-borne pathogens andcan cause a severe disease in human. The Commission Regulation (EC) N°2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs allowsthe presence of 100 CFU/g in some ready-to-eat products. Conventional bacterio-logical methods for the detection and quantification of L. monocytogenes are la-borious and time-consuming. The purpose of this study was to develop a rapid andautomated method for detection and quantification of L. monocytogenes in meat.The target of the RealTime PCR was a portion of hly gene and the procedure con-sists of two automated steps: DNA extraction, and set up of a fast RealTime PCRmix. The whole process could be completed within few hours minimizing the riskof carry-over contamination. To evaluate the inhibitor effect of food matrix on thePCR an internal amplification control was included in each reaction and coampli-fied using the same primer pair used for the target amplification. The specificity ofthe assay was confirmed on 3 L. monocytogenes strains and 26 other bacterialstrains. The method was able to detect 2 genomic equivalents (GE) in 33/42 repli-cates (LOD= 4.8 GE/reaction with a probability of 95%) and the efficiency resultedbetween 90% and 98% calculated by analyzing 7 standard curves based on six 10-fold dilutions starting from a 107 CFU/ml broth culture of L. monocytogenes. Thequantification range was linear over 6 log units with a square regression coeffi-cient > 0.99. Application of the assay was assessed with 12 artificially contami-nated samples of canned meat and with 8 real samples (smoked salmon, fresh fishand salami). The microbial load was tested in comparison with a pour plate count-ing method. The RealTime PCR was able to define an accurate quantification up to103 CFU/g both in naturally and in artificially contaminated samples.

Bibliographic study concerning procedures for preparing environmentalsamples for analyses, regarding to L. monocytogenesBarre, L., Carpentier, B. and Gnanou Besse, N.AFSSA, France

Listeria monocytogenes (L. monocytogenes) is a bacterium that can contaminatefoods and cause a mild non-invasive illness or a severe, sometimes life-threatening,illness. L. monocytogenes is widespread in the environment. It can be readily iso-lated from humans, domestic animals, raw agricultural commodities, and food pro-cessing environments. Sanitation controls include effective environmental moni-toring programs designed to identify and eliminate L. monocytogenes in and onsurfaces and areas in the plant. The conditions are likely to be present in food fac-tories that may give rise to the development of persistent L. monocytogenes strains.This persistence can be observed in spite of the well applied procedures of hy-giene. This document is a bibliographic study concerning procedures for preparingenvironmental samples for analyses, regarding to L. monocytogenes. Different pro-cedures for collecting environmental samples are described: several proceduresfor sampling methods are compared (cotton swab, sterile sponge, composite-plytissues, sonication, direct contact agar…). We have also compared different detec-tion and enumeration methods. We concluded that swabbing is known not to de-tach all micro. The removal efficiency depends on the swab material. Swabbing inmoistened condition, as processed here, is more efficient than dry swabbing. Use ofultrasound is still considered as a good method for detaching microorganisms.However, ultrasounds can be lethal to bacteria, particularly when used at low fre-quency and are more detrimental to attached bacteria than to suspended ones. Weconcluded that it is important to adapt the type of sampling tools and techniquesto the type of surfaces and sampling locations.

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Persistent L. monocytogenes isolates from Austrian and Irish dairiesshow the same phenotypic and genetic backgroundStessl, B.Veterinary University Vienna, Austria

The food borne pathogen Listeria monocytogenes continues to be of major epi-demiological interest to food industry and food sciences. The Zoonosis Report2004 of EFSA (European Food Safety Authority) listed 1267 cases of listeriosis inEurope in 2004, resultant 0.3 cases per 100 000 capita. Human infections with L.monocytogenes are rare, but are important due to the high mortality rate. Thisstudy is focused on the PFGE (Pulsed Field Gel Electrophoresis) and FTIR(Fourier Transform Infrared Spectroscopy) typing of L. monocytogenes strains iso-lated at distant sampling points (1996–2007) in the Austrian and Irish dairy chain.The aim of this research was to analyse if genetically indistinguishable (“persis-tent“) L. monocytogenes clones can be differentiated with respect to their pheno-type (adaptation towards sanitizers and hygienic measures). A set of L. monocyto-genes isolates from seven different European dairies was analysed with twoepidemiological techniques: PFGE and FTIR. The PFGE method was performedaccording the CDC PulseNet Standardized Protocol for Molecular Subtyping of L.monocytogenes. Further data analysis was completed by Fingerprinting II ClusterAnalysis using Dice coefficient and UPGMA logarithm (Biorad, Austria). The FTIRsample preparation and spectra analysis were performed as described by Ober-reuter et al. For data processing the program OPUS FT-IR (Bruker, Germany) wasused. Cluster analysis was performed using Ward’s algorithm. The PFGE as well asthe FTIR cluster analysis suggests the ability of short and long-time persistence ofL. monocytogenes isolates in soft, semi-hard and hard cheese manufacturing. Anexplanation could be that the tested persistent L. monocytogenes isolates were notexposed to selection due to sanitation (colonization of ecological niches charac-terized by a low hygienic pressure). Additional studies will concentrate on the test-ing of a L. monocytogenes strain subset against their disinfectant susceptibility.

Epidemiological data on listeriosis in Portugal: 2003 – 2008Almeida, G, Magalhães, R., Hogg, T. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

The invasive form of listeriosis although rare is a very serious disease with a highmortality rate. Young, Old, Pregnant, Immuno-compromised, sometimes referredas YOPI, are identified as at risk groups. However the infection may also occur inpeople with no known predisposing factors. In Portugal listeriosis is a not a notifi-able disease and is likely to be under-reported. In 2003 an incidence of 1.4 cases permillion inhabitants was reported. As in other European countries the number ofcases has increased in the last years. The aim of this study is to present epidemio-logical on human cases of listeriosis occurred in Portugal between 2003 and 2008.It was possible to gather 107 cases: 16% were maternal/neonatal infections and84% were non-maternal/neonatal infections. Concerning non-maternal/neonatalcases the sex ratio (M/F) was 1.73, the mean age of the patients was 62 years oldwith 46 cases (51%) in individuals aged 65 or more. The microorganism was mainlyisolated from blood (59%), from CSF (32%) and from other specimen (9%). The in-cidence of listeriosis in Portugal based on voluntary reporting is lower than the Eu-ropean notification rate (0.3/100000 inhabitants) and similar to countries like Aus-tria, Estonia, Latvia, Slovakia, Slovenia and Spain. The need of an activesurveillance system of listeriosis was clearly demonstrated.

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Diversity among Listeria monocytogenes isolated from humansKalekar, S.1*, Rodrigues, J.1, D’Costa, D.1, Malik, S. V. S.2, Kalorey, D. R.3, Chakraborty, T.4


1. ICAR Research Complex for Goa, India2. Indian Veterinary Research Institute, Índia3. Nagpur Veterinary College, India4. Institute of Medical Microbiology, Justus Liebig University, Germany

Listeria monocytogenes is a food borne pathogen associated with severe diseases inhumans and animals. It is associated with neural, visceral and reproductive clini-cal entities, leading to septicaemia, abortions, stillbirth, meningitis and menin-goencephalitis, especially in individuals at risk. We present the genotypic analysisof 17 L. monocytogenes isolates recovered from humans in India during 2006–2009by multiplex serotyping PCR allowing serovar predictions, conventional serologyand by PFGE. Multiplex-PCR serotyping assay revealed 88.24% (15/17) of thestrains belonging to serovar group 4b, 4d, 4e, 11.76% (2/17) to serovar group 1/2b,3b. Twelve (70.59%) L. monocytogenes isolates were assigned to serotype 4b, 2(11.76%) serotype 4d, one (5.88%) serotype 4e and 2 (11.76%) to serotype 1/2b byserology. Ten ApaI pulsotypes were recognized among the 17 human isolates.PFGE analysis allowed discrimination among isolates of the same serotype andamong isolates from the same sampling areas or those isolated from differentareas. Thus, PFGE together with multiplex-PCR serotyping allows rapid discrim-ination of L. monocytogenes strains. In addition, the predominance of L. monocy-togenes serotype 4b is of concern, as this serotype has been most frequently asso-ciated with human listeriosis outbreaks.


Characterization of Listeria isolated from seafoodRodrigues, J.*, Kalekar, S., Bhosle, S. N., Doijad, S. and Barbuddhe, S. B.ICAR Research Complex for Goa, India

Listeria monocytogenes, the causative organism of listeriosis, is primarily trans-mitted to humans through contaminated food. While several reports indicate thatfish and fishery products can be frequently contaminated with L. monocytogenes,no major outbreaks associated with these products have been reported. Since fishand fishery products may be a vehicle for L. monocytogenes, it is important to haveinformation on the incidence of this pathogen. The presence of L. monocytogeneshas been documented in the tropical environment but the incidence in fish andfish products is low. We examined the prevalence, molecular characteristics andvirulence potential of L. monocytogenes isolates from fishery products to gain fur-ther insights on the public health risk caused by this important food bornepathogen. A total of 221 raw seafood samples were examined for the presence ofListeria species. Of these 37 (16.74%) samples were positive for Listeria species.Out of these, 4 (1.8%) isolates were confirmed as L. monocytogenes. Other speciesdetected was L. innocua (33). The hlyA gene was detected in all the L. monocyto-genes isolates. All the L. monocytogenes exhibited phosphatidyl inositol specificphospholipase C activity on ALOA agar. Multiplex PCR based serotyping revealedthree L. monocytogenes isolates to be 1/2a, 1/2c, 3a, 3c group. One isolate belongedto 1/2b, 3b serogroup. The isolates were grouped into three AscI PFGE profiles.Prevalence of L. monocytogenes in fresh seafood is of significance as it may con-taminate and persist in the processing environment.


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Characterization of Listeria species isolated from milkD’Costa, D.1*, Bhosle, S. N.2, Dhuri, R. B.3, Kalekar, S.1, Rodrigues, J.1, Doijad, S. P.1


1. ICAR Research Complex for Goa, India2. Department of Microbiology, Goa University, India3. Goa State Cooperative Milk Producers’ Union Limited, Curti, Ponda, Goa, India

Listeria monocytogenes is a foodborne pathogen commonly found in food and en-vironment. This study aimed to determine the occurrence of L. monocytogenes inraw and market milk. Seven hundred sixty seven raw and market milk sampleswere collected at different levels of collection and processing. The samples takenincluded cow milk collected at udder level, from the milk cans, milk receiving cen-tres, processing unit and market. Samples were enriched in Listeria enrichmentbroth and incubated for 48 h, followed by plating on PALCAM agar. Presumptive L.monocytogenes isolates were purified and confirmed by PCR targeting the hly gene.Overall, 10.56% of the samples (81 of 767) were positive for Listeria species. Out ofthese 37 (4.82%) were confirmed as L. monocytogenes. Other Listeria species iso-lated were L. innocua (5.47%), L. ivanovii (0.13%) and L. grayi (0.13%). Maximumisolates were recovered from samples collected from market followed by samplesat milk processing unit. L. monocytogenes were serotyped using multiplex PCRmethod. A larger proportion of isolates (26) belonged to group corresponding toserovars 1/2a, 1/2c, 3a, and 3c. Serogroup corresponding to serovars 4b, 4d and 4ewas detected in two strains while serogroup 1/2b, 3b, 4b, 4d, and 4e was detected innine strains. . This study demonstrates the prevalence of L. monocytogenes in theraw and market milk and the need for good hygiene practices to prevent its entryinto the food chain.


Prevalence of Listeria monocytogenes in chicken production chainin ThailandKanarat, S., Nijthavorn, N. and Sukhapesna, J.Veterinary Public Helath Laboratory, Bureau of Quality Control of Livestock Products, Departmentof Livestock Development Phaya-thai Rd., Bangkok, Thailand

This study was conducted from 2004 to 2009 to investigate the prevalence of Lis-teria monocytogenes in the chicken production chain in Thailand, i.e., from pri-mary production stage to further processing plants. Samples were taken from 43breeder farms, 32 hatcheries, 1331 broiler farms, 22 slaughterhouses and 22 furtherprocessing plants. Various types of samples were collected: fecal drops on the litter,drinking water, and chicken feed from breeder and broiler farms; swabs andmecomium from hatcheries; cloacal swabs and fresh chicken meat from slaugh-terhouses; and ready-to-eat chicken products from further processing plants. Thestudy showed that there was no L. monocytogenes contamination in the chickenproduction chain except in slaughterhouses and further processing plants. Thisimplies that the chickens did not carry the organism into slaughterhouses, andconsequently primary production practices were not responsible for the the con-tamination of end products. This suggested that the observed L. monocytogenescontamination in 2.5% and 0.2% of samples of fresh chicken meat and ready-to-eat chicken products, respectively, was due to the breakdowns in the application ofgood hygienic and/or good manufacturing practices.

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Listeria monocytogenes in Ireland – Epidemiology and Molecular Typing

Cormican, M. G.1, DeLappe, N.2, McKeown, P.3 and Garvey, P.3

1. NUIGalway, Ireland2. National Salmonella Reference Laboratory Ireland, Ireland3. Health Protection Surveillance Centre, Ireland

Since January 2004 listeriosis is a notifiable disease in Ireland. Clinicians and lab-oratory directors must inform the Medical Officer of Health of all cases diagnosed.Information is collated by the national Health Protection Surveillance Centre.Isolates of Listeria spp. from human cases and from food products may be volun-tarily submitted to the National Salmonella Reference Laboratory. For the years2004 to 2007 11, 12, 7 and 21 (total =51) cases of listeriosis were reported to theHPSC. There were 37 cases categorized as adult or juvenile, 10 pregnancy-relatedand 4 neonatal. Cases were distributed through out Ireland with a slight seasonalpeak in Summer in most years. For the years 2004 to 2008 the NSRL has received4, 4, 1, 12 and 14 (total of 35) L. monocytogenes isolates from humans and 6, 34, 16,18 and 12 (total of 86) from foods. Isolates from human cases are predominantlyserotype 4b (n = 24). Isolates from food are predominantly serotype 1/2 (n= 74).Pulsed field gel electrophoresis (PFGE) performed by the Pulse Net method withApaI and AscI enzymes and analysed with Bionumerics software ahs allowed thedefinition of 10 clusters of closely related isolates (2 to 4 isolates per cluster). Theclusters link isolates from apparently sporadic human cases, including cases oc-curring in different years and link isolates from food items with human cases. Asthe clusters are small and it is not possible to perform typing in real-time epi-demiological links between cases have not been identified. The number of cases oflisteriosis in Ireland has increased in recent years. There is laboratory evidencelinking individual cases and linking cases with food. Real time molecular typing ofisolates is essential to identify linked cases in time frame that would allow timelyepidemiological investigation and intervention.

Characterization of Listeria monocytogenes isolated from human casesof listeriosis occurred in Portugal in 2008Magalhães, R.*, Barbosa, J., Santos, I., Almeida, G. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

Listeria monocytogenes is a Gram-positive foodborne pathogen that can cause se-rious infections in humans. High-risk groups include the elderly, immunocom-promised individuals, pregnant women and their neonates Clinical manifestationsof invasive human listeriosis include meningitis, encephalitis, late-term sponta-neous abortion, and septicaemia. The fatality rate is high: 20 to 30%. Foodbornetransmission of L. monocytogenes can also cause a self-limiting acute febrile gas-troenteritis in immunocompetent persons. Listeriosis is an infection of consider-able public health concern. In 2008, 27 isolates of Listeria monocytogenes were re-covered from Portuguese human cases of listeriosis and have been characterizedby biotyping (cadmium and arsenic sensitivity), genoserotyping and typing bypulsed field gel electrophoresis (PFGE) using the enzymes AscI and ApaI. Strainswere aggregated in three multiplex PCR serogroups: IVb (66.7%), IIb (25.9%) andIIa (7.4%). Concerning biotyping four groups were found: sensitive to arsenic andcadmium (51.9%), arsenic sensitive and cadmium resistant (14.8%), resistant to ar-senic and sensitive to cadmium (29.6%) and resistant to both heavy metals (3.7%).Twenty two pulsotypes were indentified. The more prevalent pulsotype aggregatesthree isolates, of these, two were time and geographical related. Two other pulso-types aggregate two time and geographical related isolates. It was shown that sevenpulsotypes (32%) were also found in previous years.


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Post-processing environmental contamination of surface-ripenedsoft cheese during affinageD’Amico, D. and Donnelly, C.University of Vermont, USA

Environmental sampling conducted in a cheese aging room identified extensivecontamination with Listeria monocytogenes. Further investigation revealed crosscontamination of washed rind cheeses aging on various racks. To determine theextent of post-processing contamination of cheeses in this facility, two commer-cially available PCR assays were investigated as tools for detection of L. monocy-togenes in naturally contaminated surface-ripened soft cheeses. Methods wereculture confirmed on CHROMagar Listeria. For analysis, cheeses were compos-ited and 10g sub-samples were assigned to an enrichment broth or Butterfield’sPhosphate buffer for enumeration. Cheeses identified as positive for L. monocyto-genes by PCR and/or culture were subsequently enumerated. To achieve a detec-tion limit of ≥5 cfu/g, composited samples were diluted 1:5 followed by direct plat-ing of 1ml on CHROMagar Listeria. Contamination levels ranged from <5 to1,600,000cfu/g and varied by cheese type with the highest contamination levels(mean ~356,000 cfu/g) observed in the smallest cheese variety (~200 g) and lowerlevels (mean ~1,500-2,500 cfu/g) in the larger cheese types (~400-2000 g). Pre-liminary ribotyping of random isolates recovered from each of the cheese vari-eties identified a single lineage II ribotype, DUP-1030B. Based on PCR and cul-tural results, 10g samples of cheese contaminated at levels as low as <5cfu/g wereidentified following enrichments of 24 or 26 hours or after secondary enrichmentfor a total of 44-48 hours. Single enrichment procedures of 24 and 26 hours inBuffered Listeria Enrichment Broth and Oxoid 24LEB, respectively, followed byplating 100µl on CHROMagar Listeria identified approximately 90% of the truepositive samples. Secondary enrichment in MOPS-BLEB increased the sensitivityto nearly 97%. PCR detection following single primary enrichment was less sen-sitive when compared to cultural techniques whereas dual enrichment PCR meth-ods were comparable to their cultural counterparts.

Genetic diversity of Listeria monocytogenes isolatedfrom Portuguese cheesesAlmeida, G., Magalhães, R., Santos, I., Barbosa, J., Hogg, T. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

The invasive form of listeriosis is a rare but serious disease with a high mortalityrate. Cheese has been associated with several outbreaks of human listeriosis in-volving large numbers of consumers, with a wide economic and public health im-pact. To reduce the incidence of listeriosis it would be necessary to reduce thecontamination level at production and at retail, or to inhibit the growth of Listeriamonocytogenes in high-risk foods. Typing of bacterial isolates reveals the relation-ship among isolates. They can be used to determine the primary sources of bacte-rial contamination and to link people who are ill following the consumption ofcontaminated foods to the sources of bacterial contamination. Eighty isolates of L.monocytogenes recovered from cheeses (40 from cheeses purchased at retail, 23from cheeses ready to go to the market, 16 from cheeses analysed at Autoridade deSegurança Alimenter e Económica laboratory and one isolate from cheese in qual-ity control routine activities) were typed by multiplex PCR serogrouping and byPFGE using AscI and ApaI restriction enzymes.Isolates were distributed by four MPCR groups: IIa (11%), IIb (30%), IIc (11%)and IVb (48%). Strains involved in human episodes in Portugal were mainly IVb(72%) followed by IIb (18%) and IIa (10%). The combination of the profiles ob-tained with both restriction enzymes (AscI and ApaI) yielded a total of 26 pulso-types. Eight of these pulsotypes were previously isolated from clinical cases of lis-teriosis in Portugal. This study highlights the possibility of cheeses beingresponsible for human cases of listeriosis in the country.

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Prevalence of Listeria spp. in retail raw ground beef in Izmir, Turkey:A comparison of standard cultural method and Fluorescentin situ Hybridization (FISH) technique for detectionHandan Baysal, A.*Izmir Institute of Technology, Department of Food Engineering, Izmir, Turkey

Fluorescent in situ hybridization (FISH) is based on the principle that hybridiza-tion of a nucleic acid sequence target of a microorganism with a specific DNAprobe labeled with a fluorochrome and imaging by a fluorescence microscope.FISH method using a fluorescent labelled oligonucleotide probe designed fromspecific DNA and RNA sequences has proven to be a useful tool for the detection ofa specific microorganism in environmental and clinical samples however the ap-plication of the method in food microbiology is investigated by the research stud-ies. It usually involves four steps: sample fixation and permeabilization; hy-bridization of the target sequence and the fluorescent probe; stringency washes;and detection of the hybridized cells. In this study the possibility to use FISHmethod for detecting a food-borne pathogen Listeria spp. that used as food safetyindex in ground meat in market place of Izmir was evaluated. The work consists oftwo steps. In the first step specified pathogen bacterium was inoculated in groundbeef and the applicability of FISH method was evaluated and in the second step thepathogen was detected in fresh ground beef by standard (conventional) culturalmethods and FISH method. High correlation (r=0.96) was found between FISHand the conventional plate count method. The FISH protocol revealed to be spe-cific for Listeria spp. detection, since all bacteria showed a positive hybridizationsignal. Results obtained from the direct application of the FISH technique to cul-tures show that it allows the detection of Listeria spp. in hours.


Using ListexP100™ for Listeria monocytogenes detection in foodsFlores Lopes, J.1, Ferreira Leite, I.1, Azeredo, J.2, Gibbs, P.1 and Teixeira, P.1

1. CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal2. IBB / Institute for Biotechnology and Bioengineering, Centre of Biological, Engineering,

Universidade do Minho, Campus de Gualtar, Braga, PortugalFood types susceptible of being contaminated with Listeria monocytogenes, such ascheese, smoked fish, salads, raw vegetables, sausages and, in general, ready-to-eatfoods, pose a serious threat to consumers as this pathogen can cause severe diseases.L. monocytogenes canmultiply evenat refrigeration temperatures,with theminimuminfective dose still remainingunknown.Development of efficient and rapidmethodsfor the a priori detection of contamination by this microorganism in foods is of greatsignificance and is required in order to ensure public health concerning consump-tion of foods that are considered to be of higher contamination risk. The vast major-ity of Listeria-specific detection methods, developed as alternatives to the standardselective plating methods, possess drawbacks such as a low specificity or efficiency(which is the caseof antibody-based assays), or alsodetect dead cells (in the caseof as-says that detect geneticmaterial, e.g. PCR, restriction-enzymepattern-based assays).The main goal of the research work described herein was to investigate the possibil-ity of usingListex™P100 (a commercial bacteriophage-basedpreparation for the con-trol ofListeria contamination in foods, gently suppliedbyEBIFoodSafetyB.V.) for thedesign of a fast-response detection method for L. monocytogenes in foods. The pro-cedure developed during our research effort encompassed a bacteriophage amplifi-cation assay based on the specificity and lytic activity of the broad-host range P100listeriaphage with plaque formation as the assay end point. Aliquots of phage P100were added to the test samples allowing all Listeria cells to be infected. After 15 min-utes of incubation at 30ºC, to allow phage adsorption and eclipse phase with the nu-cleic acid delivered into the host cell, a virucidal agent was added in order to destroyall those phages that have not effectively infected a bacterial cell. Among several ef-fective virucidal agentswe selected 4.8mMFeSO4 and 13 ppmof tannic acid and leftstanding for 5min at roomtemperature. The virucidal activitywasneutralizedby theaddition of 2% Tween-80 and the samples were then mixed with helper cells andspread over the surface of agar plates using a soft agar overlay. After 16h of incuba-tion at 30ºC, the levels ofListeria contaminationwere determinedby thepresence ofphage plaques. It was demonstrated that the method was able to detect the presenceof bacteria, however only in samples containing high numbers of L. monocytogenes.

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Mapping of molecular profiles associated to Listeria monocytogenesfood isolates circulating in ItalyDe Cesare, A.1, Parisi, A.2, Latorre, L.2 and Manfreda, G.1

1. Alma Mater Studiorum – University of Bologna, Italy2. Istituto Zooprofilattico Sperimentale della Puglia e Basilicata, Italy

Listeria monocytogenes is a bacterial pathogen transmitted to humans from a va-riety of foods. Although most human listeriosis infections in Italy are being gen-erally considered to represent sporadic cases, recent results suggest that a con-siderable number of listeriosis might occur in clusters, many of which couldrepresent single-source outbreaks. To support tracing of food sources of humanlisteriosis infections, a long term mapping of serotypes, ribotypes and AFLP typesassociated to Listeria monocytogens isolates collected from different meat andcheese products as well as food production plant environments has been per-formed. The results achieved show that AFLP profiles of Listeria monocytogensisolates from the same food source cluster together with a similarity ≥ 84%,whereas AFLP clusters grouping isolates from different sources show a similarityranging between 60 and 78%. Isolates belonging to the same AFLP cluster showcommon ribotypes and serotypes. In particular, isolates from meat based prod-ucts, such as fresh and fermented sausages, are mainly classified within EcoRI ri-botypes 14 S2 and 202 S2 and serotypes 1/2c and 1/2b. Furthermore, cheese prod-ucts are classified within the EcoRI ribotype 204 S5 and serotype 1/2a.

Zoonotic aspects of Listeria monocytogenes isolatesfrom zebu dairy animalsParihar, V. S. 1,3, Barbuddhe, S. B.1, Kalorey, D. R.2, Kotwal, S.4,Danielsson Tham, M.-L.3

and Tham, W.3

1. ICAR Research Complex for Goa, Ela, Old Goa, India2.Department of Microbiology, Maharashtra Animal and Fishery Sciences University, Nagpur, India3.Örebro University, Sweden4.Sher-e- Kashmir University of Agricultural Sciences & Technology of Jammu, J&K

Listeria monocytogenes is a non acid-fast, Gram-positive pathogen, which is con-sidered food- and feed-borne. Knowledge of the direct and indirect transmission ofL. monocytogenes between animals and humans, is however, limited. The aim ofthe current study was to characterize isolates of L. monocytogenes obtained fromzebu dairy animals with mastitis. Fifteen isolates of L. monocytogenes from thesame number of clinical and subclinical cases of mastitis were obtained from fivefarms harbouring altogether 90 zebu dairy animals. The isolates were character-ized by serotyping, multiplex PCR (plcA, hlyA, actA and iap genes) and pulsed-field gel electrophoresis (PFGE) using Asc Ι and Apa Ι enzymes. All the isolatescharacterized belonged to serovar 4b and exhibited four virulence associated genesof L. monocytogenes. PFGE revealed that all isolates belonged to the the samepulsovar. One of the reasons that all isolates belonged to the same pulsovar de-spite being from different farms may be that certain types of L. monocytogenes areadapted to specific niches. The presence of an identical pulsovar could also indi-cate a common source of infection for the different farms. All the farms deliveredmilk to the same dairy. The same pulsovar of L. monocytogenes was isolated fromzebu dairy animals with mastitis from five different farms.

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Performance of ALOA and Palcam agars for detection of Listeriamonocytogenes in naturally contaminated raw meat productsGravato Rowlands, R. E.1, Asturiano Ristori, C., Geraldes Martins, C.1,Jakabi, M.1 and Gombossy de Melo Franco, B. D.2

1. IAL, Brazil2.USP, Brazil

The correct isolation and identification of Listeria monocytogenes in foods heavilycontaminated with competing microorganisms may be difficult, especially whenL. monocytogenes is present in low numbers. ALOA (Agar Listeria Ottaviani &Agosti) is a selective chromogenic agar that minimises the growth of contaminat-ing organisms, and Listeria spp. grow on this medium producing blue/greencolonies, with pathogenic species producing a characteristic opaque halo after 24h at 37 °C. Non-Listeria spp. produce white colonies. The aim of this study was toevaluate the performance of ALOA for investigation of L. monocytogenes in 552samples of naturally contaminated raw meat products, comparing the results tothose obtained using the conventional Palcam agar. Meat samples were collected insupermarkets in the city of Sao Paulo, SP, Brazil, and comprised 138 beef sausages,138 pork sausages, 138 ground beef samples and 138 chicken leg samples. Usingthe ISO 11290-1:1996/Amd.1:2004 detection method, L. monocytogenes was presentin 234 (42.4%) samples. ALOA was more effective than Palcam agar for isolation ofL. monocytogenes (p ≤ 0.05): 82 samples (35%) were positive in ALOA only, 23(9.8%) in Palcam agar only, and 129 (55.1%) samples were positive in the twomedia simultaneously. When the ISO 11290-2:1998 enumeration method was used95 samples (17.2%) were positive for L. monocytogenes, and among them, 31 sam-ples (32.6%) were positive in ALOA only, 21 (22.1%) in Palcam agar only, and 43(45.3%) samples were positive in the two media simultaneously. However, countsof L. monocytogenes using the two media did not differ significantly (p > 0.05). Re-sults of this survey indicate that the use of ALOA both for detection and enumer-ation of L. monocytogenes increases the effectiveness of the two ISO methods whenraw meat products are tested.

Comparison of MOPS-BLEB and Fraser as secondary enrichmentbroths for Listeria monocytogenesUpham, J., Huszczynski, G., Mosher, M., Borza, A., Dorey, M., Bosley, J., Hara, K.,Mutanda, C., Liu, J., Byrne, B. and Douey, D.Canadian Food Inspection Agency

The two methods used by CFIA laboratories for detection of L.monocytogenes fromfoods employ bipartite selective enrichment procedures to increase pathogen con-centration prior to detection. The methods use the same bacteriological media forprimary enrichment but different secondary enrichment media. Secondary mediaMOPS-Buffered Listeria Enrichment Broth (MOPS-BLEB) and Fraser broth werecompared for their ability to facilitate growth of L. monocytogenes. Foods contam-inated with Listeria were subjected to a common primary enrichment followed byparallel secondary enrichment in MOPS-BLEB and Fraser broth. Listeria was iso-lated and enumerated by plating the broths on selective agar. Of 164 food samples,presumptive L.monocytogenes colonies were isolated from 164 using MOPS-BLEBand 162 using Fraser broth, demonstrating broth equivalence (p = 0.50, Chi-square). Quantitative analysis revealed significantly higher levels of L.monocyto-genes in MOPS-BLEB than Fraser broth (log10 CFU, 8.88 and 7.99, p < 0.0001 bypaired t test). Interestingly, for foods inoculated with a mixture of L.monocyto-genes and a non-pathogenic species of Listeria, L.innocua, concentrations of thelater were not significantly different in MOPS-BLEB compared to Fraser broth(log10 CFU, 8.85 and 8.47, p = 0.06), resulting in a higher ratio of L.monocytogenesto L.innocua in MOPS-BLEB. Enrichment broths used by CFIA were found to beequivalent for enabling cultural detection of L. monocytogenes, however, MOPS-BLEB produced significantly higher pathogen concentrations and pathogen tocompetitor ratios and thus has potential to enable earlier detection and facilitatedetection when competitors are present.

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Comparison of UVM, Palcam and Oxoid Novel Enrichment (ONE) brothas primary enrichment broths for Listeria monocytogenesUpham, J., Mosher, M., Borza, A., Huszczynski, G., Dorey, M., Eloranta, K. and Douey, D.Canadian Food Inspection Agency

The purpose of this study was to compare three primary enrichment broths forability to facilitate detection of Listeria monocytogenes in foods: 1) UVM I, 2) Pal-cam, and 3) a single-step enrichment broth from Oxoid Company, ONE broth.Media was directly inoculated with healthy or heat-stressed L. monocytogenes andincubated for up to 48 hr, and growth curves were constructed by diluting and plat-ing the enrichment cultures at regular time intervals to determine cell concentra-tion (cfu/ml). Growth curves were also prepared from media that had been used toenrich various food products (deli meats, dairy products, fresh produce andseafood) that were artificially contaminated with L. monocytogenes alone, or L.monocytogenes in the presence of the background organisms Escherichia coli andStaphylococcus epidermidis, or in the presence of a non-pathogenic Listeria species,L. innocua. In the absence of food matrices or competitive microorganisms, the lagphase of heat-stressed L. monocytogenes was significantly shorter in Palcam broth(6.5h) compared to UVM and ONE broth (each 7.8h), and the exponential growthrate was significantly greater in Palcam broth (0.371) and ONE broth (0.396) com-pared to UVM broth (0.277). Similar differences in growth rates were observedwhen broths were used to enrich L. monocytogenes-contaminated food products.When food were contaminated with a equal mixture of L. monocytogenes and L. in-nocua, the exponential growth rate of L. innocua was notably faster than L. mono-cytogenes in UVM and Palcam broth, whereas the opposite was true in ONE broth.In addition, the presence of L. innocua caused a decrease in L. monocytogenesgrowth rate in Palcam broth and to a lesser extent in UVM broth, but not in ONEbroth. These studies suggest that ONE broth may be preferable to UVM and Palcambroths to facilitate detection of L. monocytogenes when L. innocua is present.

Isolation and characterization of Listeria monocytogenes from asazuke(Japanese light pickles)Maklon, K., Kusumoto, A., Makino, S.-I. and Kawamoto, K.Obihiro Univ. Agri. Vet. Med., Japan

Asazuke is a ready-to-eat Japanese light pickle, mainly made of vegetables whichare known to be one of the sources of Listeria monocytogenes contamination. Al-though asazuke is a popular side dish in Japan, the hazard of bacterial contamina-tion has not been evaluated yet. In this study, we investigated the prevalence of L.monocytogenes, Salmonella spp., Escherichia coli O157 and coliforms in 108 asazukesamples that randomly collected from supermarkets in Obihiro (Hokkaido pre-fecture, Japan) during the period of June to November 2007. Twelve (11.1%) L.monocytogenes were isolated with predominant serotype 4b (7 isolates) followed by1/2a (2 isolates), 1/2b, 3b and 4c (1 isolate each) while Salmonella spp., E. coli O157and coliforms were not detected. All L. monocytogenes isolates demonstrated he-molytic activity by CAMP test and possessed all the virulence-associated genes(prfA, actA, mpl, inlA, inlC, plcA, plcB, hly, iap, clpC and opuCA) by PCR, those re-vealed their potential pathogenicity. Moreover, 7 out of 12 isolates were fromasazuke produced by one factory and pulsed-field gel electrophoresis (PFGE) pro-files suggested that 6 of them were indistinguishable. Additional investigation of L.monocytogenes contamination was performed in the asazuke factory environmentand 23 out of 60 swabs (38.33%) were isolated. Comparison of PFGE profilesshowed relatedness between food and environmental isolates. Taken together, ourresults indicated that asazuke was contaminated with L. monocytogenes, whichwas probably from vegetables, human sources or from production lines. Furtheranalysis to clarify the source of pathogen contamination is needed to establish aprevention method and consumers should be aware of asazuke consumption thatmight be contaminated with L. monocytogenes.

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Grouping of human Listeria monocytogenes isolatesLopez-Valladares, G., Danielsson-Tham, M.-L. and Tham, W.School of Hospitality, Culinary Arts & Meal Sciences, Örebro University, Grythyttan, Sweden

More than 800 clinical isolates of Listeria monocytogenes from humans in Swe-den were characterized by serotyping and pulsed-field gel electrophoresis. The re-striction enzyme used was AscI. The serovar 4b isolates were divided into 27 PFGEtypes, the serovar 1/2a and 1/2c isolates into 108, and the serovar 1/2b and 3b iso-lates into 24. PFGE types with identical profiles of small bands (< 145 kbp) weregrouped together with PFGE designations of A-Z. This grouping was based on theassumption DNA rearrangements involving insertions, deletions, or transposi-tions of genes are less common in the small fragments than in large fragments.The 27 serovar 4b PFGE types were further divided into 5 groups and the 108serovar 1/2a and 1/2c PFGE types into 16 groups. The serovar 1/2b and 3b types(n =24) were divided into five groups. Part of the collection of L. monocyto-genes isolates was analyzed with multiple-locus variable-number tandem-repeatanalysis and the clusters were generally in good agreement with both the clustersgenerated by PFGE and serotyping data. The advantage of dividing L. monocyto-genes PFGE types into groups is that the profile of every new isolate characterisedwith PFGE can easily and quickly be identified by studying the group affiliation ofthe isolate and then identifying the matching type within the group.

Characterization of Listeria monocytogenes strains isolated from foodand environmental samplesNucera, D. M.1, Lomonaco, S.1, Manila Bianchi, D.2, Decastelli, L.2, Grassi, M. A.1 and Civera, T.1

1. Università degli Studi di Torino, Italy2. Istituto Zooprofilattico del Piemonte Liguria e Valle d’Aosta, Italy

This study aimed to investigate type diversity and distribution over time andacross sources by subtyping via Pulsed Field Gel Electrophoresis (PFGE) a set of300 L. monocytogenes strains collected over a five year period. Five clinical humanstrains isolated in the same geographic area and period of the study were also in-cluded in the analysis. The isolated strains belonged to serotype 1/2a (45% of thesamples), 1/2c (22%), 4b/4e (16%); however, 5% of the strains were untypeable.Significant associations were observed between serotype 1/2a with dairy (O.R.=13.9; χ2 p < 0.05) and 1/2c with meat (O.R.= 33.3; χ2 p < 0.05). PFGE results high-lighted 152 pulsotypes shared among two or more samples: 9 pulsotypes were re-current and 6 were shared between strains isolated from different food and envi-ronmental sources. The other generated pulsotypes were source specific and/orretrieved in one year only. These findings may indicate the presence of both niche-adapted as well as ubiquitous PFGE types. Moreover, no PFGE types were sharedbetween strains collected from food/environmental isolates and human clinicalstrains. The results of this research underline and confirm the importance of im-plementing a broad typing database which could facilitate epidemiological inves-tigations and enhance novel food attribution modelling approaches for the identi-fication of listeriosis outbreaks and the related sources.These data, even if focusedon strains collected in a limited geographic area, may pose the ground to evidencehow large subtype databases may aid in the detection of common- and source spe-cific- types. However, more comprehensive databases, both at national and at Eu-ropean level, are needed to reveal L. monocytogenes strains diversity and to provideuseful data for epidemiological investigations.

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Development of a multiplex SNP-typing method to identify the fourepidemic clones of Listeria monocytogenesLomonaco, S.1, Civera, T.1, Dalmasso, A.1, Knabel, S. J.2 and Bottero, M. T.1

1. Università degli Studi di Torino, Italy2.The Pennsylvania State University, USA

The concept of epidemic clone (EC) is used to describe closely related strains of L.monocytogenes belonging to geographically and temporally distinct outbreaks. Cur-rently, four ECs have been identified: ECI, ECII, ECIII, and ECIV (also ECIa). Thecurrent classification of ECs is based on results from different subtyping techniquessuch as RFLP analysis, hybridization assays, PFGE and ribotyping. Recently, a mul-tiplex PCR able to identify ECI, ECII and ECIII was developed. In addition, se-quence-based methods, such as multivirulence-locus sequence typing, were ableto further subtype EC strains and also to highlight potentially informative singlenucleotide polymorphisms (SNPs). The aim of the present study was to develop analternative method for the identification of all four ECs by developing a SNP-typingmethod based on minisequencing. This technique simultaneously analyses severalSNPs based on a Primer-Extension Reaction (PER). Extension primers are designedimmediately adjacent to the SNPs and extended by a single [F]ddNTP. Results arethen visualized with a genetic analyzer as specific patterns. Six SNPs, yielding aspecific pattern for each EC, were selected. These SNPs were located on 4 virulencegenes (inlA, inlJ, inlB and srtA). Gene fragments were preliminary amplified with amultiplex PCR and then used as template for the PER reaction. Analyzed sampleswere 39 ECs strains, 25 strains from the ILSI Diversity Subset and 22 non EC-strains. All ECs gave a clone-specific pattern. Moreover, 9 strains not previouslyclassified as ECs showed the same profile as ECI, ECII and ECIV. These findingswere confirmed with other biomolecular tests (e.g. ECs-specific PCR) and by con-sidering available epidemiological data for these strains. The proposed SNP-typ-ing assay is potentially high-throughput and amenable to automation, allowing arapid identification of all four ECs and can therefore be used as a screening methodin the analysis of L. monocytogenes strains.

Listeria monocytogenes incidents reported to the UK Food StandardsAgency from 2000 to 2008Aish, J.Food Standards Agency, UK

The UK Food Standards Agency contributes to the management of a wide range offood related incidents involving microbiological, chemical, radiological and phys-ical hazards. One important foodborne microbiological hazard for which theAgency oversees incident investigations is Listeria monocytogenes. When incidentsare reported to the Agency the relevant data is recorded on the Agency’s IncidentsDatabase. Notification is received from a wide range of food businesses, Local Au-thorities, the Health Protection Agency and other Government Departments. In-formation held on the database was analysed to determine the number of inci-dents involving Listeria spp. that were reported to the Agency between 2000 and2008 and to identify that main food categories that were involved. During the nineyear period from 2000 to 2008 approximately 140 incidents caused by the con-tamination of food with Listeria spp. were reported to the Agency, the majority ofwhich involved L. monocytogenes. Most of the incidents involved ready-to-eatmeat/meat products, cheese, fish/shellfish and sandwiches/sandwich fillings. L.monocytogenes is an important foodborne pathogen. Although healthy people arenot usually at risk of illness, listeriosis can be an issue for vulnerable groups suchas pregnant women and the immunocompromised. In addition to overseeing inci-dent investigations the Agency’s 4C’s strategy (cleaning, cooking, chilling andavoiding cross-contamination) is important in reducing the risk from L. monocy-togenes. The Agency also provides food safety advice to vulnerable groups andcommissions research to further our understanding of this organism.

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Genetic diversity of Listeria monocytogenes in broiler flocksCourtillon, C. Toquin, M.-T., Le Nôtre, Y., Fravalo, P. and Mansour Chemaly, M.AFSSA, France

Listeria monocytogenes is a foodborne pathogen, persistent in industrial environ-ment and is responsible of listeriosis, an infectious and rare disease. The aim ofthis study was to update and create data set from broiler flocks regarding theircontamination by Listeria monocytogenes. 145 flocks have been sampled, 85 con-ventional and 60 free range flocks. Molecular characterization by PFGE with tworestriction enzymes ApaI and AscI was performed in order to establish the clonalrelationships of the isolates and to trace the contamination between the flocks. Atotal of 126 isolates were genotyped. The prevalence in broilers has been estimatedat 31.7% (46 positive samples on 145). The serotyping revealed the presence ofserotype 1/2a in majority: 50% vs. 31.7% for serogroup 4 (4e and 4b) and 18.3% for1/2b. Serotype 1/2a is the most present in conventional farms (37.3%) contrary tofree range farms (12.7%) where serogroup 4 is dominant (23% vs. 8.7%). PFGEtyping showed a high discriminatory index: 0.971 for both combined enzymes. 47genetic patterns were identified. Dendrograms, obtained with BioNumerics soft-ware, confirmed the high genetic variability between isolates (55.7% of similar-ity) especially in serotype 1/2a (65.5%). This index (0.954) was similar to the oneobtained when studying laying hen flocks. The serogroup 4 and the serotype 1/2bseemed more clonal, 0.892 and 0.798, respectively. Regarding the production sys-tem, results between conventional and free range farms are comparable, 0.959 and0.948, respectively. The PFGE showed that two identical profiles can be found indifferent flocks and different geographical locations while in a same flock, pat-terns can be very different. The breeding type is not linked to bacteria geneticidentity as are serotypes. In conclusion, this investigation brought new and up-dated data regarding Listeria monocytogenes in broiler flocks which were not avail-able before.

Tracing Listeria monocytogenes contaminations throughoutthe processing chain of a typical Italian pork meat productusing Pulsed Field Gel Electrophoresis (PFGE) characterizationAnnunziata Prencipe, V., Acciari, V., Torresi, M., Migliorati, G.,Marfoglia, C. and Valentina Rizzi, V.Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy

Pulsed Field Gel Electrophoresis (PFGE) and serotyping analyses were performedon 124 Listeria monocytogenes strains isolated from a prevalence study throughoutthe Parma dry-cured ham production, in order to evaluate contamination pat-terns. Strains had been isolated at three different points of the production process:I) fecal samples/carcasses in slaughterhouses; II) fresh hams after cutting; III)de-boned, dry-cured and packaged hams at the end of the production chain. Trace-ability to corresponding feces, fresh hams and dry-cured hams had been assuredfor all carcasses, so every strain was tested to evaluate the presence of contamina-tions in previous or next phases of the production process. Genetic relationships of56 different pulsotypes obtained from the combination of AscI and ApaI macrore-striction patterns were graphically represented and analyzed. Six different clusterswere identified. The analysis of PFGE patterns obtained from the same sampleexamined at the three different points of the production process (carcass – freshham – cured ham) did not produce identical or highly similar pulsotypes. The onlypulsotype occurring in a strain from faeces was not recovered from other materi-als. Strains from the same production plants were usually genetically homoge-neous or highly similar. In the plants where there was not genetic homogeneity,one pulsotype or cluster was always sharply prevalent over the others. The pres-ence of single or prevalent pulsotypes in the same processing plants could be as-sociated to persistent strains of Listeria monocytogenes. This is corroborated bythe highest prevalence of 1/2c serotype strains, which adhere significantly more toworking surfaces, isolated from all stages of the process. Genetically homogeneousor highly similar strains were also recovered in different establishments, at dif-ferent phases of the production process. This situation could be bounded to pre-vious sporadic contaminations between different production stages, which couldhave allowed the localization of strains into favorable niches inside the processingenvironment.

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Production and validation of capture-ELISA kit based on monoclonalantibodies specific for Listeria monocytogenes in foodstuffsPortanti, O., Di Febo, T., Luciani, M., Pompilii, C., Armillotta, G., Principe, V.,Lelli, R. and Semprini, P.Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy

A Listeria monocytogenes ELISA test was produced for use in the screening offoods. Validation was carried out according to the criteria for qualitative analysisset out in ISO 16140:2003. The ELISA kit has satisfied the norm requirements interms of relative accuracy, relative sensitivity, relative specificity, relative detec-tion level, inclusivity and exclusivity. The kit is a capture-ELISA test applied tothe sample enrichment broth, based both on a monoclonal antibody (9B8F7) toL. monocytogenes and a peroxidase-conjugated anti-Listeria monoclonal antibody(6F12C8). The method has been validated in meat, fish and dairy products bymeans of incurred and artificially contaminated samples with target and non tar-get strains. Results for relative accuracy, relative sensitivity, relative specificityand relative detection levels are in agreement with ISO 11290-1:1996 requirements.Capture-ELISA test is able to detect Listeria with 100% inclusivity and exclusivityin the above mentioned matrices. Capture-ELISA kit was produced in a ready-to-use format and can be adopted by microbiological labs that need fast analyticalmethods. It also shows the same features as the internationally recognised ISO11290 techniques, as required by UNI EN ISO 17025:2005.

Production of a reference material for microbiological testscontaining Listeria innocuaPomilio, F., Ricci, L, Di Giannatale, E., Semprini, P., Candeloro, L. and Migliorati, G.Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy

The study describes the production of a reference material constituted by glassballs, covered by skimmed milk powder micronized and artificially contaminatedwith Listeria innocua (ATCC 33090 strain). The lot of prepared material, consti-tuted by 1g unities of balls, has been made in test-tubes, submitted to a stabilityand homogeneity evaluation through the determination of the mesophilic bacter-ial count and following Listeria innocua identification. The material stability, keptat –20 °C, has been evaluated at 54, 180 and 355 days. Data pointed out the de-creasing tendency of the bacterial count, statistically significant, at 54 days. At 180and 355 days, the lot resulted stable. Regression analysis at 54 days (t-value -2,67)has underlined a significant decreasing trend (p-value < 0.05). In the observed pe-riod the decrease has been equal to 8.75 UFC/g. Samples examined in the sametime lapses resulted homogeneous in the interval between 180 and 355 days. Onlya sample out of the 105 examined resulted negative. After 355 days of preserva-tion at –20°C, the challenge test has been carried out at 5 different temperatures(-20°C; +4°C; +22°C; +30°C; +37°C). The material evaluated with non-parametrictests, for independent samples resulted stable at -20°C and +4°C. Furthermore, forthe reference value definition, a proficiency test has been organized, which al-lowed to assign the value of 3.2 UFC/g to the material. In the same study, 35 sam-ples out of 244 (15.4%) resulted negative. Used methodology could be applied toreference materials production also on other micro-organisms. The adopted tech-nology has the benefit of operating in “closed” systems, making bio-safety of op-erators and environment easily manageable.

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AREA //D // Strategies for prevention and control of Listeria monocytogenesPOSTER PRESENTATIONS // P / 126–177, 187–198

Colonization of a newly constructed commercial chicken furtherprocessing plant with Listeria monocytogenesBerrang, M. E.1, Meinersmann, R.1, Frank, J.2 and Ladely, S.1

1. USDA-Agricultural Research Service, USA2. University of Geórgia, USA

This study was undertaken to determine potential sources of Listeria monocyto-genes in a newly constructed chicken further processing plant and document theeventual colonization of the facility by this pathogen. To ascertain the colonizationstatus of the plant, floor drains were sampled after a production shift and againafter a clean up shift on a roughly monthly basis for 21 months. Potential sources ofL. monocytogenes to the plant included: incoming raw meat, incoming fresh airand personnel. Nearby environment and community samples were also examined.All L. monocytogenes detected were subjected to DNA sequence based subtyping.L. monocytogenes was not detected in the plant before the commencement of pro-cessing operations. Within four months, several subtypes of L. monocytogenes weredetected in floor drains both before and after cleaning and sanitizing operations.No L. monocytogenes was detected on filters for incoming air, samples associatedwith plant employees, or a nearby discount shopping center. One subtype of L.monocytogenes was detected in a natural stream near the plant however, this sub-type was never detected inside the plant. Eight subtypes of L. monocytogenes weredetected in raw meat staged for further processing; one of the raw meat subtypeswas indistinguishable from a persistent drain subtype recovered after cleaning oneight occasions in four different drains. Poultry further processing plants are likelyto become colonized with L. monocytogenes; raw product is an important source ofthe organism to the plant.

Episcopic differential interference contrast/epifluorescencemicroscopy to characterise in situ Listeria monocytogenes biofilmson stainless steel surfacesGião, M. S. and Keevil, C. W.University of Southampton, UK

Listeria monocytogenes is a Gram-positive pathogen associated with the contami-nation of several food products, including meat, dairy products and vegetables. Al-though this pathogen can be found in natural environments the contamination ofprocessed food can occur after contact with contaminated surfaces. Previous stud-ies routinely focused on biofilm recovery and culture quantification. In situ stud-ies utilised mostly SEM which is tedious, expensive and known to introduce arte-facts. By contrast episcopic differential interference contraste/epifluorescence(EDIC/EF) microscopy is a novel and rapid light microscopy technique ideallysuited to study biofilms in a native state. The aim of this work was to utilise the ad-vances in EDIC/EF microscopy to study the formation of L. monocytogenesbiofilms under different nutrient and temperature conditions. L. monocytogenesNCTC 13372 was suspended in Brain Heart Infusion (BHI), Tryptone Soya Broth(TSB), TSB supplemented with 0.6% of Yeast Extract (TSBYE) and filter-sterilisedtap water. Five ml of each suspension were transferred for 6 well plates containing1 cm2 stainless steel coupons and incubated at 4 °C, 22 °C, 30°C and 37°C. After 4and 24 hours 2 coupons were removed for each tested medium. One coupon wasstained in situ with SYTO 9 and observed under EDIC/EF microscopy and theother coupon was scraped and total and cultivable cells quantified by staining withSYTO 9 and plating onto BHI agar, respectively. Results showed that even for thelowest temperature, after 4 hours of incubation L. monocytogenes biofilms werealready developing on the surfaces. Except in the case of biofilms formed in tapwater where cells were in general less cultivable, the different media do not influ-ence significantly the formation of biofilm on stainless steel surfaces. Conversely,the in situ observation of the coupons demonstrated that the morphology of thebiofilm is temperature dependent with implications for food processing.

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Temperature dependent defect in biofilm formationby Listeria monocytogenesAbdalla, S.1, Glenn, S.1, Shama, G.2 and Andrew, P.1

1. Department of Infection, Immunity and Inflammation, University of Leicester LE1 9HN, UnitedKingdom

2. Department of Chemical Engineering Loughborough University, Loughborough, LE11 3TU, UnitedKingdom

Listeria monocytogenes can cause major problems in food processing industries be-cause of its ability to survive and grow over a wide range of environmental condi-tions. Furthermore L. monocytogenes can attach to and produce biofilm on widevariety of different surfaces in the food-processing environment, allowing the bac-terium to resist to antimicrobial and sanitizing agents. The aims of this study wereto investigate the ability of Listeria monocytogenes wide type strain 10403s andTn917-LTV3 transposon mutants to attach to abiotic surface at a variety of tem-peratures. A published attachment assay was modified to compare the ability of theWT strain and a number of transposon mutants to attach to polystyrene surfacesover 2 hours incubation. Attached cells were detected using crystal violet staining.Certain transposon mutants were shown to have a significant reduction in the levelof attachment, but this defect was temperature dependent, being evident at 30°Cand 18°C but not at 37 °C. The identity of the transposon insertion site in thesemutants is being actively pursued. It was concluded that there is a temperature de-pendent involvement of some gene production in surface attachment.

Mode of action of Lactococcus lactis Sa31 antimicrobial peptideon Listeria monocytogenes ½ cBarile, M.1, Mormile, A.1, Ceres, C.1, Pepe, O.2, Cortesi, M. L.1 and Murru, N.1

1. DISCIZIA, Faculty of Veterinary Medicine, Napoli, Italy2. Faculty of Agraria Università degli Studi di Napoli, Italy

Lactic acid bacteria produce antimicrobial peptides referred to as bacteriocinsthat have potential as natural food preservatives. Lactococcus lactis Sa31, isolatedfrom gilthead breams fillets packaged in modified atmospheres, normally pro-duces bacteriocin in GM17 broth with maximum activity (2.560 AU/ml) at 11h inthe late logarithmic to early stationary phase of growth. In the present work wasinvestigated the Sa 31 ability of bacteriocin production in different liquid media,the partial characterization and mode of action of peptide against Listeria mono-cytogenes ½ c strain. Overnight extracts of producer strain cultures in TriptoneSoya Broth, M17 + 0.5% glucose (GM17) and 0,5% lactose (LM17), MRS, and BHIbroth was tested against Listeria monocytogenes ½ c. To evaluate the mode of ac-tion, the crude (5.120 AU/ml-1) and partially purificated bacteriocin (10.240AU/ml-1) by precipitation with ammonium sulfate were inoculated in suspensionof 50 mM potassium phosphato buffer pH 7.0 containing Listeria monocytogenes½ c (1.4 x108 CFU/ml). Finally, for absorption study of partial purificated bacte-riocin, this was added at 10.240 AU/ml on cell of Listeria monocytogenes ½ c re-suspended in 100 mM NaCl pH 7.0 incubated for 30 min. at 20 °C. The residualbacteriocin activity was tested as described above. The strain Sa 31 was able toproduce bacteriocin in BHI broth with maximum titre 5.120 AU/ml-1 after shortincubation time. The bacteriocin, sensitive to heat and more powerful at acid pH,exhibited a bactericidal mode of action and acted rapidly. The death of Listeriamonocytogenes ½ c cells was observed within 10 min. after treatment with 10.240AU/ml of bacteriocin (reduction in viable counts from 1.4x108 to 6.5x105 CFU/ml).The bacteriocin was completely absorbed on the sensitive strain L. monocytogenes½ c at pH 7.0. These results demonstrate the effectiveness of this bacteriocin andits producer as an inhibitor of Listeria monocytogenes in vitro, and the possibility oftheir applying in a food system where post-manufacture contamination by thisorganism could be problematic as in fishery products as well as in other foods.

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Tracing the source, epidemiology and persistence ofListeria monocytogenes in Irish Farmhouse cheese processing facilitiesJordan, K., Fox, E., O’Brien, M. and Hunt, K.Teagasc, Ireland

Many outbreaks of listeriosis have been related to dairy products, although thesource of contamination has rarely been determined. Sixteen farmhouse cheese-making facilities were sampled at monthly intervals over a two-year period. Sam-ples included raw materials, food contact surfaces, non-food contact surfaces, farmsamples and product. In order to determine the source, persistence and epidemi-ology of the strains obtained from 13 cheesemaking facilities, isolates of L. mono-cytogenes were differentiated using PFGE and serotyping. At 9 of the 13 contami-nated processing facilities farm samples were the source of the environmentalcontamination. We could not determine the source of the contamination at theremaining 4 facilities. Persistent isolates (similar isolates obtained at the same fa-cility for > one year) were found at two cheesemaking facilities. Reduction of theoccurrence of L. monocytogenes in milk could reduce the contamination of cheeseprocessing facilities and therefore reduce the risk of cheese contamination. Cheeseproducers can also take steps to reduce risk by reducing milk spillage at milk in-take and sterilising any area where milk was spilt. In addition, barriers between thefarm and processing facility will help reduce the risk of contamination.

Phenotypic and genotypic characteristics of Listeria monocytogenesstrains isolated from a convenience food-processing plantBlatter, S., Stephan, R., Tasara, T. and Zweifel, C.Institute for Food Safety and Hygiene, University of Zurich, Switzerland

L. monocytogenes as a food-borne pathogen has significant public health and eco-nomic impacts. Amongst other foods, ready-to-eat products have been implicated asroutes for human infection. The aim of this study was to investigate the occurrenceand genetic diversity of L. monocytogenes isolated from the environment and foodproducts in a Swiss sandwich-producing plant. Over a 12-month period, environ-mental swabs (n=2.028) collected during the working activity and after cleaningand disinfection, as well as ingredient and sandwich samples (n=217) were col-lected. Swabs originated from the processing room and the equipment of five sand-wich production lines. Isolation of L. monocytogenes was accomplished by cultureafter enrichment (Half-Fraser/Fraser broth, Palcam and Ottaviani-Agosti agar).Isolated strains were characterized by serotyping, determination of genetic line-ages, Rep PCR typing, and PFGE analysis after macrorestriction. L. monocytogeneswere detected in 70 (3.5%) environmental swabs and 16 (7.4%) samples from in-gredients and sandwiches. Of the positive environmental samples, 40% originatedfrom three slicers, which tested repeatedly positive on several occasions. Of the 86isolated L. monocytogenes strains, 93% belonged to serotype 1/2a and genetic line-age II, whereas the remaining strains were of serotype 1/2b and genetic lineage I.Genotyping by Rep PCR and PFGE analysis yielded each six different profiles. Fifty-eight (82.9%) strains from the processing environment and 9 (56.3%) from ingre-dients and sandwiches belonged to only one genotype. Strains of this genotype werefound repeatedly on/in slicers, tables, conveyor belts, tables, a bread-feeding ma-chine, spattles, air guns, salmon and egg sandwiches. Based on the genotyping re-sults, indications of persistent contamination of the sandwich processing area witha predominant strain were evident over the whole sampling period. Moreover, infood-processing plants with low Listeria prevalence in the final product, environ-mental monitoring is more effective than end product testing.

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Influence of flow direction on the adhesion of Listeria monocytogenes tobrushed stainless steel surfacesSkovager, A.1, Whitehead, K.2, Ingmer, H.3, Verran, J.2 and Arneborg, N.1

1. Department of Food Science, Food Microbiology, Faculty of Life Sciences, University of Copenhagen,Denmark

2. Division of Biology, School of Biology, Chemistry and Health Science, Faculty of Science andEngineering, John Dalton Building, Manchester Metropolitan University, Manchester, England

3. Department of Veterinary Disease Biology, Section for Microbiology, Faculty of Life sciences,University of Copenhagen, Denmark

The ability of Listeria monocytogenes to form biofilms on the surfaces of processequipment constitutes a major health problem for the food industry. When thebacteria have succeeded in forming a biofilm, they are difficult to remove fromthe surface, due to their strong adhesion on the processing equipment. The inner-most cells of a biofilm are also protected against the action of cleaning- and disin-fection agents. It is therefore extremely important to target the bacteria beforethey adhere to the solid surface and develop into a biofilm. One factor which mayinfluence the adhesion of L. monocytogenes is the surface characteristics of pro-cessing equipment. In the present work, a method using fluorescence microscopyand a perfusion system will be developed to examine the initial adhesion of singleL. monocytogenes cells to steel surfaces. Results will be presented showing the in-fluence of flow direction on the adhesion of L. monocytogenes to brushed stain-less steel surfaces.

Occurrence of Listeria monocytogenes in raw milk and dairy productsin Kazerun, IranMehdi Mahmoodi, S. M. and Javanmardi, F.Islamic Azad University – Kazerun Branch, Iran

The presence of Listeria monocytogenes was investigated in a total of 360 raw milkand dairy product samples including white cheese, yoghurt and Iranian yoghurtdrink (Doogh) that were collected during five months from two traditional dairymanufacturer in Southern Iran. The prevalence of Listeria monocytogenes in rawmilk and white cheese samples of manufacturer A was found to be 1.7% and 3.3%respectively and of manufacturer B was found to be 3.3% and 6.7% respectively.No Listeria monocytogenes was isolated in any of the yoghurt and Doogh samples ofboth manufacturer probably because of their low pH values.

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Antilisterial mode of action of bacteriocin ST182Gu producedby Enterococcus casseliflavus isolated from guavaTodorov, S.1*, Destro, M. T.1, Chiarini, E. B.1, Vaz-Velho, M.2 and Franco, B. D. G. M.1

1. University of Sao Paulo , Brazil2. ESTG, Viana do Castelo , Portugal

This research is on the anti-Listeria bacteriocin ST182Gu and studies its mode of ac-tion. Bacteriocins of lactic acid bacteria (LAB) are ribosomally synthesized antimicro-bial peptides. Their bactericidalmechanismsmay includepore formation, degradationof cellular DNA, disruption through specific cleavage of 16S rDNA, and inhibition ofpeptidoglycan synthesis. Inourknowledge this is the first report of the isolationofbac-teriocinogenic strain of Enterococcus casseliflavus from guava. Enterococcus cas-seliflavus ST182Gu, produces a pediocin-like bacteriocin (based onhighly homology ofPCRproduct generatedwith the primers targeting pediocinPA-1 structural gene)withactivity against several LAB, Listeria spp, and some other human and foodbornepathogens.Nosignificantdifferences ingrowthandproductionofbacteriocinST182Guwere observed when the strain E. casseliflavus ST182Gu was cultured for 24h in MRSbroth at 20°C, 30°C or 37°C. At this 3 different cultivation temperatures activity was1.0x109AU/mlagainstL. ivanovii subsp. ivanoviiATCC19119and2.6x104AU/mlagainstEnterococcus faeciumATCC19443.Low levels of bacteriocinST182Guactivity (approx-imately 1.3x104AU/ml against L. ivanovii subsp. ivanovii ATCC19119 were recordedafter3hof growth inMRSbrothat37°Cand themaximalbacteriocinST182Guproduc-tion (1.0x1010AU/ml) was recorded in MRS after 27–33h of growth. A decrease in ac-tivity was observed after 33h of growth and it was reduced to 1.0x108AU/ml at 48h ofincubation. Addition of bacteriocin ST182Gu to exponential or stationary phase cul-tures of L. ivanovii subsp. ivanovii ATCC19119 inhibited growth for 24h. The effect ofbacteriocinST182Guoncells ofL. ivanovii subsp. ivanoviiATCC19119wasvisualisedbyAFM and indirectly recorded based on enzyme, protein and nucleotide material leak-age. Bacteriocin ST182Gu is highly active against L. ivanovii subsp. ivanovii ATCC19119and exhibits a synergetic effect in the inhibition of this surrogatemicroorganismwhenapplied togetherwith subletal doses of ciprofloxacin.


Control ofListeriamonocytogenes in fresh goat cheese by bacteriocinogenicstrain Lactococcus lactis subsp. lactisDF4Mi or commercial nisinNader Furtado, D., Todorov, S.*, Landgraf, M., Destro, M. T. and Franco, B. D. G. M.University of Sao Paulo , Brazil

Bacteriocins are antimicrobial peptides produced by different groups of bacteria.Foods can be supplemented with bacteriocins, or by inoculation with the bacterio-cinogenic strains, favouring the in situ production of the bacteriocin. The aim of thisstudy was to evaluate the capability of the bacteriocinogenic strain Lactococcus lac-tis subsp. lactis DF4Mi, isolated from goat milk, to control the growth Listeria mono-cytogenes in fresh goat cheese over 10 days of storage under refrigeration, and tocompare the effect that achieved in cheese added of commercial bacteriocin (nisin).Cheeses were prepared with 10 liters of pasteurized milk acidified with lactic acid(2.5% v/v) and added of calcium chloride and commercial rennet, according to thecheese manufacturer practice. Three sets of cheeses were prepared: one with pas-teurized milk with starter culture strain DF4Mi (106 UFC/ml), second with bacteri-ocin (-) strain of L. lactis subsp. lactis and other with addition of 12.5mg/l of purenisin (Fluka). L. monocytogenes 701 was added to cheeses during the manufacturing,in order to obtain 103 UFC/g. Appropriative controls such as cheese without anystarter cultures, only L. monocytogenes 701, only strain DF4Mi or only bacteriocin (-) L. lactis subsp. lactis were prepared. Cheeses were stored at 8°C and submitted tocounts of lactic acid bacteria and L. monocytogenes every other day for 10 days pe-riod. Results indicate that L. monocytogenes decreased 3-log in the cheeses contain-ing either bacteriocinogenic L. lactis subsp. lactis DF4Mi or bacteriocin (-) L. lactissubsp. lactis, showing that inhibition of L. monocytogenes might have occurred dueto another factor than the production of bacteriocin. Analysis of the cheeses con-taining nisin demonstrated that incorporation of this bacteriocin at 12.5mg/l couldinhibit the growth of L. monocytogenes allowing a decrease of 2-log counts duringstorage. The present study showed that the use of purified bacteriocin in fresh goatcheese is more effective in the control of L. monocytogenes than the direct applica-tion of the bacteriocinogenic L. lactis subsp. lactis DF4Mi.


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High pressure processing ensures elimination of Listeria monocytogenesin sliced dry cured hamStollewerk, K., Jofré, A., Comaposada, J., Aymerich, T., Ferrini, G., Arnau, J. and Garriga, M.IRTA, Spain

High pressure processing (HP) is a non-thermal treatment which inhibits or inac-tivates microorganisms and it is used to extend the shelf life of food products with-out altering their sensorial properties. Listeria monocytogenes can contaminatedifferent types of meat products, such as dry cured ham, during slicing. The aim ofthe present study was to evaluate the behavior of L. monocytogenes in three typesof boned dry cured ham (H1, H2 and H3) produced following different processes.H1 was tumble salted and smoked; H2 was brine salted and fermented; H3 wasbrine salted, fermented and smoked. Afterwards, slices of ham were spiked with amixture of 3 strains of L. monocytogenes (ca. 100 CFU/g). Drying was performed bythe Quick Dry Slice process®. Pairs of slices were vacuum packed and half of themwere submitted to a HP treatment of 600 MPa for 5 min. Packages were storedunder refrigeration for 112 days and the evolution of the pathogen was followed byplating in cromogenic agar and species-specific real time PCR. During storage ofnon-pressurized hams the levels of L. monocytogenes progressively decreased inthe three types of ham recording absence in 25g in H3 after 112 days. Pressuriza-tion produced an immediate reduction of L. monocytogenes to levels below theplate detection limit (10 CFU/g) in all the hams. In H3 the pathogen was absentduring the whole storage. After 112 days L. monocytogenes was not detected in anyof the samples (absence in 25g). The composition and processing of dry cured hamaffected the survival of L. monocytogenes. Fermentation and smoking speed up thedecrease of the pathogen, but only the application of a HP treatment enabled toachieve a product free of L. monocytogenes during the whole storage period.

Inhibition of Listeria monocytogenes by Lactococcus spp. EU2241in tropical shrimpAbdoulaye Fall, P.Ifremer, France

Shrimp is one of the most marketed seafood in the world. Due to its physicochem-ical parameters this product is very sensitive to microbial growth such aspathogens and spoiling microorganisms. Growth of these undesirable flora is oftendelayed by the use of classical techniques such as salting, low storage tempera-ture, modification of atmosphere packaging or use of preservatives. However thehuman pathogen Listeria monocytogenes (Lm) that was evidenced in raw and readyto eat shrimp by some studies can often grow in those conditions and may reachthe tolerated level of 100 Lm g-1. Biopreservation, an alternative technology forfood preservation which consists in inoculating a product with selected bacteria toeliminate or prevent growth of undesirable microorganisms, may be useful againstLm. In a previous study, a strain of Lactococcus spp. EU2241 had been isolatedfrom raw salmon and showed capacities to inhibit in model medium somepathogens isolated from seafood product. In this study the inhibition capacity ofLactococcus spp. EU2241 was tested in shrimp artificially contaminated with Lm.Four batches of cooked shrimp were prepared. 1: sterility control; 2: inoculationwith Lactococcus spp. (106 CFU g-1); 3: inoculation with Lm (103 CFU g-1); 4: co-in-oculation of Lactococcus and Lm. Shrimp were stored at 8 °C for 31 days undermodified atmosphere (CO2/N2 50-50). When inoculated alone, Lm grew very wellin shrimp reaching its maximum level, 109 CFU g-1, in 10 days. An immediate in-hibition of Lm was observed in co-inoculated batch, reaching more than 4 logs atthe end of storage with no sensory changes, confirming the interest of this bac-terium for an application in biopreservation of seafood products against Lm. Theuse of Seafood Spoilage and Safety Predictor software to predict the growth of Lmin shrimp, showed that the lactic acid produced by Lactococcus spp. (2500 ppm)and pH decrease from 6.60 to 5.91 could not totally explain the inhibition observedin this study. A chemically defined liquid medium is currently developed to studythe inhibition mechanisms.

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Listeria monocytogenes and Listeria innocua in slaughter line of a swinemeatpacking plant in Rio Grande do Sul State, BrazilSchittler, L.1 and Padilha Silva, W.2

1. UDESC, Brazil2. UFPeL, Brazil

Listeria monocytogenes is bacterium widely distributed in nature which presentshigh capacity to colonize surfaces and form biofilms in food processing plants. It isthe causative agent of listeriosis which may cause a range of diseases from gas-troenteritis to death, being the meat an important vehicle for transmission to hu-mans. The aim of the present study was to investigate the presence and charac-terize L. monocytogenes and Listeria innocua in the slaughter line of a swinemeatpacking plant, located in Missões Region, state of Rio Grande do Sul, Brazil.For this was evaluated water, cold chambers, carcasses, knives, faeces, hands anddrains and the strains obtained were serotyped and submit to molecular typingthrough the analysis by random amplification of polymorphic DNA (RAPD) withUBC 155 e UBC 127 primers. The occurrence of L. monocytogenes was of 0.95%and of L. innocua, 19.4%, in 105 samples assayed, being the cold chambers themajor point of isolation. Most of the L. innocua strains were not serotypable and,from those serotypable, the serovars 6a and 6b were prevalent. Only one L. mono-cytogenes strain was isolated, from a drain, however it belonged to serovar 4b, animportant serovar in public health, because it is frequently involved in listeriosis inhumans. RAPD of L. innocua strains produced 15 genetic combined profiles, whoseindex of discrimination was D=0.94, whereas the serotyping presented low dis-criminatory power (D=0.46). There is contamination by L. monocytogenes e L. in-occua in the slaughter line of the swine meatpacking plant assayed, and the crosscontamination in this plant is considered important, since some L. innocua strainspersist in the local.

Listeria monocytogenes in raw meat products marketed in the cityof Sao Paulo, Brazil: Incidence and counts data for risk assessmentRistori, C. A.1, Rowlands, R. E. G.1, Martins, C. G.1, Fávero, L. M.2 and Franco, B. D. G. M.3

1. Food Microbiology Laboratory, Adolfo Lutz Institute, Sao Paulo, SP, Brazil2. Food Surveillance, COVISA, Sao Paulo, SP, Brazil3. Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University

of Sao Paulo, Sao Paulo, SP, Brazil

Listeria monocytogenes is a ubiquitous bacterium frequently found in meat prod-ucts. Despite abundance of results on the prevalence of L. monocytogenes in thesefoods in different countries, quantitative data on level of contamination requiredfor appropriate risk assessments are scarce. This study aimed to evaluate theprevalence and numbers of Listeria monocytogenes in a variety of meat productsavailable in the local market in the city of Sao Paulo, SP, Brazil. Between May 2008and July 2009, 552 samples of refrigerated raw meat products (138 beef sausages,138 pork sausages, 138 ground beef samples and 138 chicken leg samples) werepurchased in 138 local supermarkets, selected after stratification by region and byrandom draw. Tests for presence and enumeration of L. monocytogenes were basedon ISO 11290-1:1996/Amd.1:2004 and ISO 11290-2:1998 methods, respectively. L.monocytogenes was detected in 48.7% of the of meat products samples. The highestprevalence occurred in ground beef (59.4%) followed by chicken legs (58%), beefsausages (37.7%), and pork sausages (39.8%). In most samples (67.4%), the level ofcontamination was lower than 102 CFU/g. Counts ranged from < 10 to 5.6x102

CFU/g in pork sausages, from < 10 to 1.9x102 CFU/g in beef sausages, from < 10 to8.9x102 CFU/g in chicken legs, and from < 10 to 4.5x103 CFU/g in ground beef. De-spite the low counts, data on prevalence of L. monocytogenes are relevant for esti-mating the risks of listeriosis associated to consumption of meat products in SaoPaulo, and for establishing science-based intervention strategies aimed at reducingthese risks, specially for pregnant women and immunocompromised individuals.

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Inhibiton of Listeria monocytogenes by Carnobacteriummaltaromaticum in combination with extract of Lippia sidoides Cham.in cold-smoked surubim fish brothBarbosa dos Reis, F., de Souza, V. M., Sousa Thomaz, M. R., Pinto Fernandes, L.,Pereira de Oliveira, W. and Pereira De Martinis, E. C.Faculdade de Ciências Farmacêuticas de Ribeirão Preto - Universidade de São Paulo, Brazil

Consumption of refrigerated cold-smoked fishes offers risk of listeriosis, the se-vere foodborne infection caused by Listeria monocytogenes (Lm). In this work, toimprove the safety ready-to-eat fish, multiple hurdle experiments were carriedout with cold-smoked surubim broth (CSSB) stored at 5 °C for 5 weeks. To inhibitLm, it was evaluated the application of a hidroalcoholic extract of the Brazilianfolk plant Lippia sidoides (ELs) (10 µl/ml) in combination with Carnobacteriummaltaromaticum (Cm) C2 and A9b+ (bacteriocinogenic strains) and A9b- (non-bacteriocinogenic). CSSB was inoculated with 102 CFU of Lm/ml and/or 105 CFUof Cm/ml, as follow: 1) Lm; 2) Cm C2; 3) Cm A9b-; 4) Cm A9b+; 5) Lm plus ELs; 6)Cm C2 plus ELs; 7) Cm A9b- plus ELs; 8) Cm A9b+ plus ELs; 9) Lm plus Cm C2; 10)Lm plus Cm A9b-; 11) Lm plus Cm A9b+; 12) Lm plus Cm C2 plus ELs; 13) Lm plusCm A9b- plus ELs; 14) Lm plus Cm A9b+ plus ELs. Bacterial populations and bac-teriocin titer were determined after 24 h and once a week for up to 35 days. Con-trol with Lm only, reached ca. 108 CFU/ml, but in co-culture with Cm, listerialpopulation was significantly lower (ca. 104 CFU/ml with Cm A9b+ or Cm A9b- and< 102 CFU/ml with Cm C2). Bacteriocin production was observed only for Cm C2alone and Cm C2 plus Lm, which may explain the stronger inhibitory effect of CmC2. ELs only did not inhibit Lm (ca. 108 CFU of Lm/ml) and its use in combinationwith any Cm strain did not present synergistic effect (ca. 104 CFU of Lm/ml). Bac-teriocin production by Cm C2 was not detected in the presence of ELs, suggestingthat ELs can adversely affect the protective culture and decrease the effectivenessof the biopreservation system studied.

Inhibition of Listeria monocytogenes in cooked hamby virulent bacteriophages and protective culturesHolck, A., Schirmer, B. C. and Berg, J.Nofima Mat AS, Norway

Listeria monocytogenes is found in a number of raw and ready-to-eat (RTE) prod-ucts, poultry, seafood and dairy products. RTE products like cooked ham have lit-tle inherent stability against the growth of L. monocytogenes. Several studies showreduction of L. monocytogenes in food when exposed to phages. Phage particles,however, become immobilised and are inactivated soon after addition to nonliquidfoods and can thus not prevent later outgrowth of surviving Listeria. Here we haveshown that protective cultures can be used successfully as an additional hurdletogether with phages to reduce growth of L. monocytogenes. Sliced cooked hamwas inoculated with cold adapted L. monocytogenes and exposed to bacteriophages.The hams were subsequently inoculated with Lactobacillus sakei TH1 protectiveculture, vacuum packed and stored at 10°C. Addition of phages gave an immediate1-2 log reduction in L. monocytogenes. After 14 – 28 days of storage, an additional 2log reduction was observed in samples with phages and protective culture com-pared to samples with phages alone. The effect of the protective culture was evi-dent both at 10 and 4 °C. Higher inoculation levels of protective culture gave astronger inhibition of L. monocytogenes. The use of phages in combination withprotective cultures is a general method that can potentially be applied to differentproducts where there is a risk for growth of L. monocytogenes.

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Surface colonization by Listeria monocytogenes: role of the flagellain the biofilm formation processDesvaux, M., Briandet, R., Renier, S., Deschamps, J., NCaccia, N., Chafsey, I. and Hébraud, M.INRA, France

The presence, contribution and/or requirement of exopolysaccharides in thebiofilm formation process in Listeria monocytogenes have not been definitively as-certained. So far, the flagellum is the only cell surface factor demonstrated as in-volved in biofilm formation within this species, even so its exact contribution androle remain controversial. This prompted us to reinvestigate the role of the fla-gella in the course of biofilm formation at growth temperatures relevant to regu-lation of genetic expression in L. monocytogenes using both static and dynamiccultures conditions. Biofilm formation was assayed at early and late stages of theprocess using paramagnetic microbeads and crystal violet methods. Cell mor-phology and biofilm architecture were determined by confocal scanning laser mi-croscopy using bacterial cells expressing fluorescent markers. Cells were grownstatically in microtiter plates and in dynamic conditions using flow cells. As re-vealed by proteolytic treatments, extracellular/cell-surface proteins rather thanexopolysaccharides are crucial determinants involved in listerial biofilm forma-tion. While L. monocytogenes was enable to swim at 20°C, no swarming could beobserved. Kinetics of biofilm formation at 20 and 40 °C revealed that unflagel-lated mutant strain formed more biofilm than the wt except at the highest growthtemperature tested. Besides crystal violet method, such results were confirmedby confocal microscopy in static and dynamic cultures conditions using fluores-cently labelled listerial cells. Biofilm formation was for the first time investigatedover a range of growth temperatures relevant to regulation of genetic expression inL. monocytogenes species. The use of complementary growth conditions, i.e. staticand dynamic cultures, allowed providing a comprehensive picture of biofilm for-mation in this species. In those tested conditions it clearly appeared that flagellaare not essential but rather detrimental to biofilm formation in L. monocytogenes.

The role of sanitizers in controlling Listeria monocytogenes on stain-less steel surfaces: Lessons learned from the 2008 Listeriosis outbreakHébert, K., Farber, J. and Pagotto, F.Health Canada

Proper maintenance and sanitization of food-contact and non-food-contact sur-faces remains a high priority for industry in ensuring a microbiologically safe finalproduct. Listeria monocytogenes is often present in factory environments andneeds to be controlled to avoid contamination of final product. The national out-break of listeriosis caused by contaminated deli-meat raised questions about theability of Listeria to survive on stainless steel surfaces. We evaluated the effec-tiveness of 11 sanitizers previously and currently used by Canadian industriesagainst strains of L. monocytogenes. Eleven sanitizers were evaluated using theAmerican Society for Testing and Materials standardized method (E2197) usingstainless steel disk coupons, according to the recommendations on the labels orfrom the manufacturer. A soil load consisting of a microbial suspension, BSA,mucin and tryptone was used to mimic environmental conditions. Bacteria wereinoculated at 107 CFU per coupon, dried and challenged against 50 µl of the sug-gested working concentration and exposure time of each sanitizer. The log reduc-tion was calculated by direct plating. Ten sanitizers met the performance criteriafor L. monocytogenes, achieving a minimal 6-log reduction. There was no signifi-cant difference observed amongst the different active ingredient formulations. Asingle, alchohol-based hand sanitizer used by the industry did not satisfy the cri-teria when tested on stainless steel coupons. This study demonstrated that sani-tizers are effective in controlling Listeria on stainless steel surfaces when properadherences to protocols were maintained. Misuse of sanitizers can allow the or-ganism to survive and possibly make its way into the final food product.

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Listeriophage ecology and diversity on dairy farmsVongkamjan, K.*, Moreno Switt, A., den Bakker, H. C., Fortes, E. D., and Wiedmann, M.Department of Food Science, Cornell University, Ithaca, NY, USA

Listeria monocytogenes is common in dairy farm environments and contaminatedsilage appears to be the major source of L. monocytogenes infections in farm ru-minants. Improved understanding of the ecology and diversity of phages infect-ing L. monocytogenes (“listeriophages”) in food associated environments is criticalto evaluate the impact, effects, and potential of phage-based pathogen controlstrategies. We collected silage samples over the course of one year on two dairyfarms to perform screening for L. monocytogenes and listeriophages, using stan-dard methods and representative host strains. L. monocytogenes isolates were char-acterized by molecular methods (e.g., sequencing of sigB) and subsequently clas-sified into lineages I and II. Listeriophages were characterized for host range usinga set of reference strains that represented 13 L. monocytogenes serotypes. Fur-thermore, phage genome size was determined by the Pulsed Field Gel Elec-trophoresis. Out of 134 silage samples tested (farm 1, n = 81; farm 2, n = 53), sixsamples were positive for L. monocytogenes; 43/81 samples from farm 1 and 40/53samples from farm 2 were positive for listeriophages. Overall 34/134 silage samplescontained >100 PFU/g silage with samples containing up to 2x104 PFU/g. For themajority of phage positive-samples, phages were detected on serotype 4a hoststrain. Phage isolates represented considerable host range diversity (12 distinctlysis groups). Most phages did not lyse lineage I strains, but lysed most lineage IIstrains and showed consistent and strong lysis patterns with all lineage III strains.Phage genome size ranged from 25 to 120 kb. Our data show considerable liste-riophage diversity in dairy farm environments, suggesting that phages play an im-portant role in the ecology of L. monocytogenes on dairy farms.


Evaluation of curative and preventive decontamination treatmentson Listeria monocytogenes biofilms with a new screening systemQuinon, E.1, Chamot, S.1, Groelly, J.2, Chavant, P.2, Bernardi, T.2, Desvaux, M.1

and Hebraud, M.1

1. INRA, France2. BioFilm Control, France

Food safety requires not only interventions on the product but also on all surfacesof the workshops from the receipt of raw materials until the processing and pack-aging of food products. All surfaces can be contaminated by microorganisms able toadhere, to form biofilms and to disseminate all along the chain. The literatureshows that bacterial cells in biofilm, compared with their planktonic counterpart,present a greater resistance to hostile environments and particularly to cleaningdisinfection procedures. The efficacy of cleaning disinfection products currentlyused for surface decontamination is generally evaluated on planktonic cells, whichdoes not predict their efficacy on bacteria in biofilm. We have implemented ascreening system, initially developed by the Biofilm Control society to evaluatebacterial ability to form a biofilm on an abiotic surface (BioFilm Ring Testâ), to testthe efficacy of 4 commercial decontamination solutions to detach biofilm from thesurface and to affect the viability of bacterial cells. Three temperatures (10°C, 20°Cand 37°C) and three Listeria monocytogenes strains were used for the tests. The ef-fect of preventive decontamination treatment of the surface on cell adhesion andbiofilm formation was also assessed. Two out of the 4 products, containing quater-nary ammonium compound (QAC) as biocide, dramatically affected the viabilityof sessile cells whatever the temperature while only one out of the two was effi-cient to detach adhered cells. The formulation of this last product also contains apolyenzymatic cocktail. The two other solutions remained inefficient on biofilms.The QAC + polyenzymatic cocktail was also the only product efficient to prevent ordelay the biofilm formation after a preventive treatment of the surface. TheBioFilm Ring Testâ can be used to sreen the efficacy of biocides on bacterial biofilmsin order to improve the microbiological status of the food processing plants.

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Pulsed Field Gel Electrophoresis, conventional and molecularserotyping on Listeria monocytogenes: An European ProficiencyTesting Inter-laboratory TrialFélix, B., Roussel, S., Tam Dao, T., Asséré, A., Lombard, B. and Brisabois, A.Agence française de sécurité sanitaire des aliments (Afssa), Laboratoire d’Etudes et de Recherches surla Qualité des Aliments et sur les Procédés agro-alimentaires (Lerqap), Maisons-Alfort, France

In Europe, the incidence of listeriosis caused by Listeria monocytogenes is rela-tively low, however an increase in cases, which differ depending of the countries,has been observed since 2000. Different methods are available for first-level char-acterization in epidemiological surveillance, in particular, conventional and mo-lecular serotyping. Regarding fine characterization techniques, macro-restrictionanalysis by pulsed-field gel electrophoresis (PFGE) has been described as the mostdiscriminatory methods for L. monocytogenes subtyping. This method is widelyused by the European National Reference Laboratories (NRLs) according to a2009 EFSA survey. AFSSA’s Laboratory for Study and Research on Food Qualityand Processing was appointed as Community Reference Laboratory (CRL) for Lis-teria monocytogenes in 2006. Recently, the CRL organized a proficiency test inorder to evaluate the ability of the NRLs to perform these three typing methods.Laboratories were free to choose either one, two or all three methods. The samepanel of six strains was used for all three methods and was sent to the 17 partici-pating NRLs. This panel included serotypes 1/2a, 4b, 1/2c, 1/2b and 3a strains. Eachlaboratory was free to choose the protocols used. All participants followed thePFGE PulseNet Europe standardized protocol. PFGE migration parameters wereset by the CRL, pattern interpretations were performed following the CRL’s in-terpretation criterion. For conventional serotyping and molecular serotyping, 86%and 93.5% of the serotypes obtained were in agreement with the CRL data. In-consistencies were mostly explained by differences observed in the use of reagents.For PFGE, 83% and 81% of profiles for AscI-PFGE and for ApaI-PFGE was undis-tinguishable of the expected profiles The inconsistencies observed were causedby slight standardization defaults or in few cases by a fault in the extraction. ThisPT trial was a valuable opportunity to improve the typing ability of NRLs and willallow PFGE pattern exchanges.

Detection of Listeria spp. in raw and pasteurized liquid egg-productsand in the egg-breaking plants environmentRivoal, K., Fablet, A., Chemaly, M., Salvat, G. and Protais, J.AFSSA, France

Listeria monocytogenes has been recognized as a human pathogen for decades andis etablished as an important food-borne pathogen. There is a lack of informationabout eggs and egg-products contamination by Listeria spp. The aim of this studywas to detect Listeria spp. in liquid egg-products (whole, yolk and white with orwithout salt and/or sugar) collected in 5 egg-breaking plants in north westernFrance, during one year. To study the seasonal variation, each egg-breaking planthas been visited during each season. For a raw egg-product sample, a pasteurizedsample has been tested and the egg shells used for theses egg-products have beenswabed to detect Listeria spp. The environment of plants was also sampled withswabs during the production of the egg-products tested. At the present time, theproject is not achieved. Three seasons have been studied and the last one is nowgoing on. The environment of the egg-breaking plants seems to be very contami-nated by Listeria spp. when the egg-products and their shells were slightly con-taminated by the bacteria. Moreover, Listeria monocytogenes has been very rarelyisolated in the egg-products as well as in the environment. At the end of the year,the study will be finished and the results could be analysed.

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Biofilm formation by Listeria monocytogenes isolates under conditionsthat mimic food and digestive tractKretli Winkelströter, L.1, Oliveira, M. A.2 and De Martinis, E. C.1

1. Universidade de São Paulo, Brazil2. Instituto Adolfo Lutz, Brazil

L. monocytogenes is ubiquitous; able to tolerate many adverse conditions found infoods and can persist in processing plants due to formation of biofilms. L. mono-cytogenes can cause severe foodborne infections in immunocompromised peopleand in pregnant women. In this work, L. monocytogenes isolates were screened forability to form biofilm under diverse conditions. Overnight cultures of 51 isolatesof L. monocytogenes from food, clinical and environmental sources were inocu-lated (1:20, v/v) in Brain Heart Infusion Broth (BHI) and under the following con-ditions: a) BHI plus 2% sucrose; b) BHI plus 5% NaCl; c) BHI plus 0.3% Oxgall; d)BHI pH 2 (adjusted with HCl) and e) BHI plus 50% (v/v) of extract from bacteri-ocin-producing cultures of Lactobacillus sakei 1, Leuconostoc mesenteroides 11A orEnterococcus faecium 130. Aliquots of inoculated broths (200µl) were transferredto wells of a polystyrene 96-wells microtiter plate and incubated at 37 °C/24h.Wells were washed 3 times with Phosphate Buffered Saline (PBS) and each wellwas stained with 200µL of 1% (v/v) crystal violet aqueous solution for 15 min.Plates were washed 3 times with PBS and wells were destained with 200µL of 95%ethanol. Concentration of crystal violet present in the destaining solution wasmeasured at 595 nm (CV-OD595) and directly correlated with biofilm formation. L.monocytogenes ATCC 19115 was used as parameter of comparison for biofilm for-mation. L. monocytogenes ATCC 19115 presented CV-OD595 in the interval of 0.69-0.87 under all conditions tested. CV-OD595 >1.1 was observed for approximately9.8%, 1.9%, 7.8% and 19.6% for L. monocytogenes isolates incubated respectively inBHI only and under presence of sucrose, NaCl and Oxgall, indicating a higher abil-ity to form biofilm. In BHI pH 2 and BHI plus 50% (v/v) of extract from bacteri-ocin-producing cultures, L. monocytogenes isolates did not show higher differencesin biofilm formation in comparison with L. monocytogenes ATCC 19115 (0.70-0.81CV-OD595). L. monocytogenes isolates showed different abilities to form biofilmunder different conditions probably due to modulation of gene expression, whichwill be further studied.

Biofilm formation of Listeria monocytogenes EGDe dependson temperature and nutrient availabilityAuchter, M.*, Endres, J., Waidmann, M. S. and Riedel, C. U.Institute of Microbiology and Biotechnology, University of Ulm, Ulm, Germany

Bioluminescent in vivo imaging revealed that in mice, Listeria monocytogenes col-onizes the gall bladder prior to systemic spread. Moreover, bacterial cells of L.monocytogenes isolated from the gall bladder display chaining morphology, a phe-notype associated with biofilm grown cells. Thus, biofilm formation in the gallbladder could be a strategy of this important pathogen to protect, establish andmultiply itself at a site inaccessible to the components of the immune system priorto causing systemic infections. To investigate the mechanisms of biofilm forma-tion, we tested L. monocytogenes EGDe and an isogenic agrD deletion mutant fortheir ability to form biofilms under different conditions. Biofilm formation wastested in a standard microtiter plate assay at different time points during biofilmgrowth of EGDe, varying temperatures (20, 30 and 37°C) and in full-strength BHI(rich broth) as well as in 10-fold diluted BHI medium (nutrient limited). Biofilmformation at 37 °C was poor in rich broth and could be significant enhanced whencells were grown under nutrient limitation. Deletion of the agrD gene resulted indefective biofilms in 10-fold diluted, as well as in full-strength BHI. The biofilmformation of the ∆agrD mutant could also be restored to wild type levels if recon-stituted cell-free supernatant (BHI added to spent EGDe supernatant, pH adjustedand filter sterilized) of EGDe wild type cells grown in full-strength BHI was addedto the ∆agrD strain. Additionally, DDAO staining and DNAse treatments revealeda potential role for extracellular DNA in the initial stages of biofilm formation.


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Antimicrobial activity of lactococcal and enterococcal strainsisolated from artisanal products from North West of Italy towardListeria monocytogenesDal Bello, B., Rantsiou, K., Ambrosoli, R., Zeppa, G. and Cocolin, L.Department of Exploitation and Protection of the Agricultural and Forestry Resources, University ofTurin, Italy

Listeria monocytogenes is a foodborne pathogenic Gram-positive bacterium that iswidely distributed in soil, sewage, fresh water sediments and effluents, and is fre-quently carried in the intestinal tract of animals and humans. Biocontrol of L.monocytogenes by bacteriocin-producing lactic acid bacteria or by bacteriocin ex-tracts has attracted great attention in recent years and new preservation strategiesto control growth of L. monocytogenes have been developed, including the appli-cation of the bacteriocin nisin (Food and Drug Administration, 1988). In this work,we have investigated the potential role as bioprotection agents of autochthonouslactococcal and enterococcal strains isolated from fresh and fermented artisanalproducts (cheese and meat) of Piedmont region (North West of Italy) determiningtheir antimicrobial spectrum of activity towards different foodborne spoilage andpathogenic microorganisms. Bacteriocin-producing strains were identified by mo-lecular methods and genetic determinants encoding the antimicrobial proteinswere targeted by PCR. Thirty-nine strains of Lactococcus lactis exhibited inhibi-tion towards L. monocytogenes NCTC 10527 and most of them (26 strains) showedthe presence of the genes responsible for the nisins A and Z production. RegardingEnterococcus spp., 31 strains showed inhibition activity towards L. monocytogenesNCTC 10527 and the presence of the genes responsible for the enterocins A and Pproduction was determined. It is interesting to underline that for some strains itwas not possible to identify any known bacteriocins. In this study a high incidenceof bacteriocin producing strains was observed. In the future, the possible use ofthese active strains could be a new way to ensure safety of foods.

Plant-based strategies for Listeria monocytogenes control in foodsPaparella, A., Serio, A., Chaves-Lopez, C. and Di Pasquale, F.University of Teramo, Italy

Essential oils (EOs) and plant extracts are considered a natural and effective al-ternative to chemical preservatives. Considering the complex chemical composi-tion of EOs, their antimicrobial action is not likely attributable to one single mech-anism, although bacterial cytoplasmic membrane seems to be a specific target.The authors evaluated the antimicrobial activity of thyme, oregano and cinnamonEOs against Listeria monocytogenes strains, isolated from the smoked salmon in-dustry and from meat products; these strains showed variable levels of antimicro-bial resistance, belonged to different serotypes and had heterogeneous molecularprofiles. Thyme and oregano EOs were particularly effective, with Minimal In-hibitory Concentrations (MICs) correlated with strains biodiversity. Automaticturbidometry was applied to evaluate the effects of EOs on cell physiology; the re-sults documented a lag phase extension and a decrease of maximum growth rateand maximum growth value. The antibacterial effect was evident even after a shortcontact time with the cells. Using flow cytometry combined with fluorescent tech-niques, the authors demonstrated that membrane disruption is the primary inac-tivation mechanism of thyme and oregano oils. A different mechanism might be in-volved in cinnamon EO, acting on cells enzymatic activity. Further studies, carriedout by means of EPR (Electronic Paramagnetic Resonance), highlighted the effectof oregano EO on membrane fluidity and order, variable with increasing EO con-centrations. These findings prove that food biopreservation with EOs involves var-ious mechanisms of action and may be considered a safe and effective measure tocontrol Listeria monocytogenes growth, although with different effects dependingon strains biodiversity.

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Scientific studies for survival of Listeria monocytogenesin dairy products and its application in practiceCabanova, L., Škuntova, O. and Kantikova, M.State Veterinary and Food Institute, Dolny Kubin, Slovakia

European reference laboratory for Listeria monocytogenes in Paris has preparedtogether with some National reference laboratories from the European MemberStates the scientific study for Listeria to prove the producer that his products aresafe till the end of the shelf- life. In our study we have developed studies for theproducer of two different dairy products after several positive findings of Listeriain the final products. As a first product the raw sheep cheese has been selectedand as a second one the steamed cheese products have been analyzed. In the firststage the physico-chemical characteristics has been determined (aw, pH) and thanthe subsamples has been prepared and artificially contaminated with a mixtureof two reference Listeria strains and third isolated strain from the previous posi-tive sample. After inoculation treated samples has been stored at 5.8–6.2 °C incu-bator till the end of the shelf-life and during this period 2-3 times has been ana-lyzed (aw, pH, Listeria detection and enumeration has been determined). At theend of the shelf – life the growth potential has been calculated and the Listerianumbers at the beginning and at the end of the shelf- life has been given.

The effect of chilling temperatures on the virulenceof Listeria monocytogenes isolates with different originsNeves, E. M.1,2, Silva, A. C.1*, Louro, P.3,4, Ferreira-Dias, S.4 and Brito, L.1

1. CBAA/ Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia,Technical University of Lisbon, Portugal

2. Instituto Superior de Estudos Interculturais e Transdisciplinares (ISEIT), Campus Universitáriode Almada, Instituto Piaget, Portugal

3. Instituto Nacional dos Recursos Biológicos, IP, Portugal4. CEER/ Departamento de Agro-Indústrias e Agronomia Tropical, Instituto Superior de Agronomia,

Technical University of Lisbon, Portugal

Although the chilling and freezing of food products are increasingly used by thefood industry, there are few studies on the effect of these temperatures on the vir-ulence of Listeria monocytogenes. The influence of 1, 7 and 30 days of storage atchilling (7 °C) and freezing temperatures (-20 °C and -76 °C) on the virulence of19 L. monocytogenes isolates was evaluated in this study. The isolates selected ac-cording to their genetic diversity and different initial levels of virulence were fromfrozen (n = 5), refrigerated (n = 3), refrigerated ready-to-eat (n = 5) and ready-to-eat foods (n = 1), from humans (n = 2), from dairy environment (n = 1) and refer-ence strains (n = 2). In the case of the chilling assays, the cells were kept in physi-ological buffer and culture medium was added just before infection. After therespective storage, the isolates were used to infect HT-29 cell monolayers in aplaque forming assay (pfa), immediately or after thawing the bacterial suspen-sions at room temperature. The pathogenic potential of the strains was expressedas the mean log of the number of plaques formed (log pfa), and one-way or multi-factorial ANOVA of the counting data was carried out. The results showed that therefrigeration temperature (7 °C) acted more significantly (P < 0.05) on the de-creasing of the virulence potential of all isolates, although time didn´t show a sig-nificant effect on this loss of virulence. In relation to freezing temperatures (-20 °C and -76 °C), the decrease in virulence was not as significant, although thetemperature of -76 °C tended to be more conservative of the initial virulence. Bet-ter elucidation of which refrigerated food matrices allow growth of the pathogenmay help to evaluate the real risk of the presence of Listeria in food.


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How to improve a sampling plan in order to better assessL. monocytogenes contamination on diced bacon at the plantBergis, H.1, Commeau, N.1, Zuliani, V.2, Cornu, M.1, Beaufort, A.1 and Garry, P.2

1. AFSSA, France2. IFIP, Institut de la filière porcine, France

Food Business Operators (FBO’s) are legally responsible for the safety of their foodproducts (Regulation CE 2073/2005). The regulation does not specify the fre-quency of sampling/ testing and it is up to the FBO’s to decide the appropriatelevel of sampling/testing to help validate and verify their food safety managementplans. In this scope, sampling plans are useful tools to control and to assess thecontamination of a given foodborne pathogen at the plant and also to help the FBOto take decisions. In the present study, a production of diced bacon in a Frenchplant was monitored to assess the contamination of this product by Listeria mono-cytogenes. The steps of the process are: tumbling, steaming, dicing, and packag-ing.The aim of this study was to try, through the elaborated sampling plan, (i) to es-tablish a link between the prevalence of L. monocytogenes in the pork breasts andin diced bacon; (ii) to determine whether the levels of contamination of the porkbreasts and in the diced bacon are related; (iii) to estimate the incidence of thetumbling step and of the dicing step on the final product contamination.The foodprocess was studied from the raw material (pork breasts) to the final product(diced bacon) and the steps possibly involved in the contamination identified. Anexperimental stratified random sampling plan was constructed in two steps of theprocess: after tumbling (a important step), and after packaging. 106 sampled tum-bling breast and 86 packages of diced bacon were analysed. Detection and enu-meration of L. monocytogenes were performed on 100cm2 of the pork breast and on100g of diced bacon. The lactic acid bacteria (LAB) were also enumerated. Fromthe obtained data on contamination (prevalence and level of contamination of L.monocytogenes and the level of LAB), it appears that the tumbling step has an ho-mogenisation effect on the contamination and sampling after this step could be agood indicator of the presence of L. monocytogenes in diced bacon. It has beenshown that over a certain level of contamination of the breast, the diced baconfrom the same batch are also contaminated with L. monocytogenes.

Contamination of Listeria monocytogenes in a cold-smokedpork processing plant using brining injectionsBerzins, A.1, Silins, I.1 and Korkeala, H.2

1. Faculty of Veterinary Medicine, Latvia University of Agriculture; University of Helsinki, Latvia2. University of Helsinki, Finland

Contamination of L. monocytogenes was studied in a cold-smoked pork processingplant using brining injections. Overall prevalence of L. monocytogenes in cold-smoked pork products during a 5-year period was 26%. Environmental samplingcombined with PFGE subtyping and serotyping was applied to investigate the ge-netic diversity of L. monocytogenes in the brining facilities and alongside prem-ises. A total 183 samples were collected for contamination analyses, including sam-ples of the product at different stages during manufacture (n = 136) andenvironmental samples (n = 47) covering most of the manufacturing surfaces dur-ing a 3-month period. Overall, the prevalence of L. monocytogenes in raw pork be-fore processing was 18%. The prevalence of L. monocytogenes in pork increasedto 60% after being in brining area and brining machine. Two different L. monocy-togenes PFGE types belonging to serotypes 1/2a and 1/2c were recovered from thedifferent sites of the brining machine (feeding teeth and smooth surfaces), thuscausing persistent contamination of RTE cold-smoked pork products over periodof 5-years. In addition, brining machine harboured two PFGE types belonging toserotypes 1/2a and 4b, which were found on different contamination sites: feedingteeth, smooth surfaces and spaces of the machine. Brining injections increased L.monocytogenes contamination in finished RTE cold-smoked pork products andprocessing environment. Brining area, and specifically, brining machine shouldbe subjected for disassembling and extensive cleaning and disinfection to elimi-nate any persistent contamination with L. monocytogenes.

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Effects of GRAS products on growth of Listeria monocytogenes duringcold storage of salmon filletsMcCarthy, S. and Johnson, D.Food and Drug Administration, USA

Microbial growth can affect the quality and safety of prepared, processed, andready-to-eat (RTE) meat, poultry, and seafood. Pathogenic bacteria, including Lis-teria monocytogenes (Lm), can be resistant to preservatives, sanitizers, and an-tibiotics. The presence of Lm on RTE products results from cross-contaminationfrom contact surfaces in food processing plants. This study examined the ability ofthree GRAS products to inhibit the growth of Lm on salmon fillets during coldstorage. Twenty-five-g portions of raw salmon fillets were inoculated externallywith six log10 CFU Lm 1a1/g. Inoculated fillets were incubated at 25°C for 20 min-utes and at 4 °C for two hours to allow attachment. Filets were then treated withsterile deionized water (SDW; control) and 3 GRAS products (IONALTM LC, MO-statinTM VS, levulinic acid; 1:5 w/v) at 25 °C for 5 min. Control and treated filetswere analyzed immediately after exposure (T0) and after storage at 4 °C for up to12 weeks. Fillets were shaken in wash buffer and cell counts in washates were de-termined by spread plating on chromogenic agar. Numbers of Lm increased byone log10 during 9 weeks of cold storage of fillets treated with SDW. In contrast,MOstatinTMVS reduced the numbers of Lm by two logs10 during storage for 9weeks. IONALTMLC and levulinic acid prevented the growth of Lm for up to fourweeks. Each of the GRAS products reduced densities of salmon bacterial flora bytwo to four logs10 at two weeks compared to SDW; however, no difference was ob-served at 12 weeks. The use of GRAS products could prevent an increase in num-bers of Lm that are associated with cross-contamination in processing plants.

Heavy-metal and detergent resistance of Listeria species isolates frommilk processing environmentsDoijad, S.*, Garg, S. and Barbuddhe, S. B.ICAR research complex for Goa, India

Listeria monocytogens is an important food-borne pathogen responsible for variedclinical forms in animal and humans. The resistance of Listeria strains to cadmium,arsenic and quaternary ammonium compounds was studied and it was correlatedwith resistance to quaternary ammonium compounds used as disinfectants in thefood-processing industry. Limited information is available on the prevalence of re-sistance among isolates from the environment of food-processing plants. In thisstudy, a total of 27 Listeria species isolates from the milk processing plants wereincluded. Out of 27, 14 (51%) found to be Listeria monocytogenes, 12 (44%) were L.innocua and 1 (3%) isolate was of L. ivanovii. All the isolates were subjected for de-termination of the resistance to cadmium, arsenic and quaternary ammonium dis-infectant (benzalkonium chloride (BC)). Isolates were not inhibited at 40µg/ml ofcadmium chloride, while 12 (44%) of them grew upto 200µg/ml. Isolates grew wellin 40µg/ml of sodium arsenite while 3 of them could tolerate 200µg/ml. The iso-lates could grew at 1µg/ml of BC while only 7 could tolerate 5µg/ml. The amount ofBC tolerated by these isolates are well above the amount used in milk processingenvironment. There was no co-relation found between resistance of isolates to cad-mium, arsenate and BC, all isolates were independently resistance to metal and de-tergent. It was interesting to note that none of the isolates contained plasmid. Thissuggests that resistance against metals and detergent in Listeria spp. is not medi-ated by the plasmid and need further investigations. Our findings suggest that themilk processing environment constitute a reservoir for L. monocytogenes and otherListeria species. Resistance to heavy metals and quaternary ammonium disinfectantis of significance to food processing industry.


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Detection of Listeria monocytogenes in lettuce sold at marketsand supermarkets in Porto, PortugalNoronha, L., Magalhães, R., Silva, J. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

Listeriosis is a severe infection caused by Listeria monocytogenes particularlyamong the elderly, very young and immunocompromized individuals and has alsobeen associated with late-term miscarriages in pregnant women. The high inci-dence of L. monocytogenes in foods and the high fatality rate associated with liste-riosis, has contributed to L. monocytogenes being considered a public health hazardand a continuing source of loss to food processors due to the large number of vol-untary and obligatory recalls. Listeriosis outbreaks have been linked to the con-sumption of raw vegetables including lettuce. According to the European FoodStandards Agency, lettuce supports L. monocytogenes growth and can be relatedwith the transmission of listeriosis. Twenty different lettuce samples collected inmarkets or supermarkets located in the North of Portugal, were analyzed for thepresence of L. monocytogenes by the VIDAS method. Of the tested lettuce samples,30% were positive for the presence of L. monocytogenes. Three isolates were col-lected from each of the positive samples. They were serotyped by Multiplex PCRand it was possible to distinguish two serogroups: 33% belonged to serogroup 4b –4d – 4e and 67% to 1/2c – 3c. The present results reinforce the importance ofwashing raw vegetables thoroughly before eating and preparing green salads andvegetable dishes shortly before eating in order to reduce the risk of listeriosis.

Preliminary analysis of structure and chemical composition of extra-cellular polymeric substance produced by Listeria monocytogenesNwaiwu, O.*, Lad, M., Davis, A., Foster, T. and Rees, C.Univeristy of Nottingham, UK

It is generally believed that, while Listeria monocytogenes is capable of formingbiofilms, it does not produced extensive amounts of extracellular polymer sub-stances (EPS) that contribute to adhesion and persistence in the environment. Wepresent here evidence that under specific growth conditions L. monocytogenes iscapable EPS synthesis and this does promote surface adhesion. After EPS extrac-tion, analysis by NMR, ATR-FTIR and SEM indicated that the material is high mo-lecular weight and the structure of Listeria EPS is different from both polysac-charide and poly-g-glutamic acid (an EPS produced by a range of Gram-positivebacteria). Further amino acid analysis also ruled out this material being a poly-mer of other amino acids, while elemental energy dispersion x-ray analysis showedvery low nitrogen content with carbon and oxygen the predominant elements. Theas yet unidentified polymer has unusual physical properties, and scanning elec-tron microscopy (SEM) showed rapid absorption of moisture by the EPS as relativehumidity increased. Hence it is likely that this polymer will also contribute to des-iccation tolerance of the bacterium.


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Ecology and persistence of Listeria monocytogenes strains in fermentedmeat sausage processors from the Northern region of PortugalFerreira, V.1, Barbosa, J.1, Vongkamjan, K.2, Moreno Switt, A.2, Hogg, T.1, Gibbs, P.1,Wiedmann, M.2 and Teixeira, P.1

1. CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal2. Cornell University, USA

In this study, 202 Listeria monocytogenes isolates were recovered from productsamples representing 7 processors of Alheira, a traditional fermented meat sausagefrom the Northern region of Portugal. Samples were collected from each processorin separate dates, either at retail establishments or at processing plants. Isolateswere characterized by molecular serotyping and DNA macrorestriction analysisby pulsed field gel electrophoresis (PFGE). Characterization by PFGE suggestedpersistence of particular strains over time in the 7 different processors, the ap-parent time of persistence ranged from 14 to 32 months. While a number of stud-ies have evaluated associations of different strain characteristics, including biofilmformation etc., with strain persistence in processing plants, no convincing andconsistent evidence for specific strain characteristics that facilitate establishmentof persistence have been found so far. We thus investigated whether phage sus-ceptibility and presence of lysogenic prophages in L. monocytogenes may be asso-ciated with strain persistence in the environment. A subset of 41 L. monocytogenesisolates representing sporadic and persistent PFGE patterns was screened forlysogeny. Twenty six prophages where induced and their lytic spectrum againstthe 41 strains of L. monocytogenes was investigated. Lysogens included both spo-radic and persistent PFGE types. While molecular serogroup D (4b, 4d, and 4e)isolates were more susceptible to phages as compared to serogroup A (1/2a and3a) or B (1/2b, 3b, and 7) isolates, there was no evidence for differences in phagesusceptibility between persistent and sporadic strains. While our findings supportthat L. monocytogenes serotypes differ in phage resistance, strain persistence doesnot seem to be associated with enhanced phage resistance.

Biofilm formation and survival of L. monocytogenes and slaughter housebacteria on surfaces at relevant environmental conditionsLangsrud, S., Møretrø, T. and Heir, E.Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway

Adhesion, biofilm formation and survival of microorganisms on food contact sur-faces are of concern in the food processing industry. Surface associated bacteriacan lead to cross contamination of food and have serious consequences for humanhealth and food quality. Persistence of Listeria monocytogenes in the food pro-cessing environment is a well known food safety concern. Also, other environ-mental bacteria may cause problems when persisting in the food industry. A betterknowledge on the ability of dominant bacteria to produce biofilm and survive onfood contact surfaces under relevant conditions is therefore important. We havestudied biofilm formation and survival of L. monocytogenes and environmentalbacteria isolated from surfaces in a meat slaughter house. Bacteria were isolatedafter cleaning and disinfection and prior to slaughter on a regular working dayand identified by 16S rDNA sequencing. Selected isolates from dominating gen-era and L. monocytogenes were tested for their ability to form biofilms under var-ious temperature conditions in a microtiter plate assay. We also studied survival onstainless steel under controlled temperature and humidity conditions. The sus-ceptibility of surface adhered bacterial cells to four commercial disinfectants com-monly used in the meat industry was investigated. Predominant bacteria in theslaughtering line belonged to the genera Pseudomonas, Serratia, Acinetobacter,Citrobacter, Aerococcus, Kocuria and Staphylcococcus. The data indicated lowbiofilm forming abilities of L. moncytogenes at relevant food industry tempera-tures, while other environmental bacteria showed variable biofilm formation at12 and 20°C. Gram-positive bacteria showed excellent survival on stainless steelduring incubation at 12 °C, 70% relative humidity. The bactericidal effects of dis-infectants on bacteria adhered to surfaces were low (< 3 log kill) for all tested dis-infectants. In conclusion, survival of bacteria after cleaning and disinfection inthe meat industry can be explained by resistance to disinfectants when dried onsurfaces and either ability to survive at dry conditions or biofilm formation in thepresence of water.

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Control of L. monocytogenes by lysozyme combined with olive leafextract in edible pullulan film coated on chicken breast filletsHandan Baysal, A.*Izmir Institute of Technology, Department of Food Engineering, Urla, Izmir, Turkey

In recent years, naturally occurring antimicrobial and antioxidant compoundshave been preferably employed in meats because of their potential health benefitsand safety compared with synthetic preservatives. The objective of this study wasto evaluate the antimicrobial effect of olive leaf extract (OLE) and lysozyme addedto pullulan film coatings against Listeria monocytogenes in raw chicken breast meatstored aerobically under refrigeration (4 °C). The effectiveness of these com-pounds in a meat model system was evaluated by surface inoculation (approxi-mately 106 CFU/g) of L. monocytogenes onto chicken breast fillets. Fresh chickenbreast fillets used in this research was purchased from a local supermarket. The in-oculated raw chicken breast fillets were treated before storage by dipping into 5%(w/v) pullulan film-forming solutions with and without the addition of antimi-crobial agents: (a) deionized water, (b) 5% (w/v) pullulan + 20% (w/v) water ex-tract of OLE, (c) 5% (w/v) pullulan + lysozyme (d) 5% (w/v) pullulan + 20% (w/v)water extract of OLE + lysozyme) for 10 min at 20°C. After removal of excess mois-ture, the samples were stored at 4 °C. The inhibitory effects of OLE and lysozymecontaining edible coatings were evaluated for 14 d. In the meat system, the L.monocytogenes population was decreased effectively after 14 d at 4 °C. This re-search has demonstrated that the use of an edible film coating containing naturalextracts or the application of a lysozyme solution is a promising means of con-trolling the growth and recontamination of L. monocytogenes on raw chickenbreast meat stored under refrigeration.


Evaluation of antilisterial activity by lactic acid bacteriaBorges, S., Barbosa, J., Albano, H., Silva, J. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

Listeriosis is an infection with a high morbidity and mortality, caused by Listeriamonocytogenes. It occurs primarily in elderly patients, immunocompromised in-dividuals, pregnant women and their neonates. Some lactic acid bacteria (LAB)are able to produce antimicrobial compounds with activity against severalpathogens, such as organic acids (lactic and acetic acids), hydrogen peroxide, an-timicrobial enzymes and bacteriocins. The objective of this work was to evaluatethe antimicrobial activity of selected LAB against L. monocytogenes. Thirty-fiveisolates of LAB, available at the culture collection of Escola Superior de Biotec-nologia, demonstrated inhibitory activity against 29 clinical isolates of L. monocy-togenes. To characterize this antilisterial activity the antagonistic spectrum of eachLAB culture, neutralized cell-free supernant and neutralized cell-free supernanttreated with catalase or trypsin were investigated. All isolates of LAB showed an-tibacterial effect probably due to the production of bacteriocins. The bacteriocinactivity (AU/mL) was determinated for three serotypes of L. monocytogenes (1/2a,1/2b, 4b) and the activity varied between 800 and 6400 AU/mL. According to thisstudy, the use of bacteriogenic LAB can be important to control L. monocytogenesof clinical origin.

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Molecular methods to assess Listeria monocytogenes routeof contamination in a dairy processing plantCocolin, L., Alessandria, V., Dolci, P. and Rantsiou, K.Univeristy of Turin, Italy

In this study we investigated the occurrence of Listeria monocytogenes in a dairyprocessing plant during two sampling campaigns in 2007 and 2008. Samples rep-resented by semi-finished and finished cheeses, swabs from the equipment andbrines from the salting step, were subjected to analysis by using traditional andmolecular methods, represented mainly by quantitative PCR. Comparing the re-sults obtained by the application of the two approaches used, it became evidenthow traditional microbiological analysis underestimated the presence of L. mono-cytogenes in the dairy plant. Especially samples of the brines and the equipmentswabs were positive only with qPCR. For some equipment swabs it was possible todetect a load of 104-105 cfu/cm2, while the ISO method employed gave negativeresults both before and after the enrichment step. The evidences collected duringthe first sampling year, highlighting a heavy contamination of the brines and ofthe equipment, lead to the implementation of specific actions that decreased thecontamination in these samples during the 2008 campaign. However, no reduc-tion in the number of L. monocytogenes positive final products was observed, sug-gesting that a more strict control is necessary to avoid the presence of thepathogen. All the isolates of L. monocytogenes were able to form biofilm, and, in-terestingly, considering the results obtained from their molecular characteriza-tion it became evident how strains present in the brines, were genetically con-nected with isolates from the equipment and from the final product, suggesting aclear route of contamination of the pathogen in the dairy plant. This study under-lines the necessity to use appropriate analytical tools, such as molecular methods,to fully understand the spread and persistence of L. monocytogenes in food pro-ducing companies.

Persistence of L. monocytogenes in artisanal cheese producing plantsAlmeida, G., Santos, I., Magalhães, R., Barbosa, J., Hogg, T. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

The consumption of raw milk or raw milk products has caused several listeriosisoutbreaks resulting in several hundred cases. This highlights the risk of these prod-ucts and led public health officials to recommend that raw milk and dairy productsprepared from raw milk should not be consumed by susceptible populations, par-ticularly pregnant women. The presence of L. monocytogenes in cheeses has beenwidely reported in the literature over the years. The prevention of Listeria con-tamination is difficult to achieve in raw milk cheeses. Also, contaminated raw milkcan contaminate the plant environment and some strains can colonize and persistfor long periods. The present work aimed to characterize L. monocytogenes isolatesrecovered between 2004 and 2007 from an artisanal ewe’s raw milk cheese pro-ducing plant. PFGE using restriction enzymes AscI and ApaI was performed inforty isolates: 18 from cheese, one from raw milk, 20 from environmental sites, andone from whey. Six combined pulsotypes were obtained, one of them aggregating 20isolates. This is an evidence of the presence of a resident clone with a persistencetime estimated in at least, 14 months. The profile obtained from cheese isolateswas also encountered in isolates from ewe’s raw milk and from raw milk reception’sfloor suggesting that raw milk could be the source of contamination. Control L.monocytogenes in the environment plays an important role in reducing its pres-ence in foods, which is necessary to reduce the incidence of listeriosis.

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Occurrence of Listeria monocytogenes in food products collected inPortugal from retail establishments and food plantsMena, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G. and Teixeira, P.CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal

Listeriosis is an infection caused by the bacterium Listeria monocytogenes. Theingestion of contaminated food with this microorganism may cause serious healthproblems for consumers. During two years (2007 and 2008) the presence of Lmonocytogenes was evaluated in a total of 1476 food samples, collected from retailand food plants. The detection of the microorganism was performed using the au-tomated VIDAS system and the positive results were confirmed following the ISO11290 standard. L. monocytogenes was detected in 134 (9.1%) of the analyzed sam-ples. Most of positive samples were from foods normally submitted to heat treat-ment before consumption such as pré-cooked foods (30.0%, 12/40; pizza, pasta,rissois, etc) and fermented meat products (20.6%, 33/160; farinheira, alheira,morcela, bacon), from raw products (16.0%, 58/362; raw meat, vegetables and fish),and from “ready-to-eat” foods: fermented meat products (8.8%, 10/114), vegeta-bles salads (1.1%, 2/174), ready cooked meals (4.5%, 6/134), cheeses (4.6%, 12/262)and fresh cheese (1.3%, 1/80). The occurrence of the microorganism in ready-to-eat foods is of more concern. L. monocytogenes grows at refrigeration tempera-tures and could achieve levels of contamination that can cause disease. Also, foodsthat will have a heat treatment at consumer’s home could represent a hazard ifcross contamination of food items that will be consumed without any further stepof destruction occurs.

Listeria monocytogenes biofilms grown at 12 °C showed reducedsusceptibility to sanitizersLourenço, A., Machado, H.* and Brito, L.CBAA – Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia, TechnicalUniversity of Lisbon, Portugal

Listeria monocytogenes may form biofilms on food contact surfaces of difficult san-itization which may lead to recurrent contamination of food products. The eradi-cation of biofilms can only be achieved by using adequate hygienization routineswhich will ultimately ensure food safety. The biofilm forming ability of four L.monocytogenes strains from different origins, cheese, dairy environment and fromhuman cases of listeriosis was evaluated, either in pure culture or in co-culturewith Pseudomonas aeruginosa, at 37 °C and 12 °C using the Calgary Biofilm De-vice® (CBD). The minimum biofilm eradication concentration (MBEC) was de-termined for four commercial dairy sanitizers (one alkyl amine acetate based, T99;two chlorine based, T66 and DD and one phosphoric acid based, BP). Co-culturebiofilms had an average total population of 7 to 8 Log10 CFU/peg. P.aeruginosawas the dominant species, either at 37°C or at 12 °C, representing 99% of the totalCFU/peg. L. monocytogenes biofilms grown, either at 37 °C or 12 °C, although withdifferent incubation times (24 hours and 7 days, respectively) reached a similarcell density (6 Log10 CFU/peg). Nevertheless, the biofilms produced at 12 °C weregenerally less susceptible to the sanitizers than when produced at 37 °C. One ofthe strains (3880) retrieved MBEC values of 30720 µg/ml and 16000 µg/ml forT99 and BP, respectively. These values were above the maximum in-use recom-mended concentrations (T99 – 29700 µg/ml and BP – 11500 µg/ml) for theseagents. The growth in co-culture also proved to be relevant regarding disinfectantsusceptibility, as the co-cultures were generally less susceptible than L. monocy-togenes pure cultures. The MBEC values obtained for the chlorine based agentswere never over recommended in-use concentrations which may indicate a moreefficient ability to eradicate biofilms, if in-use conditions are met.


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Modelling growth of Listeria monocytogenes in cheese as functionof environmental variablesSand Rosshaug, P. and Hallberg Larsen, M.Faculty of Life Science, Department of Veterinary Pathobiology, Denmark

This project is part of a larger project with the purpose to develop a predictivemodel of growth of the pathogen Listeria monocytogenes in a dairy chain producingblue/white mould cheese. This project focuses on the growth kinetics of L. mono-cytogenes as function of environmental variables. Temperature, water activity, pH,lactic acid, NaCl, and O2 are environmental factors that impact the growth kinet-ics of L. monocytogenes. The growth kinetics was investigated for 3 strains of L.monocytogenes that has been found in the dairy industry: ATCC 19111, ATCC 19113,ATCC 19115. Different growth media were tested: broth, milk, and blue/whitemould cheese. The growth kinetics of L. monocytogenes as function of these vari-ables was formulated as an ordinary differential equation using Cardinal Parame-ters to describe the influence of the environmental factors. The growth kineticswas applied in a predictive mathematical, deterministic model of L. monocyto-genes. Finally the growth kinetics of L. monocytogenes was investigated using astochastic predictive model taking into account the stochastic nature of some ofthe inputs including the lag.

Characterization of anti-Listerial bacteriocin produced by Lactobacillusplantarum ST8SH, a strain isolated from Bulgarian salamiTodorov, S. D.1* and Lemos Vaz-Velho, M.2

1. Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Brazil2. Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Portugal

Strain ST8SH, isolated from Bulgarian salami, was identified as Lactobacillus plan-tarum based on biochemical tests, sugar fermentation reactions (API50CHL), PCRwith species-specific primers and 16S rDNA sequencing. Strain ST8SH produces a3.0kDa class IIa bacteriocin, active against Listeria monocytogenes, Listeria in-nocua, Streptococcus caprinus, Streptococcus spp., Lactobacillus casei, Lactobacilluscurvatus, Lactobacillus salivarius, Lactobacillus pentosus, Enterococcus mundtii,Enterococcus faecalis and Lactococcus lactis subsp. lactis. No change in activitywas recorded after 2h at pH values between 2.0 and 12.0, and after treatment at100°C for 120 min or 121 °C for 20 min. The mode of activity against L. innocua, L.monocytogenes is bactericidal, resulting in cell lyses and enzyme- and DNA-leakageand was visualised by atomic force microscopy. The highest level of activity (25600AU/ml) was recorded when cells were grown at 37 °C or 30°C in MRS broth (pH6.5). Peptide ST8SH adsorbs at low levels (400 AU/ml) to producer cells. High cellnumbers of L. plantarum ST8SH and L. innocua LMG13568 were recorded at be-ginning when co-cultured. However, the cell numbers of L. innocua LMG13568 de-creased from 1.6x104 CFU/ml to 2.5x102 CFU/ml in 12h and to undetectable levelsafter 24h. Plantaricin ST8SH production was stimulated by presence of L. innocuaLMG13568 (102 400 AU/ml). Similar results were obtained with addition of 10%autoclaved overnight culture of L. innocua LMG13568 to MRS growth media onproduction of plantaricin ST8SH. Based on the genetic approach strain ST8SHharbours associated genetic determinants for production of a variation of the wellknown plantaricin 423. Future purification of the produced bacteriocin need tobe performed to determine if Lactobacillus plantarum ST8SH produces this bac-teriocin (plantaricin 423 – like) or harbour more then one bacteriocin operons.


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EFSA’s proposal for an EU-wide retail survey on Listeria monocytogenesin selected categories of ready-to-eat food productsBoelaert, F., Felício, T. and Makela, P.EFSA, Italy

The European Food Safety Authority and its Task Force on Zoonoses Data Collec-tion were requested by the European Commission to provide a proposal for tech-nical specifications on an EU-wide retail survey on Listeria monocytogenes in se-lected categories of ready-to-eat (RTE) food products that should take place duringthe whole year of 2010. The proposed technical specifications focus on samplingthose categories of RTE food in which the highest L. monocytogenes contaminationhave been observed in the European Union (EU): soft and semi-soft cheeses,smoked and gravad fish, and heat-treated meat products that are handled afterheat treatment. Sampling of these RTE food categories would be targeted at re-tail outlets serving the final consumer, with catering and wholesale establishmentsexcluded. Food products are suggested to be tested at the end of the shelf-life andadditionally in the case of smoked and gravad fish, immediately after sampling. Atthe Community-level a total of about 3,000 samples should be taken for each RTEfood category, per analyses stage. A specific number of samples is proposed to beproportionally allocated to the Member States according to the size of their humanpopulations. Standardised analytical L. monocytogenes detection and enumera-tion methods are proposed to be employed in the analyses of samples. In addition,water activity and pH values are to be measured in the smoked and gravad fish.This proposal for a Community-specific survey would only allow estimation of theL. monocytogenes prevalence at the Community level. A modelling and simulationapproach should be applied in the analyses of the results so that the effectivenessof the implementation of Community L. monocytogenes criteria may be assessed. Asimilar model-based approach will also be used to estimate the growth potential ofL. monocytogenes in smoked and gravad fish.

Ripening conditions: an asset to control L. monocytognes in cheesesCallon, C., Picque, D., Corrieu, G. and Montel, M.-C.INRA, France

The EC regulations for dairy products require the absence of L. monocytogenesoutput production with a possible derogation at < 100cfu/g if it is demonstratedthat there is no evolution during storage until consumption. To identify the mi-crobial populations of cheeses and environmental factors that may be barrier to L.monocytogenes in the European project Truefood an ecological approach was pre-ferred over a process of screening of strains. This approach based on the followingsteps: 1) selection of milk with anti-Listeria properties, 2) identification of micro-bial communities and simplifications of their composition, 3) cheese-making ex-periments in different conditions of ripening. This strategy was successfully ap-plied to the inhibition of L. monocytogenes in cheese core. Indeed, a microbialcommunity of raw milk, composed of 7 strains of lactic acid bacteria (Lactobacillusand Leuconostoc), in synergy with bacteria called ripening Gram positive (Staphy-lococcus, Arthrobacter, Brachybacterium, Microbacterium, Corynebacterium, Bre-vibacterium) inhibited L. monocytogenes at the same extent than the complex com-munity. The decrease of relative humidity from 98 to 93% can also act as a barrieragainst L. monocytogenes, especially at the surface of cheeses with or without mi-crobial consortium. It led also to an increase of dry matter at the surface of cheesesand a decrease of pH which may contribute to the inhibition of L. monocytogenes.Interestingly, at the beginning of ripening (8 days), a temperature of 13 °C, byfavouring the growth of Lactobacillus and Leuconostoc composing the microbialconsortium, was more effective than 9°C for inhibiting Listeria. This can be ex-plained by the highest galactose metabolism and acid production. On the contraryin the control with only S. thermophilus, the ripening temperature of 13 °Cfavoured the growth of L. monocytogenes. The ripening conditions reasoned ac-cording to the composition of microbial communities could be a promising strat-egy in the control of L. monocytogenes.

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Deterministic and stochastic behavior of Listeria monocytogenessuspended cells or detached from stainless steel surfaces duringcheese manufacturingBelessi, C.-E. A.1, Gounadaki, A. S.1, Arapakh, S.1, Schvartzman, S.2, Jordan, K.2

and Skandamis, P. N.1

1. Agricultural University of Athens, Greece2. Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland

Growth probability and kinetic models for Listeria monocytogenes in response tomultiple hurdles occurring during cheese manufacturing are mainly focused onsuspended L. monocytogenes cells. This study aimed to compared: (i) thegrowth/no growth interface of L. monocytogenes cells attached on stainless steel(SS) surfaces, or in suspension, within adjusted media and (ii) the behavior ofplanktonic and detached Listeria cells during manufacturing and ripening of twopopular Greek cheeses: Feta and Graviera. A multi-strains composite of L. mono-cytogenes isolates from cheese, factory and farm in Greece and Ireland, were grownin TSBYE, MRD, Milk, Feta and Graviera cheese in the presence of SS coupons(2x5cm2) for 3d at 20°C, to obtain the following inocula: planktonic cells (P), andcells detached from the SS coupons (D). Detachment took place by the bead vor-texing method. For growth/no growth evaluation P and D cells were inoculated inTSBYE, adjusted to 5 pH (6.8-4.8) by lactic acid and at 4 aw (0.945-0.995) by NaCl.For evaluation of L. monocytogenes kinetics in cheese, P and D cells were inocu-lated at three simulated stages of Feta and Graviera manufacture: in pasteurizedmilk, after cutting the curd and after the first ripening. The growth of D cellsslightly delayed compared to P cells while it was more affected by aw than pH. Oncheese, L. monocytogenes survived throughout the ripening at low levels. The dif-ferences in probability of growth of single cells for both inocula (P and D) wereassessed by stochastic approaches. Furthermore, PFGE analysis resulted that 91%of the cells of any tested condition belonged to the cheese factory isolate. The re-sults may address safety implications relevant to the potential of attached cells toproliferate, whereas data may contribute to filling data gaps on risk assessmentof L. monocytogenes isolates from the dairy industry.

Prevalence of Listeria monocytogenes in game meatAtanassova, V.Institute of Food Quality and Food Safety, Germany

Listeria spp. are widespread in the environment. Foods of animal origin like rawmeat, milk, soft cheese and smoked fish are often contaminated by Listeria. Duringslaughtering and food processing, these bacteria can distribute and establish asspecific contaminants in difficult to clean areas of the plant and the processingenvironment. Freshly shot game meat can be readily contaminated during the cut-ting and trimming steps. Listeria endures most non heat processing steps and willbe present in packaged retail meats. Chill storage for long periods of time can re-sult in a considerable increase in numbers. In this study 797 (roe deer, red deerand wild boar) samples of wild game from freshly shot whole carcasses and 481samples from packaged game meat stored under chill conditions (+4 °C) were an-alyzed for the presence of Listeria spp. and Listeria monocytogenes. Standard meth-ods according to ISO 11290 were applied. Enrichment of meat samples was per-formed first in half Fraser, followed by full Fraser broth. Enrichment cultures werestreaked to OCLA plates. In total 7.3% (n=58) of the meat from whole carcasseswas positive for Listeria spp., with a total of 39 (4.9%) samples being contami-nated by Listeria monocytogenes. There was no difference in the prevalence inmeat from different game species. Chill stored packaged game meat showed higherprevalence rates of 20.6% (n=99) Listeria monocytogenes positive samples. Theresults show that game meat can be contaminated by Listeria monocytogenes.Prevalence was lower at the beginning of the meat processing in carcasses still un-skinned, while during elongated storage the rate can be higher in chill stored meatcuts. To reduce the risk of infection for the consumer, proper cooking is advisedduring meal preparation.

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Relationship between pathogenic profile and in vitro biofilm formationcapacity of Listeria monocytogenes strains isolated from meat, fish andprocessing plantsMeloni, D., Mazza, R., Marceddu, M., Piras, F., Mureddu, A. and Mazzette, R.Department of Animal Biology, Faculty of Veterinary Medicine, University of Sassari

In the present survey, the relationships between pathogenic profile and in vitrobiofilm formation of 106 Listeria monocytogenes strains having no epidemiologicalcorrelation and isolated from different environmental and food sources, were ana-lyzed. The isolates were grouped into different categories based on the source ofisolation: swine and poultry carcasses (14 and 13% respectively), ground meat(7%), fermented sausages (10%), raw and smoked salmons (9 and 4% respectively),swine slaughterhouse environments (3%), fermented sausage and smoked salmonprocessing plants environments (24 and 16% respectively). The quantitative as-sessment of the in vitro biofilm formation was carried out by using a microtiterplate assay with spectrophotometric reading (OD620). The cut-off value (ODc) wasequal to three time the standard deviation of the negative controls plus the averageOD reading for the same controls. The strains were divided up into four categories,based on their ability to form biofilms: no biofilm producers (OD≤ODc), weak pro-ducers (ODc<OD≤2ODc), moderate producers (2ODc<OD≤4ODc) and strong pro-ducers (4ODc<OD). The isolates were also submitted, by multiplex PCRs, toserogrouping using the target genes lmo0737, lmo1118, ORF2819, ORF2110, prs, andto the evaluation of the presence of the following virulence genes: prfA, hlyA, rrn,inlA, inlB, iap, plcA, plcB, actA and mpl. The prfa gene was present in all the strains,while the prevalence of the other genes (except for mpl and inlB) ranged from 91-94%. The 62% of the strains showed weak or moderate in vitro ability in biofilmformation, in particular serotypes 1/2b, 1/2a and 4b, frequently associated with spo-radic or epidemic listeriosis cases. The 25% of these isolates showed polymorphismfor the actA gene, producing a fragment of 268-bp instead of the expected 385-bp.The deletion of nucleotides in this gene seems to be related to enhanced virulenceproperties among these strains.

Prevalence and molecular characterization of Listeria monocytogenesin traditional fermented pork sausages produced in ItalyMazzette, R., Meloni, D., Busia, G., Melillo, R., Mureddu, A. and Piras, F.Department of Animal Biology, Faculty of Veterinary Medicine, University of Sassari

The objective of the present study was to evaluate the occurrence of Listeria mono-cytogenes in fermented sausages produced in the Sardinia region (Italy). The en-vironments and products from two large and two small processing plants, repre-sentative of the regional supply chain, were sampled to investigate prevalence andgenetic profile of Listeria monocytogenes. A total of 170 samples were collected:132 environmental samples, 12 from swine carcasses, 16 from ground meat and 10from fermented sausages at the end of ripening. Detection and enumeration ofListeria monocytogenes were carried out by using the ISO 11290-1:1996 and 11290-2:1998 protocols respectively. A subset of 28 strains, randomly selected betweenthose isolated from the positive samples, were further characterised by multiplexPCR-based serogrouping. The contamination routes of Listeria monocytogenes inthe plants were traced by use of PFGE restriction analysis. In addition, a quanti-tative assessment of the in vitro biofilm formation was also carried out. The re-sults underlined the presence of Listeria monocytogenes in all the fermentedsausages processing plants (overall prevalence in the environments, 15%). Theprevalence of Listeria monocytogenes was 37% in ground meat and 80% in the drysausages. These products did show detectable levels, always ranging below100 CFU/g. No specific serotype was recovered during the processing and ripeningof the dry sausages. The serotypes 1/2b - 1/2a showed better ability in biofilm for-mation. A high heterogeneity of pulsotypes (n= 14) occurred within the plants,with subtypes appearing to be specific to each processing plant. These results maybe due to the great diversity of the Listeria monocytogenes strains collected fromraw meat, but also to the quantities used by each plant. The large plants exhibitedthe highest PFGE profiles heterogeneity, supporting this hypothesis, since theseplants also used the greatest amount of raw meat from several different sources.

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Minimum Biofilm Eradication Concentration (MBEC)of different antimicrobials on Listeria monocytogenesand Salmonella enterica biofilmsRodrigues, D.1, Teixeira, P.1, Oliveira, R.1, Ceri, H.2 and Azeredo, J.1

1. Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade doMinho, Campus de Gualtar, Braga, Portugal

2.Biofilm Research Group, Department of Biological Sciences, University of Calgary, Canada

Inadequate disinfection of food processing environments contributes to foodbornedisease outbreaks, particularly those concerning L. monocytogenes and Salmonellaenterica. The ability of these bacteria to adhere and form biofilms on several sur-faces makes sanitation more difficult and challenging, reason why susceptibilitytests must cover not only planktonic cells but also adhered cells and biofilms.Through MBEC assessment using Calgary Biofilm Device (CBD), this work aimedat comparing the performance of four antimicrobials on L. monocytogenes and Sal-monella enterica biofilms and the disinfection efficiency between strains andspecies. Three L. monocytogenes strains (994, 1562 and CECT 4031T) and five S.enterica strains (355, CC, NCTC 13349, LT2 and ATCC 140285) were used. Biofilmswere grown on CBD for 24 hours, in Mueller-Hinton Broth, at 37°C with shaking at125 rpm. Disinfection was performed with sodium hypochlorite, benzalkoniumchloride, hydrogen peroxide and triclosan, while bacterial death was assessed bystandard plate method on Trypticase Soy Agar after sonication. Results showedthat sodium hypochlorite had the lowest MBEC values while triclosan had theworst performance, since no S. enterica biofilm eradication was achieved even atthe maximum concentration used. It was also found that, except for hydrogen per-oxide, MBEC values for L. monocytogenes biofilms were similar or inferior to thosefound for S. enterica. Significant differences on minimum survival biomass resultswere also observed between strains of the same species. Summarizing, this workhas pointed out chlorine agents as the most effective on disinfecting biofilms ofboth species used and revealed a higher susceptibility of L. monocytogenes biofilmsto disinfection in general when compared with S. enterica biofilms. Moreover, notonly interspecies but also intraspecies variability were found to influence disin-fection efficacy.

Examination of the ability of adherence, biofilm formation and sensi-tivity to some disinfectants of different Listeria monocytogenes strainsMilanov, D.1, Vidić, B.1, Petrović, J.1, Bugarski, D.1 and Ašanin, R.2

1. Scientific Veterinary Institute “Novi Sad”, Novi Sad, Republic of Serbia2. Faculty of Veterinary Medicine, Republic of Serbia

The objective of this work was to examine the capabilities of 14 Listeria monocy-togenes strains to attach on glass and to form biofilm on stainless steel surfaces.The strains originated from animals (8 strains), food (4 strains) and feed (1 strain).The referent strain was a human isolate (ATCC 19115). The number of L. monocy-togenes was determined after 3-hours of attachment and after 48-hour incubationin tryptone soy broth at 25 °C and 37 °C by the use of standard count techniqueon blood agar from tenfold dilution. Three days old biofilm formed on glass sur-faces of the selected L. monocytogenes strains was treated with paracetic acid andphenol disinfectants for 5 and 10 minutes. On the stainless steel surface thebiofilms were formed during 7 days of incubation in a tryptone soy broth supple-mented with 0.6% yeast extract (TSB-YE) at the temperature of 25 °C. The devel-oped structures were examined using scanning electron microscopy. For all theexamined L. monocytogenes strains the number of attached bacteria to glass slidesfor 3 hours of incubation in static conditions at 25 °C and 37°C ranged from 102-104 cfu/cm2. After 48 h of incubation the number of cells that grow on glass slidesdid not depend on initial attachment and for all the strains it was 105-107 cfu/cm2.Among tested Listeria monocytogenes strains, significant differences in terms oftheir ability to form biofilm were found. Seven of 14 investigated strains of Listeriamonocytogenes did not form biofilm, and only individual bacterial cells were dis-tributed over the stainless steel surface. The strains classified as biofilm producersformed structures of different appearances, from a uniform, confluent monolayerof bacterial cells to individual large, three-dimensional cell aggregates. L. mono-cytogenes cells attached to glass surface expressed higher resistance to disinfec-tants than the cells in suspension.

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Evolution of Listeria monocytogenes contaminationin poultry production: from the farms to the processing levelsMansour Chemaly, M., Toquin, M.-T., Courtillon, C., Le Nôtre, Y., Rivoal, K. and Fravalo, P.AFSSA, France

This investigation is a part of a large french program aiming to assess and updatedata regarding L. monocytogenes in poultry flocks (laying hens, broilers, turkeys)and the impact of their introduction in the slaughtering process. Samples werecollected from the flocks (5 bootswabs) which were followed up to the slaughter-houses where sampling consisted of swabs from the environment (before and aftercleaning and desinfecting procedures), caeca and products (neck skins and fillets).The isolation of L. monocytogenes was performed according to the NF ENISO11290-1 method and the identification included serotyping. RFLP-PFGE wascarried out in order to establish the clonal relationships and to trace the contam-ination between the farms and the slaughterhouses. 32% of the flocks were testedpositive for Listeria monocytogenes. The serotyping of L. monocytogenes strainsshowed that the majority belonged to the type 1/2a which presented high geneticdiversity (Simpson Index: 0.95). At the slaughterhouses, the contamination variedbetween 4 and 26% of the total sampling. Positive samples were found in the en-vironment after cleaning and desinfection and the same isolates were found in theenvironment between the slaughtering of other batches and on the products.Strains found at the farm level were clearly different from those found at theslaughterhouses. Our work highlighted the spreading of L. monocytogenes in poul-try flocks, residual and cross contamination at the slaughterhouses. Despite a highlevel of faecal contamination at the farm level, no relation could be establishedwith the contamination of slaughterhouses. This suggests that the primary pro-duction is not the main source of contamination of poultry products. Effortsshould be focused on the slaughterhouses where inefficient C&D procedures andcross contamination were the main identified sources of contamination.

A regular survey of Listeria in ready-to-eat foods (2004 – 2009)Furtado, R., Loreto Campos, M., Correia, C., Ferreira, I., Maia, C., Rosa, N., Santos, S.,Santos, M. I. and Saraiva, M.Departamento de Alimentação e Nutrição, Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P.,Lisboa, Portugal

Listeria monocytogenes is one of the most important pathogens found in food. Theconsumption of food products contaminated with this bacterium can cause liste-riosis, a disease with a high mortality rate that can affect especially vulnerablegroups. The ubiquitous nature of Listeria, its resistance to several environmentalconditions and the ability to grow at refrigeration temperatures, promote the oc-currence of contamination in any stage of the food chain. Taking simple precau-tions like thoroughly cooking foods, respecting the chill chain, proper washingfruit and vegetables eaten raw and washing hands repeatedly, reduce contamina-tion chances. The main activity of our Laboratory, it is the control of the microbi-ological quality of ready-to-eat foods served in canteens. This regular survey in-cludes the evaluation of the lay out of the physical facilities based on Reg. (EU)n. º 852/2004 and Codex Alimentarius, and the collection of food samples andswabs in surfaces and utensils. This work evaluate the presence of Listeria mono-cytogenes (VIDAS LMO2 Bio 12/11-03/04) and quantify the level of Listeria spp.(ISO 11290-2:1998/Amd:2004), in food samples collected in the establishmentssurveyed by INSA Lisboa, between 2004 and 2009. In a total of 5450 ready-to-eatfoods analysed, Listeria monoytogenes was present in 73 samples, in which 8.2%exceeded the 100 cfu/g limit. Considering that daily thousands of meals are con-sumed by risk populations, such as those of kindergarten, schools, hospitals andhomes for the elderly, and that our positive results correspond to 58.9% of samplescollected in these type of establishments, they could stand as a useful tool, demon-strating that if additional precautions are not implemented, Listeria monocyto-genes can be a potential risk for these particularly susceptible groups, even at con-centrations less than 100 cfu/g.

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Incidence of Listeria monocytogenes in Queijo FrescoRosa, N., Campos, L., Correia, C., Ferreira, I., Furtado, R., Maia, C., Santos, S.,Cunha, C. I. and Santos, M. I.Departamento de Alimentação e Nutrição, Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P.,Lisboa, Portugal

Listeria monocytogenes is a ubiquitous bacterium responsible for inumerous casesand outbreaks of listeriosis in humans, usually transmitted by the consumptionof contaminated foods, mainly those “ready-to-eat”. Being “Queijo Fresco” a ready-to-eat product, it’s one of those which have driven Listeria monocytogenes to be amajor concern to Public Health. It was the purpose of this study to evaluate thepresence of Listeria monocytogenes in “Queijo Fresco” from a number of brandswith the most commercial significance in the Lisbon area. The “Type of Commer-cial Presentation” was also studied for its influence in the occurrence of the mi-crorganism in the above mentioned food products. A total of 125 samples were ex-amined for the presence of Listeria monocytogenes. Secondary enrichments, inFraser broth, were analysed by the mini-VIDAS LMO® (bioMérieux, Durham,France), enzyme-linked fluorescent immunoassay method for Listeria monocyto-genes detection. Positive samples were confirmed by isolation on ALOA® (AESCHEMUNEX) selective agar followed by biochemical characterization with APILISTERIA® (bioMérieux, Durham, France). Statistical analysis was performedusing the Statistical Package for the Social Sciences (SPSS v13.0) software. Of 125samples, 13 (10.4%) were positive for Listeria monocytogenes. It was statisticallyproved that the Commercial Presentation influences the presence of Listeriamonocytogenes, being the cheeses sold packed less contaminated than those soldunpacked (p= ,049). This study demonstrates that Listeria monocytogenes is pres-ent in “Queijo Fresco” from a range of brands commercialized in Lisbon. The con-tamination observed, represents a potential risk for the Portuguese consumer dueto his natural taste for this product.

Risk factors for Listeria monocytogenes contaminationin French broiler flocksAury, K., Le Bouquin, S., Toquin, M.-T., Petetin, I., Le Nôtre, Y., Allain, V.,Fravalo, P. and Mansour Chemaly, M.AFSSA, France

Despite a decreasing incidence of Listeriosis in France since 1999, Listeria mono-cytogenes continue to be a public health concern. In order to update data relatingto L. monocytogenes in France in poultry production, an epidemiological study wasconducted in broiler chicken flocks between October 2005 and September 2006aiming to identify the potential risk factors associated to the presence of L. mono-cytogenes. 142 broiler chicken flocks were included in this study. The L. monocyto-genes status of these flocks was based on 5 samples of bootswabs per farm. Theflocks were considered positive if at least one sample was tested positive for L.monocytogenes. Information on potential risk factors was collected by question-naire at the same time as sample collection. The association between characteris-tic management practices and L. monocytogenes status was assessed by logistic re-gression. The prevalence of L. monocytogenes contamination in these flocks was31.7%. The risk of L. monocytogenes contamination was increased when farmerdid not respect the principle of the two areas (clean and dirty) at the poultry houseentrance. The absence of spraying at the first disinfection and/or pest control ofthe poultry house before the arrival of the next flock was found to increase therisk of being infected. When litter storage was not protected and when farm stafftake care of the other broiler chicken houses of the holding, the risk of L. monocy-togenes contamination increased significantly. For the watering system, pipetteswithout recuperator were found to be associated with a higher risk of contamina-tion than pipettes with recuperator or drinkers. This study brings new insightsfor the risk management of L. monocytogenes infection in broiler flocks.

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Portuguese sushi: is it contaminated with L. monocytogenes?Mendes, D., Furtado, R., Maia, C., Correia, C., Campos Cunha, I., Pedroso, L. and Santos, M. I.Instituto Nacional de Saúde Doutor Ricardo Jorge

Healthier nutritional lifestyles and cultural globalization have popularized theconsumption of ready-to-use products of seafood, like sushi, that were previouslyrestricted to oriental countries. Sushi is a traditional Japanese food, mostly com-posed of rice and raw fish. The sushi trend involves a higher consume of raw fish,particularly among young, urban people including pregnant women. Although fishis considered a healthy food, as with other animal products, consumption of rawmuscle incurs potential health risks such as ingestion of pathogenic bacteria. Ad-ditionally, the preparation practices involved in the production of sushi has thepotential to allow contamination, namely with Listeria monocytogenes, the agentof listeriosis, an infection that targets mainly pregnant women (and their fetuses),children, the elderly and immunocompromised individuals. In spite of the numberof cases per annum is relatively low, these infections can be acute, with mortalityup to 30%. Due to the seriousness of clinical manifestations and high rates of mor-tality in populations at risk, control and prevention of this disease, represents animportant challenge to sanitation authorities and deserves the attention of foodmicrobiologists and health professionals. In this study, 90 samples of sushi col-lected from different establishments in Lisbon, Portugal, were analyzed for theirmicrobiological status and the prevalence of pathogenic bacteria. For L. monocy-togenes detection the ISO 11290-2:1998/Amd 1:2004 method was performed. Theresults obtained showed that all samples were negative for L. monocytogenes andjust one of them revealed the presence of Listeria seeligeri. Although the number ofsamples studied was small, we believe this study provides important informationconcerning the incidence of L. monocytogenes in Portuguese sushi and also evi-dences that the consumption of this type of food probably is not a major problemin relation with this pathogen.

Prevalence of Listeria monocytogenes throughout the productionprocess of Parma ham: tracing contaminations from slaughterhousesto the final productPrencipe, V. A.1, Rizzi, V.1, Iannetti, L.1, Serraino, A.2, Calderone, D.3, Rossi, A.4, Morelli, D.1,Marino, L.1 and Migliorati, G.1

1. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy2. University of Bologna - Veterinary Medicine, Italy3. Consorzio del Prosciutto di Parma, Italy4. Centro Ricerche Produzioni Animali S.p.A., Italy

In order to evaluate Listeria monocytogenes prevalence and contamination levelsthroughout the Parma Ham production chain, 774 swine carcasses were tracedalong the whole process until the final product. Analyses were carried out on sam-ples originated from the same carcass but taken at three different points of theprocess (as carcass, fresh ham and dry-cured ham). Fecal samples were also takenfrom 498 carcasses (about 2/3 of the whole sample). The prevalence of Listeriamonocytogenes, calculated at 2.9% in the carcasses swabbed in slaughterhouses,increased up to 12.5% in fresh hams sampled when entering the manufacturingplants and eventually fell to 2.0% at the end of the production chain (de-bonedand packaged dry-cured hams). Listeria monocytogenes was isolated from only onefecal sample (prevalence 0.2%), corroborating the evidence of the low importanceof primary production as a source of contamination from this bacteria. Contami-nation levels ranged from 0.10 to 0.33 MPN/cm2 in carcasses, 0.04 to 2400 UFC/gin fresh hams, 0.04 to 100 UFC/g in dry-cured hams sampled at the end of the pro-duction chain. Only the 3.2% (n=3) of contaminated fresh hams came from con-taminated carcasses, no one among the 14 contaminated dry-cured hams camefrom contaminated fresh hams. Certainly, cutting was the stage with the highestcontamination risk throughout the Parma Ham production process. However, be-side the sharp reduction of contaminations subsequent to dry-curing, seasoningenvironment should be addressed as the critical step of the whole process for thecontaminations found in the final product. The significant differences betweenplants corroborates the importance of the processing environment as source ofcontamination from Listeria monocytogenes. Further biomolecular analyses areneeded in order to confirm the marginal role of transferring contaminations be-tween different stages of the production chain and to assess the presence of per-sistent strains inside the processing plants.

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Effect of the inoculum size on growth of L. monocytogenesin dices of poultry breastLardeux, A.-L., Gnanou-Besse, N., Doux, C. and de Courseulles, E.AFSSA, France

Traditionally the microbiological safety of foods has been established via challengetests. Since the contamination level attained by Listeria monocytogenes may dependon the initial bacterial concentration, it is important to take into account this effectwhen performing challenge tests. To date, there are, to our knowledge, few studieswhich have examined its impact in food. The effect of the inoculum size on growthof L. monocytogenes in a matrix of dices of poultry breast has been studied. This ma-trix is interesting because it is located near the interface growth/no growth of L.monocytogenes, it has a natural microflora and contains lactic acid. Challenge testshave been carried out for three batches, according to technical guidance documentof Community Reference Laboratory for Listeria monocytogenes. Thus, the fasteststrain of L. monocytogenes has been selected by a study of strains growth withpotassium lactate at different concentrations. Then, L. monocytogenes growth stud-ies were carried out at 8°C, using two contamination levels (1 cfu/g and 100 cfu/g),in vacuum-sealed bags or under modified atmosphere (50% nitrogen – 50 % car-bon dioxide). Follow-ups of natural microflora (mesophilic and lactic) have beenperformed, as well as the measurement of physical-chemical parameters (pH, awand lactic acid). L. monocytogenes was able to grow in all conditions. However,growth was higher under vacuum packaging conditions, and with a high inoculationlevel. Moreover, important differences were observed between batches. In parallel,an important growth of background microflora was observed, which probably had astrong impact on L. monocytogenes growth.

Investigation into the mechanisms of detergent induced changesin disinfectant susceptibility of attached ListeriamonocytogenesWalton, J., Hayes, R., Protheroe, R., Hill, D. and Gibson, H.University of Wolverhampton, UK

The control of Listeria through effective cleaning and disinfectant strategies iscrucial to maintaining the safety of food. The aim of this work is to investigate theeffect of detergent treatments on the susceptibility of attached Listeria monocyto-genes to subsequent disinfectant treatments. While the detergents were preparedto working concentrations, the disinfectants were lower than the recommended inuse concentration to allow quantification of changes in susceptibility. Results sofar have shown that L. monocytogenes, attached to stainless steel surfaces, becamesignificantly less susceptible (up to 3.5 log10 difference) to benzalkonium chlo-ride (BAC, 0.005% v/v) following treatment with the anionic detergents sodiumalkyl sulphate (SAS, 0.2% v/v), sodium dodecyl sulphate (SDS, 0.2% w/v) andsodium lauryl ether sulphate (SLES 0.2% v/v). L. monocytogenes also became sig-nificantly less susceptible (0.4 log10 difference) to sodium dichloroisocyanurate(NaDCC, 0.0008% w/v) following treatment with SAS but significantly more sus-ceptible (1 log10 difference) following treatment with SLES. The non-ionic deter-gent, fatty alcohol ethoxylate (FAE, 0.1% v/v), had no effect on susceptibility toeither BAC or NaDCC. The changes in susceptibility may be due to effects on cellmembrane permeability, cell surface hydrophobicity and/or reduced uptake due toefflux. Flow cytometry using the fluoresceine propidium iodide revealed signifi-cant increases in cell membrane permeability by SAS and FAE while no changewas observed with SDS. Hydrophobic interaction chromatography showed that L.monocytogenes became less hydrophobic following treatment with SAS and SDSbut FAE had no effect. Current work using ethidium bromide has shown a reduc-tion in fluorescence following treatment with SAS and SDS suggesting that the de-tergents may trigger an efflux mechanism resulting in reduced uptake of disinfec-tant. To conclude, detergents can influence the susceptibility of L. monocytogenesto BAC and NaDCC which does not appear to be related to changes in cell mem-brane permeability. However, susceptibility does appear to correlate with changesin cell surface hydrophobicity and enhanced efflux.

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Prevalence of Listeria monocytogenes in raw milk soldat vending machines in Abruzzo regionPrencipe, V. A., Scattolini, S., Sperandii, A. F. and Migliorati, G.Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy

A survey was carried out in Abruzzo region for assessing the microbial quality ofraw milk sold at self-service automatic vending machines. From January to Octo-ber 2008, 299 raw milk samples, collected at 20 distributors, were tested to de-tect Listeria monocytogenes. Moreover, the presence of the compulsory informa-tion for consumer and the observance of the proper storage temperature wereverified. 31 samples (10.5%) positive for Listeria monocytogenes were found, takenfrom 6 distributors supplied by 3 farms. 29 over the 31 positive samples were col-lected at 4 vending machines supplied by only one farm. All the Listeria monocy-togenes strains were identified as serotype 4 b. The resistance to two antimicro-bials was detected in 26.6% of the strains tested showing two different patterns(OXCC, OXL), in 74.2% to three antimicrobials with only one profile (OXCCL).PFGE analysis identified a single pulsotype specific for each farm, whose sampleshad been found contaminated by Listeria monocytogenes. The combination of theresistance patterns and restriction profile of the 29 samples isolated from A farm,identified three different subtypes. The persistence of Listeria monocytogenes inraw milk samples, supplied by A farm, confirmed that an inadequate hygiene man-agement exposes the consumer to a real risk of infection by Listeria monocyto-genes. The absence of information for the consumer and the lack of cold chainmaintenance during the distribution step could increase this risk. Therefore, it isnecessary that farmers and distributors’ managers apply continuously the owncheck and its efficacy should be verified by the Competent Authority implement-ing specific surveillance plans. However it is fundamental to inform the consumercorrectly and on continuous basis about the potential risks associated to foodproducts and on the right handling procedures during transport, storage and treat-ment of food products.

Characterization of Listeria monocytogenes strains isolated from softand semi soft cheeses sampled at retail levelAcciari, V., Torresi, M., Migliorati, G., Di Giannatale, E., Semprini, P. and Prencipe, V.Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy

The aim of this study was to characterize the 47 Listeria monocytogenes strainsisolated during a survey carried out on cheeses sampled at retail level. Five semi-soft and soft cheeses (gorgonzola, taleggio, asiago, crescenza and brie) were se-lected among the higher consumption dairy products in Italy and the most fre-quently contaminated by Listeria monocytogenes. Each strain was serotyped, testedfor susceptibility to antimicrobials and pulsotyped using AscI and ApaI PulsedField Gel Electrophoresis (PFGE). The main serotypes were 1/2a (76.6%) and 1/2c(21.3%), the 1/2b was isolated in only one sample. The antimicrobial resistancepatterns showed that most Listeria monocytogenes strains were resistant tooxacillin (97.6%), lincomicin (80.9%) and clindamycin (78.7%). The resistance totwo antimicrobials with two different resistance patterns (OXCC, OXL) was de-tected in 17% of the strains tested, to three antimicrobials with one resistanceprofile (OXCCL) in 70.2%. No strains were sensitive to all antimicrobials tested.Combination of AscI and ApaI macrorestriction patterns yielded 11 different pul-sotypes clustering in three groups. Two main pulsotypes were found by grouping21.3% and 57.4% of the strains isolated. Evaluation of PFGE profiles showed no re-lationship between pulsotypes and cheese type, manufacturer or retail outlet.Temporal distribution of the prevailing pulsotypes showed a persistent profilethroughout most of the study period, except from August to September, when adifferent pulsotype was found. Therefore the temporal variation in the prevalenceof specific strains, as reported in this survey, could be the consequence of factorsable to influence the product contamination pattern. Large scale studies will con-tribute to assess the dynamics of Listeria monocytogenes contamination.

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Preliminary report on the organisation of a food microbiologyproficiency testing program as a tool to guarantee the equivalenceof the US and IT official control systemsDi Giannatale, E., Marfoglia, C., Prencipe, V., Salini, R., Migliorati, G. and Ricci, L.Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy

After Listeria monocytogenes was isolated in products imported from Italy, in 2002the Italian Ministry of Health developed an integrated approach to assure theequivalence between the Italian and the US official control systems. A key part ofthe ongoing program has been carried out thought an ad hoc laboratory proficiencytesting with the aim of monitoring the technical skills of the official laboratories incharge of testing Listeria monocytogenes on meat products to be exported in theUSA. This study describes the organization of a proficiency testing and the protocolexperimented for preparation and verification of samples consisting in food matrixwith a basic microflora contaminated with Listeria monocytogenes at two differentlevel of contamination. The proficiency testing is an indispensable tool to assessthe performance level of a testing laboratory and to demonstrate its reliability atnational and international framework. In our specific context, the proficiency pro-gram has also concurred as one of the effective measures to guarantee the equiva-lence within the certifications issued by the Italian and US official laboratories.

High nisin susceptibility of Listeria spp. wild-type strains isolatedfrom dairies with traditional cheese preservation in PortugalPintado, C. M. B. S1,2 and Ferreira M. A. S. S.2

1. Escola Superior Agrária, Instituto Politécnico de Castelo Branco, Castelo Branco, Portugal2. Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Lisboa, Portugal

Evaluation of nisin susceptibility of 219 Listeria spp. wild-type strains isolated frommilk, cheese and cheese processing environment at given conditions was the aim ofthis work. Minimum inhibitory concentrations (MIC) were evaluated by the agarincorporation method, on TSYEGA medium. The variability of Listeria spp. suscep-tibility was very low especially at pH 5.5, with 98% of isolates showing MIC valuesbetween 10 and 50 IU ml-1 at 37°C. The increase of pH from 5.5 (average of cheesecore pH) to 6.8 (average of cheese rind pH) at 37°C resulted in an increase in theMIC values by 5 to 20 times (from 10 – 50 to 50 – 100 IU) or more (> 100 IU ml1) in68% of isolates. An increase in temperature from 20°C to 37°C at pH 5.5 resultedin an increase on MIC values only in 8% of all isolates tested. Nisin spontaneous re-sistance at a frequency of 10-3 to 10-4 was observed more frequently at pH 6.8 and37 °C. Exposure to combined stress conditions different from the ones testedherein was previously reported to increase cell resistance mechanisms and indi-cates that changes in virulence may occur concomitantly. Nevertheless, the major-ity of strains tested were able to grow at 10 IU nisin ml-1 but not at 50 IU ml-1 at pH5.5, showing a very susceptible profile. The lower pH (5.5) and temperature (20°C)tested, offered better conditions to the inhibitory action of nisin which demon-strated a good potential for use in bioactive coatings as a combined hurdle effect tocontrol L. monocytogenes strains in dairy products.

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AREA //E // Communication, risk perception and consumer practices – Social sciences in Listeria controlPOSTER PRESENTATIONS // P / 178–181

What is an appropriate level of protection for Listeria monocytogenesin foodstuffs consumed by vulnerable groups?Little, C., Gillespie, I., Grant, K., Gormley, F., Mook, P. and McLauchlin, J.Health Protection Agency Centre for Infections, UK

It is widely recognised that the consumption of contaminated food is an impor-tant route of transmission of listeriosis and a wide range of food products havebeen shown to be associated with both outbreaks and sporadic cases. Food safetycriteria for L. monocytogenes in Regulation (EC) No. 2073/2005 are applicable tothree categories of ready-to-eat (RTE) foods: absence of L. monocytogenes in 25g isrequired in RTE foods intended for infants and those for special medical purposes;while for all other RTE foods L. monocytogenes should not exceed 100 cfu/g withinshelf-life. However, vulnerable groups (pregnant women, the immunosuppressed,the elderly, and many patients in hospitals) are at particular risk of infection, andconsumption of low levels of L. monocytogenes may be of greater risk when eatenby these groups. Source attribution of L. monocytogenes infections in England andWales to RTE food sources has shown that the most important source for the over-all population were multi-component foods (e.g. sandwiches, pre-packed mixedsalads) (23.1%). Attribution of major sources was similar for the elderly population(multi-component foods (22.0%)). However, for pregnant women, beef (12.3%),milk/milk products (11.8%) and fish (11.2%) were more important sources of in-fection. Concurrently, a number of outbreak investigations in England, Wales andNorthern Ireland have revealed that infections were linked to consumption ofsandwiches served to the vulnerable group population in hospital settings. Sand-wiches tested in these investigations contained the outbreak strain of L. monocy-togenes at very low concentrations (present at <10/g) and legally would be deemedsafe by the Regulation. However, the legal limit of 100 cfu/g is not based upon for-mal dose-response formulas and given the recognised rise in listeriosis observed inthe EU, it is questionable whether the legal limit affords the level of protectiondeemed appropriate to protect the vulnerable group population.

ILCD: An Interactive Listeria culture diversity knowledgebaseAshok Kumar, J.1, Barbuddhe, S. B.1, Kalekar, S.1, Rodrigues, J.1, Chopade, N. A.2, Hain, T.3

and Chakraborty, T.3

1. ICAR Research Complex for Goa, Ela, Old Goa2.Department of Pathology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences

University, Nagpur, India3. Institute of Medical Microbiology, Justus Liebig University, Giessen, Germany

Indian Listeria Culture Database (ILCD) is an online databank of profiles devel-oped for the Listeria strains isolated in India from various sources. ILCD is basedon a relational database management system that is hyperlinked to visualize phe-notyping and genotyping results in the form of tables and DNA fingerprint imagesfor individual strains. The system has been developed using open source LAMP(Linux, Apache, MySql and PHP) tools. The database contains geographical sourceof the strain, its lineage, serotype, source of isolation (animal/human), year of iso-lation, phenotypic and genotypic characteristics, antibiotic sensitivity patterns,and DNA fingerprint (PFGE image). This is an interactive web based database sothat the data can be exchanged between laboratories electronically. A flexiblesearch system based on systematic comparisons of the database entries allowsinter-laboratory comparison of profiles. There are search options, state wise,serotype wise and so on. The parameters can be selected to search the informationrequired. Whole description of a particular strain can be visualized by clicking onidentity number of the strain. The data can be stored and accessed via internetbrowser. The web interface of ILCD allows users with no special programmingbackground or bioinformatics experience to examine the results. The user is ableto view the description of any particular strain e.g. isolated from human or ani-mal or food source; or retrieve all information about a particular strain. Being thefirst of its kind, ILCD is expected to be a very helpful tool in strengthening theconcept of ‘geographic genomics’ and will be very helpful to molecular epidemiol-ogists and those interested in research on Listeria.

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AREA //E // Communication, risk perception and consumer practices – Social sciences in Listeria controlPOSTER PRESENTATIONS // P / 178–181

Listeria spp. and the domestic environment: consumer knowledge,attitudes, risk perceptions and food-handling behavioursRedmond, E. C.Cardiff School of Health Sciences, UK

In Europe, since 2000 incidence of listeriosis has increased by >59%. This in-crease has occurred almost exclusively in adults aged > 60 years with listerial bac-teremia. The importance of the home as location for acquiring foodborne diseasehas prompted numerous studies to understanding of consumer food safety prac-tices in the domestic environment. Cognitive and behavioural data is required todevelop effective communication initiatives to improve food-handling practicesand decrease incidence. This study aims to review microbiological studies and de-termine consumer risk perceptions, knowledge and domestic food-handling be-haviours of that may contribute to the risk of listeriosis. Electronic searches ofthe Internet and library databases, personal-communication with food safety pro-fessionals and attendance at international conferences has facilitated the collec-tion of 143 cognitive and behavioural consumer food safety studies and 16 micro-biological surveys of the domestic environment carried out over the past 35years.Listeria has been isolated from up to 62% domestic kitchens and frequently con-taminated items include refrigerators (2-10%isolations) and kitchen dish-cloths/sinks (10-84%isolations). Cumulatively, 8% of all consumer food safetystudies investigated food safety of older adults; 4% of pregnant women and 16%other consumer groups. Data indicates fewer (17%) older adults are aware of Lis-teria compared to other consumer groups (32-87%). Similarly, 83% adults (aged >60years) lack knowledge of correct refrigerator temperatures, with > 80% tem-peratures exceeding recommendations. Observed risk-related handling malprac-tices implemented by adults aged 60-75years are linked to pathogenic contami-nation of kitchen surfaces and ready-to-eat (RTE) foods. Considerableinadequacies in food safety practices and behavioural influences have been iden-tified that may increase risk of listeriosis. Data comparisons between consumergroups will be presented, including likelihood of consuming RTE foods past use-by-dates, frequency of checking refrigerator temperatures and cleaning of refrig-erators. Findings will be discussed in the context of strategy development and fu-ture consumer food safety communication iniatitives designed to decreaseincidence of listeriosis.

Do you know the temperature in your refrigerator?Røssvoll, E.1, Jacobsen, E.2, Ueland, Ø.1, Einar Granum, P.3 and Langsrud, S.1

1. Norwegian Institute of Food, Fisheries and Aquaculture Research2.SIFO, National Institute for Consumer Research, Norway3.Norwegian School of Veterinary Science

The use of legislation to reduce risk is at present used at all steps of the food pro-duction chain from the primary producer to the stores. It is, however, both difficultand undesirable to manage consumer food handling through legislation. As soon asthe products are bought by the consumer, the knowledge about how the product istreated further terminates. The aim of this study was to improve food safety inthe domestic environment by risk analysis of consumer food handling and evalua-tion of risk-reducing measures. We wanted to get a better understanding of theinteraction between consumer food handling practices and the potential growth ofpathogens under domestic conditions. To investigate this we conducted a con-sumer survey, a case study, and a microbial experiment and showed that 1) con-sumers were ignorant to or unaware of the temperature in their refrigerator, 2)ready-to-eat (RTE) foods were exposed to great temperature variations by con-sumers, and 3) the fluctuations in temperature resulted in enhanced growth ofpsychrophilic pathogens as Listeria monocytogenes. In conclusion, consumers ex-pose themselves to increased risk of contracting food borne diseases by being ig-norant to or unaware of the temperature in their refrigerators. The temperaturefluctuations found in the case study enhanced pathogenic growth, and can in worstcases lead to outbreaks of food borne diseases.

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AREA //A // Biology of Listeria monocytogenesPOSTER PRESENTATION//A/ 47

Listeria monocytogenes agr system: Quorum sensing, or maybe not?Garmyn, D., Révelin, C. and Piveteau, P.Université de Bourgogne – INRA, France

Communication, often referred as Quorum Sensing, is involved in the adaptationof most bacteria to their environment. So far, the agr system is the sole communi-cation system described in the genus Listeria. The agr communication system af-fects the biology of Listeria monocytogenes during saprophytic life (biofilm for-mation) and during infection. We have recently demonstrated that agr expressionwas heterogeneous during growth of L. monocytogenes EGDe as biofilm. Our ob-servations did not fit the criteria of the Quorum Sensing paradigm. Indeed, Quo-rum sensing means that the whole population presents the same phenotype oncethe quorum is reached. In order to investigate whether the agr system fits the Quo-rum Sensing paradigm, or not, we investigated agr expression in situ by combininggfp reporters, flow cytometry and fluorescent microscopy. This experimental setup was used to revisit auto-induction, Quorum Sensing and more recent theoriesof bacterial communication during the saprophytic life of Listeria monocytogenes.

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AREA //D // Strategies for prevention and control of Listeria monocytogenesPOSTER PRESENTATIONS // P / 199–200

Utilization of Lactococcus lactis M104, a wild nisin-producing raw milkisolate, as an antilisterial adjunct in traditional Greek Graviera cheeseprocessingSamelis, I., Pappa, E., Bogovic-Matijasic, B. and Rogelj, I.National Agricultural Research Foundation, Dairy Research Institute, Greece

Recent validation studies have shown that growth inhibition of listerial contami-nation is assured during processing and storage of traditional Greek Gravieracheese in compliance with the current E.U. regulatory criteria for a maximum Lis-teria monocytogenes population of 100 cfu/g allowable in ready-to-eat foods. Since,however, the pathogen survived well during ripening, this study aimed at enhancingL. monocytogenes inactivation by using Lactococcus lactis M104, a nisin-producing(Nis+) raw milk isolate, as an antilisterial adjunct at cheese manufacture. FreshGraviera curds with added a commercial starter culture (SC), or the SC plus theNis+ strain (SC+M104), were inoculated (3 log cfu/g) with a 3-strain cocktail ofavirulent L. monocytogenes/innocua before molding in a commercial plant. Afterbrining, cheeses were ripened at 17-18ºC and 90% RH for 20 days and stored at 4ºCin vacuum. The fate of Listeria was monitored during cheese ripening (0, 1, 3, 5, 11,17, and 23 days) and storage (40 and 60 days). Changes in lactic acid bacteria (LAB)populations and main chemical parameters were also monitored, while the Nis+gene was detected in ripened cheeses and their LAB consortia by PCR. Populationsof Listeria indicators did not grow in the cheeses at any stage; they declined 10-fold(P < 0.05) within the first 5 days and survived with little death thereafter. AlthoughNis+ colonies and the Nis+ gene were isolated from the SC+M104 cheeses only, nomajor differences in Listeria survival occurred between cheeses with or withoutthe Nis+ strain. All cheeses contained enterocin (A, B and P) plus plantaricin Agenes associated with Enterococcus faecium and Lactobacillus plantarum of theNSLAB flora, and showed similar chemical changes. Thus, conditions prevailingduring Graviera cheese processing might suppress nisin (Nis+) expression by L.lactis M104 at levels insufficient to deliver or enhance Listeria inactivation.

The European Project BASELINE “Selection and improving of fit-for-purpose sampling procedures for specific foods and risks”Manfreda, G. and De Cesare, A.Department of Food Science – Alma Mater Studiorum University of Bologna, Italy

Food Safety Objectives (FSOs) and Performance Objectives (POs) are new criteriacomplementing the existing concepts of microbiological criteria. To achieve theseobjectives it is critically important a harmonisation of food safety control proce-dures. The EU project “Selection and improving of fit for purpose sampling pro-cedures for specific foods and risks”-BASELINE, funded under the FP7 pro-gramme, intends to obtain the following objectives: 1) to review the samplingschemes currently available for food authorities and food producers to collect datafor quantitative risk assessment at EU level; 2) to assess the relevance and suitablelimit values of POs and FSOs for biological risks, including Listeria monocytogenes;3) to evaluate the need for new or adapted methods for sampling and testing theidentified biological risks; 4) to develop predictive mathematical models for thetarget biological risks; 5) to validate and harmonise the sampling schemes and thealternative detection methods developed in the project; 6) to share and dissemi-nate the scientific knowledge coming out from the project to stakeholders. Theproject results will be translated in clear recommendation to the EC as well as endusers and they will have a significant impact on protection of human health. Aposter detailing the project objectives, strategies and expected impact will be pre-sented at the conference.

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AUTHORS INDEX

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Abdalla, S. D/P/128Abee, T. A/O/05Acciari, V. C/P/184, D/P/196Achtman, M. A/P/27Agyekum, K. B/O/14Aharonowitz, Y A/O/11Aish, J. C/P/125Albano, H. D/P/163Alessandria, V A/P/32, A/P/38, B/P/64, D/P/164Allain, V. D/P/190Allen, V. A/P/29Almeida, G. A/P/30, A/P/44, C/P/105, C/P/111,

C/P/113, D/P/166, D/P/165Almeida, M. T. B/P/66Alonzo, F. B/P/56Alvarez-Dominguez, C. A/P/00, B/O/19, C/PL/09,Amagliani, G. C/P/93Amar, C. C/P/91, C/P/92Amar, C. F. C/P/85Ambrosoli, R. D/P/150Ancora, M. C/P/102Andersen, J. B. B/P/182Andrew, P. B/P/54Andrew, P. D/P/128Andrew, P. W. A/P/10Anita, B. A/P/12, A/P/19Arapakh, S. D/P/172Armillotta, G. C/P/185Arnau, J. D/P/136Arneborg, N. D/P/132Asakura, H. A/P/22Ašanin, R. D/P/177Ashok Kumar, J. C/O/25, E/P/179Asséré, A. D/P/146Atanassova, V. D/P/173Aubry, C. B/O/21Auchter, M. B/P/50, D/P/148Aury, K. D/P/190Aymerich, T. D/O/43, D/P/136Azeredo, J. C/P/115, D/P/176Azevedo, I. A/P/30Bai, F. A/P/36Barbosa, J. A/P/30, C/P/111, C/P/113, D/P/160,

D/P/163, D/P/165Barbuddhe, S- B. A/P/06, A/P/07, B/P/49, C/O/25,C/P/106, C/P/107, C/P/108, C/P/117, D/P/157, E/P/179.

Barile, M. D/P/129Barre, L. C/P/103Batista, S. A/O/02Baysal, A.H. C/P/114Beaufort, A. D/O/40, D/P/154Belessi, C. A. D/P/172Belletti, N. D/O/43Benjamin, F. D/P/146Berg, J. D/P/141Bergis, H. D/O/40, D/P/154Bergmann, S. B/P/76Bergstrøm, A. B/P/182Bernardi, T. D/P/145Berrang, M. E. D/P/126Berzins, A. D/P/155Besse, N. G. C/P/103Bhosle, S. N. C/P/107, C/P/108Bianchi, D. M C/P/123Bielecka, M.K. C/O/24Bitar, A. P. B/P/58, B/P/59Blatter, S. D/P/131Block, C. S. C/O/29Boelaert, F. C/O/32, D/P/170Boone, R. C/P/94Boor, K. J. A/O/13, A/P/31Borges, S. A/P/30, D/P/163Borza, A. C/P/119, C/P/120Boscher, E. A/P/04Bosley, J. C/P/119Botello-Morte, L. A/P/43Bottero, M. T. C/P/124Boucheix, C. B/O/20Bover-Cid, S. D/O/43

Bowman, J. P. A/O/03, A/P/17Boye, M. B/P/182Brandi, G C/P/93Braun, E C/O/29Bremont, S. C/P/90Brennan, O. A/O/09Briandet, R. D/P/142Briers, Y. A/O/07Brisabois, A. A/P/01, B/P/47, C/P/82, D/P/146Brisse, S. A/O/06Brito, L. A/O/02, D/P/153, D/P/167Bruno, J. C. B/O/22Bugarski, D. D/P/177Burall, L. A/P/26Burgess, S. C. B/P/48Busia, G. D/P/175Butler, F. D/O/42Byrne, B. C/P/119Cabanes, D. B/O/23, B/P/65, B/P/66, B/P/68, B/P/70Cabanova, L. D/P/152Cabo, M.L. A/P/45Cabrita, P. A/O/02Caccia, N. D/P/142Calderone, D. D/P/194Calendar, R. A/O/10Calvo, E. A/P/43Camejo, A. B/O/23, B/P/70Cammá, C. C/P/102Campos, L. D/P/189Campos, M. L. D/P/188Candeloro, L. C/P/186Cantinelli, T. A/O/06Carneiro, L. A/P/44, D/P/166Caro, V. A/O/06Carpentier, B. C/P/103Carranza-Cereceda, C. A/P/00, B/O/19Carrasco-Marin, E. A/P/00, B/O/19Carrera, S. A/P/45Cartinhour, S. A/O/13Carvalho, F. B/O/23Carvalho, F. B/P/65Casey, P. G. B/P/69Castle, M. E/O/49Cécile, C. D/P/171Ceres, C. D/P/129Ceri, H. D/P/176Chafsey, I. A/P/08, D/P/142Chakraborty, T. A/PL/01, B/P/77, B/P/78,

C/O/25, C/P/106, E/P/179Chambom, C. A/P/08Chamot, S. D/P/145Chaturongakul, S. A/P/31Chaudhuri, S. B/P/71Chavant, P. D/P/145Chaves-Lopez, C. D/P/151Chemaly, M. D/P/147Chemaly, M. M. C/P/183, D/P/187, D/P/190Chen, J. A/O/04, A/P/36Chen, Y. A/P/16Chenal-Francisque, V. A/O/06, C/P/100, D/O/46Chiarini, E. D/P/134Chopade, N. A. E/P/179Christensen, B. B. B/P/182Civera, T. C/P/123, C/P/124Clark, A. D/O/38Cocolin, L. A/P/32, A/P/38, D/P/150, D/P/164Colaneri, C. A/P/01Comaposada, J. D/P/136Commeau, N. D/P/154Corbett, D. B/P/63Corde, Y. B/P/47Cormica, M. G. C/P/110Cornu, M. D/O/40, D/P/154Correia, C. D/P/188, D/P/189, D/P/191Cortesi, M. L. D/P/129Cossart, P. B/O/20Costas, B. D/O/37Courvalin, P. A/P/01, C/P/90Courtillon, C. C/P/183, D/P/187

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Couture, H. C/P/97Crerar, S. E/O/49Cross, L. C/P/92Cummins, J. B/P/62Cunha, C. I. D/P/189, D/P/191Czuprynski, C. B/P/60D’Orazio, V. A/P/43da Silva Felício, M.T C/O/32da Silva, M. B/O/16Dal Bello, B. D/P/150Dalgaard, P. D/O44Dalmasso, A. C/P/124D’Amico, D. C/P/112Daneshvar, M. I. A/P/33Daniel, P. D/P/171Danielsson-Tham, M.-L. C/O/30, C/P/122Datta, A. A/P/26, B/P/67David, C. B/P/54Davis, A. D/P/159D’Costa, D. C/P/106, C/P/108De Cesare, A. C/P/116de Courseulles, E. D/P/192De Martinis, E. C. D/P/148De Valk, H. C/O/31, C/P/100Dear, P. C/P/85Decastelli, L. C/P/123DeLappe, N. C/P/110Dell’Era, S. A/O/07Den Bakker, H.C. A/P/33, D/P/144Deng, X. A/O/08Desai, M. C/P/92Deschamps, J. D/P/142Destro, M. T. D/P/134, D/P/135Desvaux, M. A/P/08, D/P/142, D/P/145Desvaux, M. D/P/142Dhuri, R. B. C/P/108di Febo, T. C/P/185di Giannatale, E. C/P/186, D/P/196, D/P/197di Pasquale, F. C/P/101, D/P/151Disson, O. C/O/24Doijad, S. C/P/107, C/P/108, D/P/157Dolci, P. D/P/164Donaldson, J. R. B/P/48Donnelly, C. C/P/112Dorey, M. C/P/119, C/P/120Douey, D. C/P/119, C/P/120Doux, C. D/P/192Dowd, G. B/P/69Duarte Cruz, C. B/P/53Dumas, E. A/P/08Dusch, V. D/O/46, C/P/100Dussurget, O. B/P/55Ebersbach, T. B/P/182Egorova, I. C/P/95, C/P/96Einar Granum, P. E/P/181Eiriz, E. A/P/45Eisenreich, W. B/O/18Elemam M. M. C/P/88Eleni, S. A/P/12Eliav, H. C/O/29Ells, T. C. A/P/40Eloranta, K. C/P/120Endres, J. D/P/148Engeljohn, D. D/PL/11Ermolaeva, S. A/P/34, B/O/15, B/P/73Eugster, M. R. A/O/10, A/P/28Eusébio, C. A/P/44Eylert, E. B/O/18Fablet, A. D/P/147Faith, N. B/P/60Faith, N. G. B/P/74Faleiro, M. L. A/P/10Fall, P. A. D/P/137Fang, W. A/O/04, A/P/36Farber, J. A/P/09, C/PL/08, C/P/97, D/P/143Fávero, L.M. D/P/139Favretti, M. C/P/84Felício, T. D/P/170Félix, B. C/P/82

Fernandes, L. P. D/P/140Fernandez Guerrero, M. L. B/P/75Fernandez-Prieto, L. A/P/00, B/O/19Ferreira, I. D/P/188, D/P/189Ferreira, M.A.S.S. A/P/42, D/P/198Ferreira, P. B/O/23Ferreira, R.B. A/O/02Ferreira, V. A/P/30, D/P/160Ferreira-Dias, S. D/P/153Ferrini, G. D/P/136Fertikov, V. C/P/96Filiatrault, M. J. A/O/13Fletcher, G. B/P/53Flieger, A. A/P/03Foglini, M. C/P/93Forster, B. M. B/P/58Fortes, E. D. A/P/33, D/P/144Foster, T. D/P/159Fox, E. D/P/130Fraccalvieri, R. A/P/37Franco, B. D. G. M. C/P/118, D/P/134,

D/P/135, D/P/139Frank, J. D/P/126Fravalo, P. C/P/183, D/P/187, D/P/190Freitag, N. B/P/56Freitag, N. E. B/O/22Frewer, L.J. E/PL/14Furtado, D. N. D/P/135Furtado, R. D/P/188, D/P/189, D/P/191Fuss, A. A/O/11Gaedt Kastbjerg, V. A/P/23, B/P/72Gahan, C.G. B/P/61, B/P/62, B/P/69Galander, S. A/P/03Gallagher, D. C/O/28García-del Portillo, F. A/P/43Garg, S. D/P/157Garland, C. D. D/O/38Garriga, M. D/O/43, D/P/136Garry, P. D/P/154Garvey, P. C/P/110Gendel, S. C/P/97George, C. D/P/171Gião, M. S. D/P/127Gibbs, P. C/P/115, D/P/160Gibson, H. D/P/193Gillespie, I. C/O/26, C/O/27, C/P/91, E/P/178Gillespie, I. A. C/O/33,C/P/98Gilmartin, N. C/P/99Gilmour, M. A/P/29, C/PL/08Glenn, S. B/P/54, D/P/128Gloria, L. C/P/122Gnanou-Besse, N. D/P/192Goebel, W. B/O/18Gopal, S. A/O/11Górgolas, M. B/P/75Gormley, F. E/P/178Gotz, A. B/O/18Gouin, E. B/O/20Goulão, M. M. A/P/42Goulet, V. C/O/31, D/O/46, C/P/100Gounadaki, A. S. D/P/172Graham, S. A/P/29Gram, L. A/P/23, B/P/72Granier, S. A. A/P/01Grant, K. C/O/26, C/O/27Grant, K. C/O/33, C/P/85, C/P/91, C/P/92,

C/P/98, E/P/178Grassi, M. A. C/P/123Graves, L. C/O/36, A/P/33Grépinet, O. B/P/47Groelly, J. D/P/145Guevara, R. E. C/P/86, C/P/89, E/O/47Guillet, C. C/O/24Gunjal, P. A/P/06Haahr Kallipolitis, B. A/P/24Haase, J. A/P/27Hagen, N. A/O/11Hain, T. B/P/77, B/P/78Hain, T. E/P/179

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Halbedel, S. A/P/03Halberg Larsen, M. A/P/15Handan Baysal, A D/P/162Hara, K. C/P/119Hayes, R. D/P/193Hearty, S. C/P/99Heavin, S. A/O/09, A/P/13Hébert, K. D/P/143Hebraud, M. D/P/145Hébraud, M. A/P/08, D/P/142Heir, E. D/P/161Helsel, L. O. A/P/33Henriques, A. B/P/65Herrera, J.J.R. A/P/45Hershko-Klement, A. C/O/29Herve-Bazin, M. C/P/90Higuchi, Y. A/P/35Hill, C. B/PL/06, B/P/50, B/P/69Hill, D. D/P/193Hof, H. A/P/27Hogg, T. C/P/105, C/P/113, D/P/160, D/P/165Holch, A. B/P/72Holck, A. D/P/141Holman, D. B. A/P/40Hormazábal, J.C. D/PL/12Hunt, K. D/P/130Huszczynski, G. C/P/119, C/P/120Huwiler, S. A/P/28Hyytia-Trees, E. C/O/36Iannetti, L. D/P/194Ida, M. A/P/35Igimi, S. A/P/18, A/P/22Imhof, R. D/O/41Ingmer, H. A/P/15, A/P/23, D/P/132Ioannis, D. A/P/12, A/P/19Iugovaz, I. C/P/94Jablonska, J. B/P/81Jacobsen, E. E/P/181Jakabi, M. C/P/118Javanmardi, F. D/P/133Jean, A. C/P/97Jen, C. B/P/54Jenö, P. A/O/02Jiang, L. A/P/36Jofré, A. D/P/136Johansson, J. A/O/12, A/P/46Johnson, D. D/P/156Join-Lambert, O. C/O/24Jordan, K. D/O/42, D/P/130, D/P/172Joyce, S. A. B/P/61, B/P/69Jusdado, J. J. B/P/75Kai, A. A/P/35Kalebar, S. C/P/106, C/P/107, C/P/108, E/P/179Kallipolitis, B. A/P/02Kalorey, D. R. C/P/106Kalorey, D. R. A/P/06, A/P/07, B/P/49,

C/O/25, C/P/117Kaminskaya, A. A/P/34Kanarata, S. C/P/109Kaneko, S. A/P/35Kantikova, M. D/P/152Karatzas, K. A/O/09Kathariou, S. B/P/74Kawamoto, K. B/O/16, C/P/121Kearney, A. A/P/29Keevil, C. W. D/P/127Keich, U. A/O/13Kent, H. A/P/29Kerouanton, A. B/P/47Kiil Nielsen, P. A/P/02, A/P/24Kim, J. B/P/74King, L. C/O/31, C/P/100Klumpp, J. A/O/01Knabel, S. J. C/O/35, C/P/124Knoll, L. B/P/60Knudsen, G. B/P/182Kolbasov, D. C/P/95, C/P/96Korikanthimath, V. S. C/O/25Korkeala H. A/P/11, D/P/155

Koshy, A. A/O/03Kotwal, S. C/P/117Koutsoumanis, K. D/O/37Kreft, J. A/O/11Krishnendu, M. B/P/77Kurkure, N. V. A/P/07, B/P/49, C/O/25Kusumoto, A. C/P/121Lad, M. D/P/159Ladely, S. D/P/126Laksanalamai, P. A/P/26Laksanalamai, P. B/P/67Landgraf,M. D/P/135Langsrud, S. D/P/161, E/P/181Lardeux, A,-L. D/O/40, D/P/192Larios, O. A/P/29Larsen, M. H. A/P/23, D/P/168Larsson, J.T. C/O/34Latorre, L. A/P/37, C/P/87, C/P/116Laurent, E. C/P/100, D/O/46Lawrence, M. L. B/P/48Le Bouquin, S. D/P/190Le Monnier, A. C/O/24, C/P/90Le Nôtre, Y. C/P/183, D/P/187, D/P/190Leclercq, A. A/O/06, C/O/24, C/P/90,

C/P/100, D/O/46Lecuit, M. A/O/06, B/PL/03, C/O/24, C/P/83,

C/P/90, D/O/46Lee Chang, K. J. A/O/03Lee, B. A/P/29Leisner, J.J. A/P/15Leite, I. F. C/P/115Lelli, R. C/P/185Lemos Vaz-Velho, M D/P/169Lengeling, A. B/P/76Li, Z. A/O/08Licht, T. R. B/P/182Lienenklaus, S. B/P/81Lindbäck, T. B/P/79Lindström, M. A/P/11Little, C. C/P/91, E/P/178Little, C. L. C/O/33, C/P/98Liu, J. C/P/119Lobacz, A. D/O/45Loessner, M. J. A/O/01, A/O/07, A/O/10, A/P/28Lombard, B. D/P/146Lomonaco, S. C/P/123, C/P/124Loose, M. B/P/78Lopes, J.F. C/P/115Lopez, J. A/O/06Lopez-Valladares, G. C/O/30Lortholary, O. C/O/24Lourenço, A. A. D/P/167Louro, P. D/P/153Luber, P. C/PL/10Luchansky, J. B. B/P/74Luciani, M. C/P/185MacDonald, D. C/PL/08Machado, H. D/P/167Maciag, P. C. B/O/17Madarame, H. B/P/52Madrazo-Toca, F. A/P/00, B/O/19Magalhães, R. A/P/30, A/P/44, C/P/105,

C/P/111, C/P/113, D/P/166, D/P/165Magnani, M. C/P/93Mahmoodi,S. M. M. D/P/133Maia, C. D/P/188, D/P/189, D/P/191Maia, C. H. A/P/42Maia, R. L. E/O/48Mailles, A. C/P/83Makela, P. C/O/32Makela, P. D/P/170Makino, S.I . B/O/16,C/P/121Maklon, K. C/P/121Malik, S.V. S. C/O/25, C/P/106Mamzer-Bruneel, M.F. C/O/24Manfreda, G. C/P/116Marault, M. C/P/82Marceddu, M. D/P/174Marfoglia, C. C/P/184, D/P/197

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Marie-Christin, M. D/P/171Marino, L. D/P/194Mariscotti, J. A/P/43Marques, N. A/P/10Marquis, H. B/P/58, B/P/59Martins, C. G. D/P/139, C/P/118Martins, M. B/P/68Maruyama, T. A/P/22Mascola, L. C/P/86, C/P/89Mastronicolis, S. A/P/12, A/P/19Mateus, T. E/O/48Matos, J. C/P/91, C/P/92Matsubara, S. B/O/16Mattila, M. A/P/11Mazza, R. D/P/174Mazzette, R. D/P/174, D/P/175McCarthy, S. D/P/156McIlwham, S. A/P/09McKeown, P. C/P/110McLauchlin, J. C/P/98, E/P/178Meinersmann, R. D/P/126Mejlholm, O. D/O/44Mele, P. C/P/87Meloni, D. D/P/174, D/P/175Mena, C. D/P/166Mendes, D. D/P/191Mereghetti, L. B/P/47Miccolupo, A. A/P/37Migliorati, G. C/P/102, C/P/184, C/P/186,

D/P/194, D/P/195, D/P/196, D/P/197,Milanov, D. D/P/177Milillo, S. R. A/P/33Milohanic, E. A/P/20Moes, S. A/O/02Moller Nielsen, E. C/O/34Monden, S. A/P/18, A/P/22Monk, I. R. B/P/50, B/P/51Mook, P. C/O/26, C/O/27, C/O/33, C/P/91,

C/P/98, E/P/178Morax, L. A/P/28Morelli, D. D/P/194Morey, R. E. A/P/33Mormile, A. D/P/129Morrissey, J. A/P/13Morvan A. C/P/90Mosher, M. C/P/119, C/P/120Moubareck, A. C/P/90Moubarek, C. A/P/01Moussan Ake, F. A/P/21Mraheil, M. A. B/P/77Müller, S. A/P/25Mureddu, A. D/P/174, D/P/175Murray, C. C/P/97Murru, N. D/P/129Mutanda, C. C/P/119Nadon, C. A/P/29, C/PL/08Nakama, A. A/P/22, A/P/35Nanduri, B. B/P/48Netterling, S. A/P/46Neuhaus, K. A/P/25Neves, E. D/P/153Nightingale, K. A/P/16Nijthavorn, N. C/P/109Nilsson, R. E. A/P/17Nir-Paz, R. C/O/29Normanno, G. A/P/37Noronha, L. D/P/158Nucera, D. M. C/P/123Nwaiwu, O. A/P/39, D/P/159O’ Kennedy, R. C/P/99O’Brien, M. D/P/130O’Brien, S. J. C/O/26O’Byrne, C. A/O/09, A/P/13,A/P/41Oevermann, A. B/P/52Okada, N. A/P/18Okada, Y. A/P/18, A/P/22Okutani, A. A/P/22Oliveira, M. A. D/P/148Oliveira, R. D/P/176

Oliveira, W. P. D/P/140Oliver, H.F. A/O/13Omiccioli, E. C/P/93Ondrusch, N. A/O/11Orsi, R. H. A/P/33, A/O/13Padilha Silva, W. A/P/05Pagotto, F. A/P/09, C/PL/08, C/P/94, D/P/143Paitan, Y. C/O/29Paparella, A. C/P/101, D/P/151Parihar, V.S. C/P/117Parisi, A. A/P/37, C/P/87, C/P/116Pasche, B. B/P/76Paterson, Y. B/O/17Patil, V. A/P/07Paz, R.-N. A/O/10Pedroso, L. D/P/191Pepe, O. D/P/129Pereira de Martinis, E. C. D/P/140Petetin, I. D/P/190Petrovic, J. D/P/177Phillippy, A.M. A/O/08Pihier, N. C/P/100, D/O/46Pillich, H. B/P/78Pinfold, T. A/O/03Pintado, C.M.B.S. A/P/42, D/P/198Pinto, E. A/P/10Piras, F. D/P/174, D/P/175Pittman, J.R. B/P/48Pizarro-Cerdá, J. B/O/20Plaza, B. M. B/P/75Pompilii, C. C/P/185, C/P/186Ponnala, L. A/O/13Portanti, O. C/P/185Portnoy, D. B/PL/04Pouillot, R. C/O/28, C/P/97Poulsen, K. P. B/PL/05, B/P/60Preising, N. P. B/P/50Prencipi, V. C/P/102, D/P/196, D/P/197Prencipe, V. A. C/P/184, D/P/194, D/P/195Principe, V. C/P/185Protais, J. D/P/147Protheroe, R. D/P/193Pucciarelli, M.G. A/P/43Pushkareva, V. B/O/15Quinon, E. D/P/145Raengpradub, S. A/P/31Ramos-Vivas, J. A/P/00Rantsiou, K. A/P/32, A/P/38, D/P/150, D/P/164Rawool, D. B. C/O/25Redmond, E. C. E/P/180Rees, C. D/P/159Rees, C.E.D. A/P/39Reis, F. B. D/P/140Renier, S. D/P/142Ribot, E. C/O/36Ricci, L. C/P/186, D/P/197Riedel, C. U. B/P/51, B/P/50, D/P/148Ristori, C.A. D/P/139, C/P/118Rivoal, K. D/P/147, D/P/187Rizzi, V. C/O/32, C/P/102, C/P/184, D/P/194Roberts, I. S. B/P/54, B/P/63Roche, S. M. B/P/47Rodrigues, D. D/P/176Rodrigues, J. A/P/06, C/P/106,C/P/107,

C/P/108, E/P/179Rodriguez-Del Rio, E. A/P/00, B/O/19Ronholm, J. A/P/14Roof, S. E. A/P/33Rørvik, L. M. B/P/79Rosa, N. M. C/P/87Rosa, N., D/P/188, D/P/189Ross, T. A/P/17Ross, W. C/P/97Ross, W. H. A/P/16Rosshaug, P. S. D/P/168Rossi, A. D/P/194Røssvoll, E. E/P/181Roussel, S. A/P/01, C/O/34,C/P/82Rousselm, S. D/P/146

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Rowlands, R. E. G. C/P/118, D/P/139Rubinstein, E. B/O/20Saá Ibusquiza, P. A/P/45Sabol, A. C/O/36Sahaghian, R. B/P/74Sahu, S. B/P/67Salah, S. D/O/46Salem, S. C/P/100Salini, R. D/P/197Salvat, G. D/P/147Salzberg, S. L. A/O/08Santagada, G. A/P/37Santos, I. A/P/30, A/P/44, C/P/111, C/P/113,

D/P/166, D/P/165Santos, M. I. A/P/42, D/P/188, D/P/189, D/P/191Santos, S. D/P/188, D/P/189Saraiva, M. D/P/188Sauders, B.D. A/P/33Savelli, C. C/PL/08Scattolini,S. D/P/195Schechner, V. C/O/29Scherer, S. A/P/25Schirmer, B. C. D/P/141Schittler, L. D/P/138Schlech, W. B/P/80Schlech, W.F. C/PL/07Schughart, K. B/P/76Schuler, S. B/P/54Schuppler, M. A/O/07Schvartzman, S. D/O/42, D/P/172Scortti, M. C/O/24Scott, V. N. A/P/16Seavey, M. M. B/O/17Secic, I. B/P/79Selyaninov, Y. C/P/95Semprini, P. C/P/185, C/P/186, D/P/196Serio, A. C/P/101, D/P/151Serraino, A. D/P/194Seuberlich, T. B/P/52Sewell, D. B/O/17Shama, G. D/P/128Shaw, I. A/P/41Shimojima, Y. A/P/35Silins I. D/P/155Silva, A. D/P/153Silva, J. A/P/44, D/P/158, D/P/163Silva, W. P. D/P/138Silveira Naleiro, E. A/P/05Simpson, J. M. A/P/16Skandamis, P. N. D/P/172Skovager, A. D/P/132Škuntova, O. D/P/152Slepkov, E. R. B/P/58, B/P/59Smith, M. A. B/O/14Solodova, E. B/P/81Somervuo, P. A/P/11Somov, G.P. B/P/73Sonegaonkar, A. A/P/07Sorvillo, F. C/P/86Sousa, S. B/O/23, B/P/66, B/P/68Souza, V. M. D/P/140Sperandii, A. C/P/102Sperandii, A. F. D/P/195Stabler, R. A/P/05Stahl, J.-P. C/P/83Steigerwalt, A. G. A/P/33Stephan, R. A/O/01, D/P/131Stessl, B. C/P/104Stoll, R. A/O/11, B/O/18Stollewerk, K. D/P/136Sukhapesnaa, J. C/P/109Sun, Q. A/O/13Suzuki, H. A/P/18, A/P/22Swain, B. K. C/O/25Swaminathan, B. A/P/33Switt, A. M. D/P/144, D/P/160Tam Dao, T. C/P/82, D/P/146Tamburro, M. C/P/85Tasara, T. A/O/01, D/P/131

Teixeira, A. P. B/P/47Teixeira, P. A/P/30, A/P/44, C/P/105, C/P/111,

C/P/113, C/P/115, D/P/158, D/P/160, D/P/163,D/P/165, D/P/166, E/O/48

Teixeira, P. D/P/176Témoin, S. B/P/47Tenenhaus-Aziza, F. D/O/42Tham, M. D. C/P/117Tham, T. N. B/O/20Tham, W. C/O/30, C/P/117Thierry-Bled, F. D/O/46Thomaz, M. R. S. D/P/140Todorov, S. D/P/134, D/P/135, D/P/169Tola, S. C/P/87Tonucci, F. C/P/93Toquin, M. -T. C/P/��, D/P/��, D/P/��Torres, R. B/P/75Torresi, M. C/P/184, D/P/196Tortorello, M. L. A/O/08Tran, C. A/O/06Tresse, O. D/O/39Trigo, M.J. A/O/02Trottier, Y. C/P/94Trout-Yakel, K. A/P/29Truelstrup Hansen, L. A/P/40Tyler, S. A/P/29Ueland, Ø. E/P/181Umap, S. A/P/07Upham, J. C/P/119, C/P/120Utratna, M. A/P/41Vaikousi, H. D/O/37Vaillant, V. C/O/31, C/P/83, C/P/100Valinsky, L. C/O/29van der Veen, S. A/O/05Van Domselaar, G. A/P/29Van Stelten, A. A/P/16Vandevelde, M. B/P/52Varfolomeev, A. A/P/34Vasantrao Kurkure, N. A/P/06Vázquez, D. A/P/45Vazquez-Boland, J. C/O/24Vaz-Velho, M. D/P/134Velge, P. B/P/47Verran, J. D/P/132Vidić, B. D/P/177Vignaud, M. C/P/82Virgilio, S. C/P/87Vishal Singh, P. C/O/30Vongkamjan, K. D/P/144, D/P/160Voronin, M. C/P/95Wadge, A. E/PL/13Waidmann, M. S. B/P/50, B/P/51, D/P/148Walton, J. D/P/193Wang, J. B/P/63Wang, W. A/O/13Warke, S. A/P/07, B/P/49Weiss, S. B/P/81Whitehead, K. D/P/132Whiting, R. C. A/P/16Wiedmann, M. A/PL/02, A/O/13, A/P/16,

A/P/31, A/P/33, D/P/160, D/P/144Wilhelm, T. C/P/122Williams, D. B/O/14Winkelströter, L. K. D/P/148Wong, H. T. L. A/P/39Wren, B.W. A/P/05Xayarath, B. B/P/57Yurov, D. A/P/34Zahle Andersen, A. A/P/24Zaytseva, E.A. B/P/73Zeiman, E. A/O/10Zeppa, G. D/P/150Zhang, W. A/O/08Zuliani, V. D/P/154Zurbriggen, A. B/P/52Zweifel, C. D/P/131

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Universidade Católica Portuguesa – Escola Superior de BiotecnologiaPorto, 2010

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