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International Symposium on Problems of Listeriosis Ficha Técnica

LIVRO DE ACTAS DO CONGRESSO: ISOPOL XVII Intrenational 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 WELCOMETOISOPOLXVII

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

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

More than 350 delegates from the clinical, veterinary, and public health areas as well as the food industry, will be present, representing ca. 45 countries. This diversity will certainly provide for a very stimulating and plural atmosphere which is sure to promote rich discussions and exchanges of ideas.

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

We are also thankful to our sponsors, which, in many diﬀerent ways, have greatly 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 history. It is best met in person; take the opportunity to stay awhile, to meet the people, to enjoy the charms of this city of contrasts – you may ﬁnd the need to return again to enjoy it all!

Paula Cristina Maia Teixeira

TABLE OF CONTENTS

KEYNOTESPEECH

Listeria monocytogenes: a multifaceted model 21 Cossart, P.

AREA //A // Biology of Listeria monocytogenes

PLENARYLECTURES A / PL/ 01 The pangenome of Listera spp. 22 Chakraborty, T. A / PL/ 02 Ecology of L. monocytogenes and Listeria spp. in natural 22 and food associated environments Wiedmann, M.

ORALPRESENTATIONS A/O/ 01 The cold shock associated proteins (Csps) promote tolerance 33 of different environmental stresses and host cell invasion of Listeria monocytogenes Tasara, T., Klumpp, J., Loessner, M. J. and Stephan, R. A/O/ 02 Different levels of flagellin detected during growth of 33 Listeria monocytogenes strains at low temperature Cabrita, 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 34 Listeria monocytogenes strains in the presence of unprotonated organic acids Lee Chang, K. J., Pinfold, T., Koshy, A. and Bowman, J. P. A/O/ 04 Internalin profiling, multilocus sequence typing and 34 virulence assesments suggest evolutionary history of the Listeria monocytogenes-Listeria innocua clade Chen, J. and Fang, W. A/O/ 05 The SOS response of Listeria monocytogenes is involved 35 in stress resistance, mutagenesis, and biofilm formation van der Veen, S. and Abee, T. A/O/ 06 Clonal diversity of Listeria monocytogenes, a worldwide perspective 35 Chenal-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 36 feature a unique mode of division Briers, Y., Dell’Era, S., Schuppler, M. and Loessner, M. J. A/O/ 08 Pangenomic analysis of Listeria monocytogenes 36 Deng, 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 media on GAD system activity Karatzas, K.-A., Brennan, O., Heavin, S. and O’Byrne, C. P. A/O/ 10 Role of Listeria monocytogenes tyrosine phosphatases 37 in conferring listeriophage resistance Paz, 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 38 Ondrusch, N., Gopal, S., Fuss, A., Hagen, N., Stoll, R., Aharonowitz, Y. and Kreft, J. A/O/ 12 RNA-structures acting at a distance 38 Johansson, J. A/O/ 13 Deep RNA sequencing of Listeria monocytogenes revealsoverlapping 39 and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs Oliver, H. F., Orsi, R. H., Ponnala, L., Keich, U., Wang, W., Sun, Q., Cartinhour, S., Filiatrault, M. J., Wiedmann, M. and Boor, K. J.

POSTERPRESENTATIONS A/P/ 00 CompartmentalizationofIFN-gammaandIL-6receptorssignalling 62 in Listeria monocytogenes phagosomes: immune vesicles Ramos-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 63 from non human sources in France over a ten year period Granier, 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 63 of Listeria monocytogenes to cell wall acting antibiotics Kiil Nielsen, P. and Kallipolitis, B. A/P/ 03 Phenotypic analysis of selected listerial secretion mutants 64 Halbedel, S., Galander, S. and Flieger, A. A/P/ 04 Distribution of serotypes and pulsotypes of L. monocytogenes 64 in pig farms (France 2008) Boscher, E. A/P/ 05 Comparative phylogenomics of Listeria monocytogenes reveals 65 an adaptation profile Silveira Nalério, E., Padilha Silva, W., Stabler, R. and Wren, B. W. A/P/ 06 Serotyping and PFGE patterns of Listeria monocytogenes isolated 65 from poultry meat Vasantrao Kurkure, N., Kalorey, D. R., Rodrigues, J., Gunjal, P.1 and Barbuddhe, S. B. A/P/ 07 Genotypic characterization of Listeria monocytogenes isolated 66 from fresh leafy vegetables Warke, 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 66 by genomic and proteomic analyses Desvaux, M., Dumas, E., Chafsey, I., Chambon, C. and Hébraud, M. A/P/ 09 Investigating differences in lineages of Listeria monocytogenes 67 using comparative genomics McIlwham, S., Farber, J. and Pagotto, F. A/P/ 10 Autolysis in Listeria monocytogenes – a proteomic approach 67 Pinto, E., Marques, N., Andrew, P. W. and Faleiro, M. L. A/P/ 11 Role of flhA, cheR and motA in growth of Listeria monocytogenes 68 at low temperature Mattila, M., Lindström, M., Somervuo, P. and Korkeala, H. A/P/ 12 Theeffectofaceticacid(atpH5.5)orbenzoicacid(atneutralpH)onlipid 68 composition and fluidity of Listeria monocytogenes membrane Ioannis, D., Anita, B., Eleni, S. and Mastronicolis, S. A/P/ 13 Elucidation of the responses to weak acids in the human pathogen 69 Listeria monocytogenes using gene microarrays O’Byrne, C., Heavin, S. and Morrissey, J. A/P/ 14 The immunogenic surface protein IspC acts as an N-Acetylglucosaminidase 69 in Listeria monocytogenes serotype 4b Ronholm, J. A/P/ 15 Listeria monocytogenes EGD chitinolytic activity is regulated 70 by carbohydrates but also by the virulence regulatory gene, PrfA Halberg Larsen, M., Leisner, J. J. and Ingmer, H. A/P/ 16 Infectious dose curves for guinea pigs challenged with a Listeria 70 monocytogenes epidemic clone strain and a strain carrying a naturally-occurring virulence-attenuating mutation in inlA show a significant shift in median infectious dose Nightingale, K., Van Stelten, A., Simpson, J. M., Chen, Y., Scott, V. N., Ross, W. H., Whiting, R. C. and Wiedmann, M. A/P/ 17 MudPITbasedproteomicanalysisofalkalineadapted,environmentally 71 persistent Listeria monocytogenes strains Nilsson, R. E., Ross, T. and Bowman, J. P. A/P/ 18 RpoN,thealternativesigmafactor,isassociatedwiththegrowthphase 71 transition and pathogenesis in Listeria monocytogenes Okada, Y., Suzuki, H., Monden, S., Igimi, S. and Okada, N. A/P/ 19 Cellular lipid fatty acid pattern differences between reference 72 and ice-cream isolate of Listeria monocytogenes as response to cold stress Anita, B., Ioannis, D. and Mastronicolis, S. A/P/ 20 Role of the dihydroxyacetone metabolism in the resistance 72 of Listeria innocua to pediocin Milohanic, E. A/P/ 21 Glucose transport system in Listeria monocytogenes 73 and their impact on virulence gene expression Moussan Ake, F. A/P/ 22 Antimicrobial susceptibilities of Listeria monocytogenes isolatedinJapan 73 Monden, 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 74 on Listeria monocytogenes adhesion and invasion in Caco-2 cells Gaedt Kastbjerg, V., Halberg Larsen, M., Ingmer, H. and Gram, L. A/P/ 24 The SOS response in Listeria monocytogenes –astresssurvivalmechanism 74 Kiil Nielsen, P., Zahle Andersen, A. and Haahr Kallipolitis, B. A/P/ 25 Acid shock triggers heavy metal detoxification in Listeria monocytogenes 75 Müller, S., Neuhaus, K. and Scherer, S. A/P/ 26 Listeria monocytogenes mutants defective in growth at 5°C 75 and in high salt environment Burall, L., Laksanalamai, P. and Datta, A. A/P/ 27 Revival of 5,000 Listeria strains from Seeliger’s historical collection 76 with a semi-automated microbiological pipeline Haase, J., Hof, H. and Achtman, M. A/P/ 28 Two point mutations are responsible for the lack of glycosidic substition 76 in 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 77 clinical isolates during a large foodborne outbreak Gilmour, 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 77 and clinical Listeria monocytogenes isolates Barbosa, 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, 78 but only sigma B appears to be important for survival of acid, alkaline and oxidative stress Chaturongakul, S., Raengpradub, S., Wiedmann, M. and Boor, K. J. A/P/ 32 Virulence gene expression in Listeria monocytogenes strainsisolated 78 from different sources Alessandria, V., Rantsiou, K. and Cocolin, L. A/P/ 33 Differentiation of Listeria monocytogenes, Listeria innocua 79 and Listeria marthii, a novel Listeria species isolated from the natural environment, Finger Lakes National Forest Graves, 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 79 and lmo1638 genes Yurov, D., Varfolomeev, A., Kaminskaya, A. and Ermolaeva, S. A/P/ 35 Antimicrobial susceptibilities of Listeria monocytogenes isolated 80 from retail beef, pork and poultry in Japan Ida, M., Shimojima, Y., Kaneko, S., Higuchi, Y., Nakama, A. and Kai, A. A/P/ 36 Genetic basis of two low pathogenic L. monocytogenes strains 80 with apparent phospholipase C activity Jiang, L., Bai, F., Chen, J., and Fang, W. A/P/ 37 Molecular genotyping and antimicrobial resistance 81 of Listeria monocytogenes from foods and the environment Parisi, A., Miccolupo, A., Fraccalvieri, R., Latorre, L., Normanno, G. and Santagada, G. A/P/ 38 Virulence transcriptome analysis of Listeria monocytogenes 81 by application of microarrays in vitro and in situ Rantsiou, K., Alessandria, V. and Cocolin, L. A/P/ 39 Effects of growth conditions on surface properties of Listeria; 82 a proposed role for AI-2 Wong, H. T. L., Nwaiwu, O. and Rees, C. E. D. A/P/40 Stress behaviour of a Listeria monocytogenes 568 Lmo1634 transposon mutant 82 Truelstrup Hansen, L., Holman, D. B. and Ells, T. C. A/P/41 Investigation of the conditions that trigger the activation 83 of the alternative sigma factor σB in Listeria monocytogenes Utratna, M., Shaw, I. and O’Byrne, C. A/P/42 Characterization of Listeria monocytogenes 1/2a, 1/2b, 1/2c and 4b 83 by amplified fragment length polymorphism and evaluation of their geographical distributions in Portugal Maia, 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 84 of the LPXTG family Botello-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 84 derived from food and food-processing bakery plant Eusébio, 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 85 to obtain L. monocytogenes cells adapted to BAC Saá 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 85 Listeria monocytogenes Netterling, S. and Johansson, J. A/P/47 Listeria monocytogenes agr system:Quorumsensing,ormaybenot? 162 Garmyn, D., Révelin, C. and Piveteau, P.

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

PLENARYLECTURES B / PL/ 03 How Listeria monocytogenes breaches host barriers 23 Lecuit, M. B / PL/ 04 Secretion of a novel L. monocytogenes cyclic dinucleotide 23 into the cytosol of infected host cells activates an innate immune pathway Portnoy, D. A. B / PL/ 05 Diagnosis andclinical management of listeriosis in ruminants and camelids 24 Poulsen, K. P. B / PL/ 06 Listeriolysin S – a second haemolysin with a role 24 in the virulence of Listeria monocytogenes Hill, C.

ORALPRESENTATIONS B/O/ 14 Probiotics reduce Listeria monocytogenes-induced tissue invasion 40 and stillbirths in pregnant guinea pigs Smith, M. A., Agyekum, K. and Williams, D. B/O/ 15 Listeriolysin O favors Listeria monocytogenes growth 40 in co-culture with the ciliate Tetrahymena pyriformis Ermolaeva, S. and Pushkareva, V. B/O/ 16 Atopy is a risk factor for listeriosis 41 Kawamoto, K., Matsubara, S., Da Silva, M. and Makino, S.-I. B/O/ 17 Cancer immunotherapy using novel Listeria monocytogenes-bacterial 41 vectors to target the vasculature of progressive tumors Paterson, Y., Seavey, M. M., Maciag, P. C. and Sewell, D. B/O/ 18 The intracellular carbon metabolism of Listeria monocytogenes 42 in comparison to that of other intracellular bacterial pathogens replicating in mammalian host cells Gotz, A., Eylert, E., Stoll, R., Eisenreich, W., and Goebel, W. B/O/ 19 LIMP2 links late phagosomal trafficking with the onset of the innate 42 immune response to Listeria monocytogenes: a role in macrophage activation Fernandez-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 43 in mammalian cells Tham, T. N., Gouin, E., Rubinstein, E., Boucheix, C., Cossart, P. and Pizarro-Cerdá, J. B/O/ 21 Listeria innate immune evasion by peptidoglycan modification 43 Aubry, C. B/O/ 22 Constitutive activation of the central virulence transcriptional regulator 44 PrfA enhances Listeria monocytogenes pathogenesis but reduces bacterial fitness outside of the host Freitag, N. E. and Bruno, J. C. B/O/ 23 The lvfH gene of Listeria monocytogenes encodes a novel 44 virulence factor highly activated during infection Carvalho, F., Camejo, A., Ferreira, P., Sousa, S. and Cabanes, D. POSTERPRESENTATIONS B/P/ 47 A Listeria monocytogenes strainisstillvirulentdespitenon-functional 86 major virulence genes: Optical Mapping shows a potential mechanism Roche, 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 86 strains in murine macrophages Donaldson, 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: 87 Possible source of food borne infection Kalorey, D. R., Warke, S., Kurkure, N. V. and Barbuddhe, S. B. B/P/ 50 Agr-dependent peptide sensing in L. monocytogenes – Effects on 87 biofilm formation, virulence and global gene expression Waidmann, 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 88 a temperature-dependent and -independent mechanism Waidmann, M. S., Monk, I. R. and Riedel, C. U. B/P/ 52 Bovine cranial nerve Schwann cells express E-cadherin, 88 a candidate key-player in the brainstem invasion of Listeria monocytogenes in cattle Madarame, H., Seuberlich, T., Vandevelde, M., Zurbriggen, A., and Oevermann, A. B/P/ 53 Pathogenic potential of Listeria monocytogenes isolates 89 from New Zealand seafood premises: implications for control Durante Cruz, C. and Fletcher, G. B/P/ 54 Copper homeostasis and virulence in Listeria monocytogenes 89 David, C., Schuler, S., Glenn, S., Jen, C., Andrew, P. and Roberts, I. S. B/P/ 55 Pattern of cytokine production during murine listeriosis 90 Dussurget, O. B/P/ 56 Analysis of the post-translocation chaperone PrsA2 and its unique 90 role in facilitating Listeria monocytogenes pathogenesis Alonzo, F. and Freitag, N. B/P/ 57 CtaP is a multifunctional cysteine-transport associated protein 91 required for Listeria monocytogenes pathogenesis Xayarath, B. B/P/ 58 Enzymatic activity of the metalloprotease of Listeria isregulatedbypH 91 Forster, B. M., Pavinski Bitar, A., Slepkov, E. R. and Marquis, H. B/P/ 59 Identification of propeptide residues regulating the compartmentalization, 92 maturation, and activity of the broad-range phospholipase C of Listeria monocytogenes Slepkov, E. R., Pavinski Bitar, A. and Marquis, H. B/P/ 60 A mouse model of fetoplacental Listeria monocytogenes 92 infection and abortion Poulsen, K. P., Faith, N., Laura Knoll, L. and Czuprynski, C. B/P/ 61 Listeria infection of the insect model system Galleria mellonella 93 Joyce, S. A. and Gahan, C. G. B/P/ 62 Construction of a murinised Listeria monocytogenes H7858 (4b) 93 strain for improved murine infection Cummins, J. and Gahan, C. B/P/ 63 Theroleofaphosphoinositidephosphataseintheintracellularsurvival 94 of Listeria monocytogenes Wang, J., Corbett, D. and Roberts, I. S. B/P/ 64 Virulence gene expression in Listeria monocytogenes strainsisolated 94 from different sources Alessandria, V. B/P/ 65 Targeted Signature-tagged mutagenesis for phenotype screening 95 of Listeria monocytogenes mutants Henriques, A., Carvalho, F. and Cabanes, D. B/P/ 66 Listeria monocytogenes cellular infection triggers 95 tyrosine-phosphorylation of Myosin IIA, a new protein involved in invasion Almeida, M. T., Cabanes, D. and Sousa, S. B/P/ 67 Invasion profile of Listeria monocytogenes strains involved 96 in invasive and gastroenteritis listeriosis outbreaks Laksanalamai, P., Sahu, S. and Datta, A. B/P/ 68 Molecular characterization of the Vip-Gp96 interaction 96 Martins, M., Cabanes, D. and Sousa, S. B/P/ 69 Investigation of the molecular mechanisms by which 97 Listeria monocytogenes grows in the mammalian gall bladder Dowd, G., Joyce, S., Casey, P. G., Hill, C. and Gahan, C. G. B/P/ 70 Role of cadmium efflux system in Listeria monocytogenes virulence 97 Camejo, A. and Cabanes, D. B/P/ 71 Investigation of chitinase as a potential virulence factor 98 in Listeria monocytogenes Chaudhuri, S. B/P/ 72 Sub-lethal concentrations of common disinfectants do not 98 influence survival and growth of Listeria monocytogenes in whole blood Holch, A., Gaedt Kastbjerg, V. and Gram, L. B/P/ 73 Internalin LRR domain variability in Listeria monocytogenes 99 isolated from different hosts Zaytseva, E. A., Ermolaeva, S. A. and Somov, G. P. B/P/ 74 Reduced virulence of an adenylosuccinate lyase transposon mutant 99 of a serotype 4b strain of Listeria monocytogenes Faith, N. G., Kim, J.-W., Kathariou, S., Sahaghian, R. and Luchansky, J. B. B/P/ 75 Manifestations and outcome of listeriosis in adult patients 100 Fernández Guerrero, M L., Mancebo Plaza, B., Torres, R., Górgolas, M. and Jusdado, J. J. B/P/ 76 Model for human Listeriosis: in vivo monitoringoforallyinfectedmice 100 using bioluminescent Listeria monocytogenes Bergmann, S., Lengeling, A., Pasche, B. and Schughart, K. B/P/ 77 Galleria mellonella as model system to study Listeria species-specific 101 and Listeria monocytogenes serotype-specific pathogenesis Mraheil, M. A., Krishnendu, M., Hain, T. and Chakraborty, T. B/P/ 78 Listeria monocytogenes ActA is a key player in evading autophagic recognition 101 Pillich, H., Loose, M., Hain, T. and Chakraborty, T. B/P/ 79 Non-haemolytic and hypovirulent Listeria monocytogenes became 102 haemolytic and virulent after passage through mice Secic, I., Lindbäck, T. and Rørvik, L. M. B/P/ 80 Mutants of Listeria monocytogenes (Lm)resistanttothepolycationic 102 peptide protamine appear to be attenuated for virulence Schlech, W. B/P/ 81 The role of plasmacytoid dendritic cells in the course 103 of Listeria monocytogenes infection Solodova, E., Lienenklaus, S., Jablonska, J. and Weiss, S. B/P/ 182 Oxygen restriction increases the infection potential of 103 Listeria monocytogenes – a transcriptional analysis Andersen, 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 monocytogenes and listeriosis

PLENARYLECTURES C / PL/ 07 Update on clinical aspects of listeriosis: What’s new? 25 Schlech, W. F. C / PL/ 08 A Canadian outbreak of listeriosis due to deli-meat: 25 Driving change in the food safety system Farber, J. M., Pagotto, F., Gilmour, M., Nadon, C., Savelli, C., and MacDonald, D. C / PL/ 09 Listeria monocytogenes phagocytic strategy 26 Alvarez-Dominguez, C.

ORALPRESENTATIONS C/O/ 24 Human listeriosis due to Listeria ivanovii 45 Guillet, 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 45 Barbuddhe, 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: 46 quantifying the risk Mook, P., Grant, K., O’Brien, S. J. and Gillespie, I. C/O/ 27 Risk factors for death in Listeria monocytogenes infection, 46 England and Wales, 1990 to 2008 Mook, P., Grant, K. and Gillespie, I. C/O/ 28 A discrete event model to track Listeria monocytogenes 47 in the retail environment Pouillot, R. and Gallagher, D. C/O/ 29 Clinical and epidemiological aspects of listeriosis in Israel 47 Hershko-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 duringhalfacenturyinSweden 48 Lopez-Valladares, G., Danielsson-Tham, M.-L., Vishal Singh, P. and Tham, W. C/O/ 31 Estimated incubation periods for listeriosis vary according 48 to clinical form of disease Goulet, V., King, L., Vaillant, V. and De Valk, H. C/O/ 32 Listeria monocytogenes infoodandanimalsintheEuropeanUnionin2008 49 da Silva Felício, M. T., Rizzi, V., Boelaert, F. and Makela, P. C/O/ 33 Listeria monocytogenes infection in the over 60s in England 49 between 2005 and 2008: a retrospective case-control study utilising market research panel data Gillespie, I. A., Mook, P., Little, C. L. and Grant, K. C/O/ 34 Better and faster typing, MLVA – shall we play together? 50 Larsson, J. T., Roussel, S. and Moller Nielsen, E. C/O/ 35 comK prophage junction fragments in Listeria monocytogenes contain 50 SNPs that differentiate subclones of ECII and ECIII that are unique to individual meat and poultry processing plants in the U.S. Knabel, S. J. C/O/ 36 Genetic diversity of Listeria monocytogenes measuredbymultiple-locus 51 variable-number tandem repeat analysis (MLVA) Hyytia-Trees, E., Sabol, A., Graves, L. and Ribot, E.

POSTERPRESENTATIONS C/P/ 82 Semi-automated repetitive sequenced-based PCR compared 104 to pulsed field gel electrophoresis for Listeria monocytogenes sub-typing Roussel, 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 104 with high case fatality Mailles, A., Vaillant, V., Lecuit, M. and Stahl, J.-P. C/P/ 84 Listeria monocytogenes: identification and subtyping 105 Favretti, M. C/P/ 85 Virulotyping of Listeria monocytogenes byhighresolutionmeltanalysis 105 Amar, C. F., Tamburro, M., Dear, P. and Grant, K. C/P/ 86 Risk factors for nonperinatal listeriosis mortality in Los Angeles County, 106 California, 1992–2004 Guevara, R. E., Mascola, L. and Sorvillo, F. C/P/ 87 Molecular typing of Listeria monocytogenes isolatedfromovinesausage 106 Nives, 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 107 Listeria monocytogenes in food within 24 h Elemam, M. M. C/P/ 89 Perinatal listeriosis in Los Angeles County, California, 1992–2004 107 Guevara, R. E. and Mascola, L. C/P/ 90 Antimicrobial resistance of Listeria monocytogenes human strains 108 isolated since 1926 in France Morvan, 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 108 Grant, K., Amar, C., Matos, J., Mook, P., Little, C. and Gillespie, I. C/P/ 92 Use of Fluorescent Amplified Fragment Length Polymorphism 109 for improved typing of Listeria monocytogenes Matos, J., Amar, C., Desai, M., Cross, L. and Grant, K. C/P/ 93 Towardsanapplicationforfieldsamplesofamultipathogenplatform 109 for molecular detection of raw milk pathogens Omiccioli, E, Amagliani, G., Brandi, G., Tonucci, F., Foglini, M. and Magnani, M. C/P/ 94 Detection and recovery of Listeria species from stainless steel 110 Boone, R., Iugovaz, I, Trottier, Y.-L. and Pagotto, F. C/P/ 95 Natural carriage of Listeria in fresh-water fish in Russia 110 Egorova, I., Voronin, M., Selyaninov, Y. and Kolbasov, D. C/P/ 96 Prevalence of Listeria in wild fauna in Russia 111 Egorova, I., Fertikov, V. and Kolbasov, D. C/P/ 97 Quantitative assessment of the exposure to Listeria monocytogenes 111 from soft-ripened cheese consumption in North America: a joint FDA/Health Canada project Gendel, 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 112 neighbourhood deprivation Gillespie, 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 112 for on-site detection of L. monocytogenes Gilmartin, N., Hearty, S. and O’ Kennedy, R. C/P/ 100 Food Investigation of sporadic cases of neuroinvasive listeriosis 113 Goulet, 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 Vaccinium myrtillus, 113 Prunus domestica and Myrtus communis against Listeria monocytogenes Serio, A., Di Pasquale, F. and Paparella, A. C/P/ 102 High throughput quantitative detection of Listeria monocytogenes 114 in food by RealTime PCR Cammà, C., Ancora, M., Rizzi, V., Sperandii, A., Prencipe, V. and Migliorati, G. C/P/ 103 Bibliographic study concerning procedures for preparing 114 environmental samples for analyses, regarding to L. monocytogenes Barre, L., Carpentier, B. and Gnanou Besse, N. C/P/ 104 Persistent L. monocytogenes isolatesfromAustrianandIrishdairies 115 show the same phenotypic and genetic background Stessl, B. C/P/ 105 Epidemiological data on listeriosis in Portugal: 2003–2008 115 Almeida, G, Magalhães, R., Hogg, T. and Teixeira, P. C/P/ 106 Diversity among Listeria monocytogenes isolated from humans 116 Kalekar, 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 116 Rodrigues, J., Kalekar, S., Bhosle, S. N., Doijad, S. and Barbuddhe, S. B. C/P/ 108 Characterization of Listeria species isolated from milk 117 D’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 117 in Thailand Kanarat, S., Nijthavorn, N. and Sukhapesna, J. C/P/110 Listeria monocytogenes in Ireland – Epidemiology and Molecular Typing 118 Cormican, M. G., DeLappe, N., McKeown, P. and Garvey, P. C/ P/111 Characterization of Listeria monocytogenes isolated 118 from human cases of listeriosis occurred in Portugal in 2008 Magalhães, R., Barbosa, J., Santos, I., Almeida, G. and Teixeira, P. C/ P/112 Post-processing environmental contamination of surface-ripened 119 soft cheese during affinage D’Amico, D. and Donnelly, C. C/ P/113 Genetic diversity of Listeria monocytogenes isolated 119 from Portuguese cheeses Almeida, G., Magalhães, R., Santos, I., Barbosa, J., Hogg, T. and Teixeira, P. C/ P/114 Prevalence of Listeria spp.inretailrawgroundbeefinIzmir,Turkey: 120 A comparison of standard cultural method and Fluorescent in situ Hybridization (FISH) technique for detection Handan Baysal, A. C/ P/115 Using ListexP100™ for Listeria monocytogenes detection in foods 120 Flores Lopes, J., Ferreira Leite, I., Azeredo, J., Gibbs, P. and Teixeira, P. C/ P/116 Mapping of molecular profiles associated to Listeria monocytogenes 121 food isolates circulating in Italy De Cesare, A., Parisi, A., Latorre, L. and Manfreda, G. C/ P/117 Zoonotic aspects of Listeria monocytogenes isolates 121 from zebu dairy animals Parihar, 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 122 Listeria monocytogenes in naturally contaminated raw meat products Gravato Rowlands, R. E., Asturiano Ristori, C. A., Geraldes Martins, C., Jakabi, M. and Gombossy de Melo Franco, B. D. C/ P/119 ComparisonofMOPS-BLEBandFraserassecondaryenrichmentbroths 122 for Listeria monocytogenes Upham, 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) 123 broth as primary enrichment broths for Listeria monocytogenes Upham, J., Mosher, M., Borza, A., Huszczynski, G., Dorey, M., Eloranta, K. and Douey, D. C/ P/121 Isolation and characterization of Listeria monocytogenes 123 from asazuke (Japanese light pickles) Maklon, K., Kusumoto, A., Makino, S.-I. and Kawamoto, K. C/ P/122 Grouping of human Listeria monocytogenes isolates 124 Lopez-Valladares, G., Danielsson-Tham, M.-L. and Tham, W. C/ P/123 Characterization of Listeria monocytogenes strains isolated 124 from food and environmental samples Nucera, 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 125 the four epidemic clones of Listeria monocytogenes Lomonaco, S., Civera, T., Dalmasso, A., Knabel, S. J. and Bottero, M. T. C/ P/125 Listeria monocytogenes incidents reported to the UK 125 Food Standards Agency from 2000 to 2008 Aish, J. C/ P/183 Genetic diversity of Listeria monocytogenes in broiler flocks 126 Courtillon, C. Toquin, M.-T., Le Nôtre, Y., Fravalo, P. and Mansour Chemaly, M. C/ P/184 Tracing Listeria monocytogenes contaminations throughout 126 the processing chain of a typical Italian pork meat product using Pulsed Field Gel Electrophoresis (PFGE) characterization Annunziata Prencipe, V., Acciari, V., Torresi, M., Migliorati, G., Marfoglia, C. and Valentina Rizzi, V. C/ P/185 Productionandvalidationofcapture-ELISAkitbasedonmonoclonal 127 antibodies specific for Listeria monocytogenes in foodstuffs Portanti, 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 127 containing Listeria innocua Pomilio, F., Ricci, L, Di Giannatale, E., Semprini, P., Candeloro, L. and Migliorati, G.

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

PLENARYLECTURES D / PL/ 10 Risk assessment using the microbiological criteria: arguments for 27 zero tolerance (USDA) other viewpoint (Europe): Risk-based microbiological criteria for Listeria monocytogenes in RTE foods Luber, P. D / PL/ 11 USDA regulatory approach and considerations for the control 27 of Listeria monocytogenes Engeljohn, D. D / PL/ 12 Listeria monocytogenes,anemergentpathogeninChileandLatinAmerica 28 Hormazábal, J. C.

ORALPRESENTATIONS D/O/ 37 Time Temperature Indicators can be used as an effective 52 Risk Management Tool for Listeria monocytogenes in Ready-To-Eat foods Koutsoumanis, K., Vaikousi, H. and Costas, B. D/O/ 38 Safety of Salad Leaves and Herbs 52 Garland, C. D. and Clark, A. D/O/ 39 Environmental factors affect adhesion of Listeria monocytogenes 53 to inert surfaces through flagellum expression Tresse, O. D/O/ 40 Shelf-life laboratory durability and challenge studies for Listeria 53 monocytogenes in ready-to-eat foods: a presentation of the European technical guidance document intended for laboratories Beaufort, A., Bergis, H., Cornue, M. and Lardeux, A.-L. D/O/ 41 Successful strategies against Listeria monocytogenes in Switzerland 54 Imhof, R. D/O/ 42 Absence of Listeria monocytogenes growthduringrawmilkcheesemaking: 54 a modelling approach Jordan, K., Schvartzman, S. Butler, F. and Tenenhaus-Aziza, F. D/O/ 43 A predictive model to set high pressure processing criteria 55 for Listeria monocytogenes inactivation on dry-cured ham Bover-Cid, S., Belletti, N., Garriga, M. and Aymerich, T. D/O/ 44 Application of a validated predictive model to prevent growth 55 of Listeria monocytogenes in ready-to-eat foods – importance for product development and risk management Mejlholm, O. and Dalgaard, P. D/O/ 45 Application of predictive microbiology to control the growth 56 of Listeria monocytogenes – dairy products as an example Lobacz, A. D/O/ 46 Characterization of Food Alert for Listeria monocytogenes in France in 2008 56 Leclercq, A., Dusch, V., Salah, S., Laurent, E., Chenal-Francisque, V., Thierry-Bled, F., Lecuit, M., Goulet, V. and Pihier, N.

POSTERPRESENTATIONS D/P/ 126 Colonization of a newly constructed commercial chicken further 128 processing plant with Listeria monocytogenes Berrang, M. E., Meinersmann, R., Frank, J. and Ladely, S. D/P/ 127 Episcopic differential interference contrast/epifluorescence microscopy 128 to characterise in situ Listeria monocytogenes biofilms on stainless steel surfaces Gião, M. S. and Keevil, C. W. D/P/ 128 Temperature dependent defect in biofilm formation 129 by Listeria monocytogenes Abdalla, S., Glenn, S., Shama, G. and Andrew, P. D/P/ 129 Mode of action of Lactococcus lactis sa31 antimicrobial peptide 129 on Listeria monocytogenes ½ c Barile, 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 130 in Irish Farmhouse cheese processing facilities Jordan, K., Fox, E., O’Brien, M. and Hunt, K. D/P/ 131 Phenotypic and genotypic characteristics of Listeria monocytogenes 130 strains isolated from a convenience food-processing plant Blatter, S., Stephan, R., Tasara, T. and Zweifel, C. D/P/ 132 Influence of flow direction on the adhesion of Listeria monocytogenes 131 to brushed stainless steel surfaces Skovager, A., Whitehead, K., Ingmer, H., Verran, J. and Arneborg, N. D/P/ 133 Occurrence of Listeria monocytogenes inrawmilkanddairyproducts 131 in Kazerun, Iran Mehdi Mahmoodi, S. M. and Javanmardi, F. D/P/ 134 Antilisterial mode of action of bacteriocin ST182Gu produced 132 by Enterococcus casseliflavus isolated from guava Todorov, 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 132 strain Lactococcus lactis subsp. lactis DF4Mi or commercial nisin Nader 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 133 in sliced dry cured ham Stollewerk, 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 133 in tropical shrimp Abdoulaye Fall, P. D/P/ 138 Listeria monocytogenes and Listeria innocua in slaughter line of 134 a swine meatpacking plant in Rio Grande do Sul State, Brazil Schittler, L. and Padilha Silva, W. D/P/ 139 Listeria monocytogenes in raw meat products marketed in the city 134 of Sao Paulo, Brazil: Incidence and counts data for risk assessment Ristori, 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 135 in combination with extract of Lippia sidoides Cham. in cold-smoked surubim fish broth Barbosa 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 135 by virulent bacteriophages and protective cultures Holck, A., Schirmer, B. C. and Berg, J. D/P/ 142 Surface colonization by Listeria monocytogenes: role of the flagella 136 in the biofilm formation process Desvaux, 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 136 on stainless steel surfaces: Lessons learned from the 2008 Listeriosis outbreak Hébert, K., Farber, J. and Pagotto, F. D/P/ 144 Listeriophage ecology and diversity on dairy farms 137 Vongkamjan, K., Moreno Switt, A., den Bakker, H. C., Fortes, E. D., and Wiedmann, M. D/P/ 145 Evaluationofcurativeandpreventivedecontaminationtreatments 137 on Listeria monocytogenes biofilms with a new screening system Quinon, E., Chamot, S., Groelly, J., Chavant, P., Bernardi, T., Desvaux, M. and Hebraud, M. D/P/ 146 Pulsed Field Gel Electrophoresis, conventional and molecular 138 serotyping on Listeria monocytogenes: An European Proficiency Testing Inter-laboratory Trial Félix, B., Roussel, S., Tam Dao, T., Asséré, A., Lombard, B. and Brisabois, A. D/P/ 147 Detection of Listeria spp.inrawandpasteurizedliquidegg-products 138 and in the egg-breaking plants environment Rivoal, K., Fablet, A., Chemaly, M., Salvat, G. and Protais, J. D/P/ 148 Biofilm formation by Listeria monocytogenes isolatesunderconditions 139 that mimic food and digestive tract Kretli Winkelströter, L., Oliveira, M. A. and De Martinis, E. C. D/P/ 149 Biofilm formation of Listeria monocytogenes EGDe depends 139 on temperature and nutrient availability Auchter, M., Endres, J., Waidmann, M. S. and Riedel, C. U. D/P/ 150 Antimicrobial activity of lactococcal and enterococcal strains 140 isolated from artisanal products from North West of Italy toward Listeria monocytogenes Dal Bello, B., Rantsiou, K., Ambrosoli, R., Zeppa, G. and Cocolin, L. D/P/ 151 Plant-based strategies for Listeria monocytogenes control in foods 140 Paparella, A., Serio, A., Chaves-Lopez, C. and Di Pasquale, F. D/P/ 152 Scientific studies for survival of Listeria monocytogenes 141 in dairy products and its application in practice Cabanova, L., Škuntova, O. and Kantikova, M. D/P/ 153 The effect of chilling temperatures on the virulence 141 of Listeria monocytogenes isolates with different origins Neves, 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 142 L. monocytogenes contamination on diced bacon at the plant Bergis, H., Commeau, N., Zuliani, V., Cornu, M., Beaufort, A. and Garry, P. D/P/ 155 Contamination of Listeria monocytogenes in a cold-smoked pork 142 processing plant using brining injections Berzins, A., Silins, I. and Korkeala, H. D/P/ 156 Effects of GRAS products on growth of Listeria monocytogenes 143 during cold storage of salmon fillets McCarthy, S. and Johnson, D. D/P/ 157 Heavy-metal and detergent resistance of Listeria species isolates 143 from milk processing environments Doijad, S., Garg, S. and Barbuddhe, S. B. D/P/ 158 Detection of Listeria monocytogenes in lettuce sold at markets 144 and supermarkets in Porto, Portugal Noronha, L., Silva, J. and Teixeira, P. D/P/ 159 Preliminary analysis of structure and chemical composition of 144 extracellular polymeric substance produced by Listeria monocytogenes Nwaiwu, O., Lad, M., Davis, A., Foster, T. and Rees, C. D/P/ 160 Ecology and persistence of Listeria monocytogenes strainsinfermented 145 meat sausage processors from the Northern region of Portugal Ferreira, 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 andslaughterhouse 145 bacteria on surfaces at relevant environmental conditions Langsrud, S., Møretrø, T. and Heir, E. D/P/ 162 Control of L. monocytogenes bylysozymecombinedwitholiveleafextract 146 in edible pullulan film coated on chicken breast fillets Handan Baysal, A. D/P/ 163 Evaluation of antilisterial activity by lactic acid bacteria 146 Borges, S., Barbosa, J., Albano, H., Silva, J. and Teixeira, P. D/P/ 164 Molecular methods to assess Listeria monocytogenes route 147 of contamination in a dairy processing plant Cocolin, L., Alessandria, V., Dolci, P. and Rantsiou, K. D/P/ 165 Persistence of L. monocytogenes inartisanalcheeseproducingplants 147 Almeida, G., Santos, I., Magalhães, R., Barbosa, J., Hogg, T. and Teixeira, P. D/P/ 166 Occurrence of Listeria monocytogenes in food products collected 148 in Portugal from retail establishments and food plants Mena, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G. and Teixeira, P. D/P/ 167 Listeria monocytogenes biofilms grown at 12°C showed reduced 148 susceptibility to sanitizers Afonso Lourenço, A., Machado, H. and Brito, L. D/P/ 168 Modelling growth of Listeria monocytogenes in cheese as function 149 of environmental variables Sand Rosshaug, P. and Hallberg Larsen, M. D/P/ 169 Characterization of anti-Listerial bacteriocin produced by 149 Lactobacillus plantarum ST8SH, a strain isolated from Bulgarian salami Todorov, S. D. and Lemos Vaz-Velho, M. D/P/ 170 EFSA’s proposal for an EU-wide retail survey on Listeria monocytogenes 150 in selected categories of ready-to-eat food products Frank Helena Boelaert, F. H., Felício, T. and Makela, P. D/P/ 171 Ripening conditions: an asset to control L. monocytognes incheeses 150 Callon, C., Picque, D., Corrieu, G. and Montel, M.-C. D/P/ 172 Deterministic and stochastic behavior of Listeria monocytogenes 151 suspended cells or detached from stainless steel surfaces during cheese manufacturing Belessi, 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 151 Atanassova, V. D/P/ 174 Relationship between pathogenic profile and in vitro biofilmformation 152 capacity of Listeria monocytogenes strains isolated from meat, fish and processing plants Meloni, D., Mazza, R., Marceddu, M., Piras, F., Mureddu, A. and Mazzette, R. D/P/ 175 Prevalence and molecular characterization of Listeria monocytogenes 152 in traditional fermented pork sausages produced in Italy Mazzette, R., Meloni, D., Busia, G., Melillo, R., Mureddu, A. and Piras, F. D/P/ 176 Minimum Biofilm Eradication Concentration (MBEC) of different 153 antimicrobials on Listeria monocytogenes and Salmonella enterica biofilms Rodrigues, D., Teixeira, P., Oliveira, R., Ceri, H. and Azeredo, J. D/P/ 177 Examination of the ability of adherence, biofilm formation and sensitivity 153 to some disinfectants of different Listeria monocytogenes strains Milanov, D., Vidić, B., Petrović, J., Bugarski, D. and Ašanin, R. D/P/ 187 Evolution of Listeria monocytogenes contamination in poultry production: 154 from the farms to the processing levels Mansour 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) 154 Furtado, 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 155 Rosa, 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 155 in French broiler flocks Aury, 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? 156 Mendes, 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 156 in dices of poultry breast Lardeux, A.-L., Gnanou-Besse, N., Doux, C. and de Courseulles, E. D/P/ 193 Investigation into the mechanisms of detergent induced changes 157 in disinfectant susceptibility of attached Listeria monocytogenes Walton, J., Hayes, R., Protheroe, R., Hill, D. and Gibson, H. D/P/ 194 Prevalence of Listeria monocytogenes throughouttheproductionprocess 157 of Parma ham: tracing contaminations from slaughterhouses to the final product Prencipe, 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 inrawmilksoldatvendingmachines 158 in Abruzzo region Prencipe, V. A., Scattolini, S., Sperandii, A. F. and Migliorati, G. D/P/ 196 Characterization of Listeria monocytogenes strainsisolatedfromsoft 158 and semi soft cheeses sampled at retail level Acciari, 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 159 proficiency testing program as a tool to guarantee the equivalence of the US and IT official control systems Di 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 159 from dairies with traditional cheese preservation in Portugal Pintado, C. M. B. S and Ferreira M. A. S. S. D/P/ 199 Utilization of Lactococcus lactis M104, a wild nisin-producing 163 raw milk isolate, as an antilisterial adjunct in traditional Greek Graviera cheese processing Samelis, I., Pappa, E., Bogovic-Matijasic, B. and Rogelj, I. D/P/ 200 The European Project BASELINE “Selection and improving 163 of 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

PLENARYLECTURES E / PL/ 13 How can the social sciences help us understand the prevalence of 29 listeriosis in the UK? Wadge, A. E / PL/ 14 Consumer perceptions, behaviour and microbial food safety; 29 implications for Listeria control Frewer, L. J.

ORALPRESENTATIONS E/O/ 47 Efforts to update and perform health education on listeriosis in 57 Los Angeles County, California, by the County of Los Angeles Department of Public Health Guevara, R. E. E/O/ 48 Awareness of listeriosis among Portuguese pregnant women 57 Mateus, T., Maia, R. L. and Teixeira, P. E/O/ 49 The development and progress of a risk-based strategy 58 for the control of Listeria monocytogenes in New Zealand Castle, M. and Crerar, S.

POSTERPRESENTATIONS E/P/ 178 What is an appropriate level of protection for Listeria monocytogenes 160 in 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 160 Ashok 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, 161 attitudes, risk perceptions and food-handling behaviours Redmond, E. C. E/P/ 181 Do you know the temperature in your refrigerator? 161 Røssvoll, E., Jacobsen, E., Ueland, Ø., Einar Granum, P. and Langsrud, S.

Authors Index 165

PLENARY LECTURES

KEYNOTESPEACH

Listeria monocytogenes: a multifaceted model Cossart, P.* Institut Pasteur, Unité des Interactions Bactéries cellules, Inserm U604, INRA USC2020 Listeria monocytogenes is an intracellular pathogen responsible for severe human food-borne infections characterized by gastroenteritis, materno-fetal infections and brain infections with a mortality rate of 30%. The disease is mainly due the capacity of Listeria to cross three host barriers: the intestinal barrier, the maternofetal barrier and the blood brain barrier. It is also due to the capacity of the organism to survive and replicate in macrophages and to enter and replicate in non phagocytic cells. We use a combination of approaches to understand the mechanisms which allow establishment and maintenance of a Listeria infection. We then investigate if our findings have a general significance. In nearly three decades of molecular and cellular investigations, the study of Listeria momocytogenes and of listeriosis has led to new concepts in infection biology and in several other areas of biology including cell biology, microbiology, molecular medicine and genomics.

* Plenary Supported by FCT

21 AREA // A // Biology of Listeria monocytogenes PLENARYLECTURES // PL/ 1–2

REFERENCE The pangenome of Listera spp. A / PL Chakraborty, T.* 01 Institute of Medical Microbiology, Justus-Liebig University of Giessen, Germany Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has served as an invaluable model for intracellular parasitism. We have sequenced representative genomes comprising all species for the genus Listeria as well as strains representing clonal lineages of the pathogenic species of L. monocytogenes. Comparative genome analysis now provides clear evidence indicating that the various 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 strain that harboured many of the currently known virulence factors. Thus, non-pathogenic Listeria are compromised with regard to their ability to live in the cytoplasm of infected host cells but have acquired genes enabling their growth in soil and decaying vegetation. Comparative transcriptome analysis of intracellular growth has also uncovered additional levels of adaptive evolution in growth among the different lineages of L. monocytogenes. Analysis of the pan-genome of L. monocytogenes strains revealed an extensive, as yet unexplained gene-repertoire in these genomes, and provides evidence for the evolution of these strains by gathering genes from organisms in different environmental and host niches.

* Plenary Supported by FCT

REFERENCE Ecology of L. monocytogenes and Listeria spp. in natural A / PL and food associated environments 02 Wiedmann, M.* Department of Food Science, Cornell University, Ithaca, NY, USA Listeria spp., including the pathogen L. monocytogenes, can be isolated from a variety of environments, often at considerable frequency. A number of our studies have specifically shown that Listeria spp. can often be isolated from > 20% of samples collected from natural, urban, and farm environments and can also be commonly isolates from food associated environments (e.g., processing plants, retail environments). Molecular characterization and subtyping tools not only typically reveal considerable diversity among Listeria spp. isolates from different environments, but also provide evidence for (i) association between certain subtypes or species and specific environments (suggesting existence of specific Listeria eco- types) as well as (ii) long-term persistence of specific Listeria strains in different environments. For example, in one of our studies, L. seeligeri and L. welshimeri were significantly associated with natural environments (p<0.0001), while L. innocua and L. monocytogenes were significantly associated with urban environments (p<0.0001). Increased efforts to characterize Listeria isolates from various sources 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 from pathogenic clades to virulence attenuated clades, including a number of distinct clades which contain homologues for some but not all of the genes critical for virulence 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 associated environments. Strain persistence in these environments appears to be an important contributor to food contamination and foodborne listeriosis cases. Identifi- cation of persistent strain and their elimination in food associated environments thus has become a critical component in efforts to reduce human listeriosis cases.

* Plenary Supported by FLAD

22 AREA // B // Listeria monocytogenes as a human and animal pathogen PLENARYLECTURES // PL/ 3–6

How Listeria monocytogenes breaches host barriers REFERENCE B / PL Lecuit, M.* 03 Institut Pasteur, Inserm, Paris, France Listeria monocytogenes (Lm) is a human foodborne pathogen that causes listeriosis, a systemic infection leading to meningitis, encephalitis and feto-placental infection. To reach its target organs, Lm crosses the intestinal, blood-brain and placental barriers. We will present the results of our investigations regarding the molecular mechanisms underlying Lm crossing of these three barriers.

* Plenary Supported by FCT

REFERENCE Secretion of a novel L. monocytogenes cyclic dinucleotide into the / cytosol of infected host cells activates an innate immune pathway B PL Portnoy, D. A.* 04 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 aspects of intracellular parasitism and cell-mediated immunity. Because L. monocytogenes induces a robust CD8+ T-cell response, attenuated strains of L. monocytogenes are being developed as live, attenuated vaccine vectors for infectious disease and malignancies. What makes L. monocytogenes such a strong inducer of cellular immunity? As everyone in this audience appreciates, virulent strains of L. monocytogenes secrete a pore-forming cytolysin (LLO) that allows bacteria access the host cell cytosol. We previously discovered that vacuolar and cytosolic bacteria induced distinct host transcriptional responses, the latter leading to the expression of beta interferon and a host of co-regulated genes. To understand the microbial components that activate the cytosolic pathway, we used a forward genetic screen to identify bacterial mutants that induced an enhanced or diminished host transcriptional response to cytosolic bacteria. Most of the mutants identified mapped to genes encoding or regulating multidrug efflux pumps. We now have a series of strains that induce beta interferon over a 60-fold range. These data were 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 the L. monocytogenes molecule as a cyclic dinucleotide. The precise structure of this novel bacterial molecule, identification of the cyclase and phosphodiesterase will be presented at the meeting.

* Plenary Supported by FLAD

23 AREA // B // Listeria monocytogenes as a human and animal pathogen PLENARYLECTURES // PL/ 3–6

REFERENCE Diagnosis and clinical management of listeriosis B / PL in ruminants and camelids 05 Poulsen, K. P.* School of Veterinary Medicine, University of Wisconsin, Madison, USA Listeria monocytogenes is a successful intracellular pathogen of domestic and food producing animals. Ruminant (cattle, sheep, goats) and camelid (llama and alpaca) species are constantly exposed to this gram-positive pathogen. It is ubiquitous in the environment and tends to overgrow in poorly prepared silage (pH>5.0). Lis- teriosis manifests in these species as three distinct clinical syndromes including abortion, neonatal sepsis, and meningioencephalitis (circling disease). Infection in food producing animals, and subsequent shedding of L. monocytogenes in milk and meat 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 presented including video clips of clinically affected animals.

* Plenary Supported by FLAD

REFERENCE Listeriolysin S – a second haemolysin with a role in the virulence B / PL of Listeria monocytogenes 06 Hill, C.* Alimentary Pharmabiotic Centre and Microbiology Department, University College Cork, Ireland Listeria monocytogenes require listeriolysin O (encoded by llo or hlyA) to escape the vacuole and initiate intracellular growth and intercellular spread. Llo- mutants are essentially avirulent and so listeriolysin O is regarded as the primary virulence factor in L. monocytogenes. Given that all strains and serotypes possess this virulence factor, it is unlikely to explain the increased incidence of particular serotypes (such as 4b) in listeriosis epidemics. Many laboratories have sought to solve the Listeria conundrum of why certain strains may be more likely to cause disease in humans. We have identified a second haemolysin, which we have named listeriolysin S, which is associated with epidemic strains. Listeriolysin S is very different from listeriolysin O in that it is a highly modified peptide, which resem- bles (in probable structure if not in primary sequence) the streptococcal virulence factor, streptolysin S. In fact, a family of these modified virulence peptides can be inferred from genomic data of other pathogens, including Staphylococcus aureus and Clostridium botulinum. listeriolysin S is not expressed under laboratory conditions, but can be induced with a number of reagents in vitro. In addition, a mutant in which listeriolysin S is constitutively expressed is hyper-hemolytic in comparison to wildtype strains. We have also demonstrated a small, but significant, role for listeriolysin O in murine models of infection and in polymorphonuclear leucocyte survival assays. Listeriolysin S provides a possible explanation for the enhanced virulence of certain strains in human listeriosis, but much remains to be done to confirm or refute this hypothesis.

* Plenary Supported by FEMS

24 AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis PLENARYLECTURES // PL/ 7–9

Update on clinical aspects of listeriosis: What’s new? REFERENCE C / PL Schlech, W. F.* 07 Dalhousie University, Canada Listeriosis remains a challenging problem for clinicians. Diagnosis may be delayed and treatments prolonged although decreases in mortality have been noted in some surveillance programs. New syndromes, such as necrotizing fasciitis, have been described as well as further outbreaks of febrile gastroenteritis. Sepsis and rhomboencephalitis remain the most common presenting syndromes for invasive listeriosis. Genetic markers for infection have also been uncovered. Infection in the immunocompetent host still occurs but risk factors for the disease primarily points to abnormalities in cell-mediated and innate immunity as major predispo- sitions to listeriosis. Use of TNF-alpha inhibitors and corticosteroids are of particular concern. Some newer antibiotics active against L. monocytogenes have been studied but ampicillin and gentamicin remains the treatment of choice. There is further evidence that trimethoprim-sulfamethoxazole has a strong protective effect against listeriosis in the compromised host receiving this drug for protection against other pathogens.

* Plenary Supported by FCT

A Canadian outbreak of listeriosis due to deli-meat: REFERENCE C / PL Driving change in the food safety system 08 Farber, J. M.1*, Pagotto, F.1, Gilmour, M.2, Nadon, C.2, Savelli, C.3, and MacDonald, D.3 1. Food Directorate, Health Canada 2. National Microbiology Laboratory, Public Health Agency Canada 3. 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 invasive listeriosis. During this outbreak, 57 cases of illness, in 7 provinces, resulted in 23 deaths from listeriosis, which was traced back to contaminated deli-meat from 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 underlying medical conditions. Fifty (88%) cases reported deli-meat consumption. Addition- ally, 84% of cases had institutional exposure. Investigators confirmed Company A deli meat was served to 27 cases. The major probable cause of the Listeria contamination in Company A’s plant was related to meat slicing equipment. High-throughput genome sequencing of two serotype 1/2a Listeria monocytogenes isolates was completed during the outbreak. By screening for genetic traits specific to the outbreak genomes, examination of clinical, environmental and food isolates associated with the outbreak revealed that three distinct, but highly-related strains may have been involved in this nationwide outbreak. As a result of the outbreak, the Public Health Agency of Canada (PHAC), the Canadian Food Inspection Agency (CFIA) and Health Canada (HC) conducted independent lessons-learned exercises. In addition, the CFIA developed mandatory new Directives for federally-registered meat and poultry plants, and Health Canada started updating their policy on Listeria monocytogenes in RTE foods. Furthermore, a federal review by an independent in- vestigator (Ms. Sheila Weatherill) into the outbreak resulted in 57 recommendations directed at food processors, regulators, public health professionals and individual consumers. Work is currently underway at PHAC, HC and CFIA to directly address the recommendations coming out of the Weatherill report.

* Plenary Supported by FCT

25 AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis PLENARYLECTURES // PL/ 7–9

REFERENCE Listeria monocytogenes phagocytic strategy C / PL 09 Alvarez-Dominguez, C.* Hospital Santa Cruz de Liencres-IFIMAV and IES Zapaton, Spain Listeria monocytogenes enters macrophages and resides for a short period within the phagosomal compartment before escaping and replicating in the cytosol. Lis- teria has evolved a fine phagosomal strategy to survive the microbicidal machinery of macrophages and avoid eliciting a strong innate immune response. First, Listeria-GAPDH enzymatic modification and inactivation of Rab5a caused a delay on phagosome maturation. Second, Listeria interferes with the trafficking of two lysosomal proteins, cathepsin-D and LIMP2. Cathepsin-D enzymatic action blocks the pore-forming function of Listeria-LLO within the phagosomes. Therefore, inhibiting the transport of this lysosomal protease to the phagosomes increases Lis- teria viability. Finally, the Listeria interference with LIMP2 trafficking disrupts the connection between late endosomal events and the onset of innate immunity. In brief, Listeria phagosomal strategy has the purpose to avoid the phagosomal transformation into fully competent bactericidal and antigen-processing compartments. Deciphering Listeria phagosomal strategy to subvert the host immune response might help to design better therapies against listeriosis.

* Plenary Supported by FCT

26 AREA // D // Strategies for prevention and control of Listeria monocytogenes PLENARYLECTURES // PL/ 10–12

REFERENCE Risk assessment using the microbiological criteria: D / PL Arguments for zero tolerance (USDA) other viewpoint (Europe): Risk-based microbiological criteria for Listeria monocytogenes 10 in RTE foods Luber, P.* Federal Office of Consumer Protection and Food Safety, Berlin, Germany The control of Listeria monocytogenes in foods has been a focus activity ever since the link between listeriosis in vulnerable populations and exposure via foods became clear. Listeria spp. originate from soil and can contaminate many different foods during food production or via processing environments. Once Listeria bacteria get in a food plant, they might survive in the processing environment over long time periods and could pose a contamination hazard to produced foods. More- over, the ability of L. monocytogenes to multiply under refrigeration temperatures and without oxygen, for example in vacuum packs or in foods which are packed in modified atmospheres, makes it challenging to control the bacteria further up in the 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 foods which can become contaminated with L. monocytogenes, ready-to-eat (RTE) foods which are consumed without further heat treatment are associated with the greatest listeriosis risk. Regulators in the European Community and the Codex Alimen- tarius Commission have developed risk-based microbiological criteria for L. monocytogenes in foods. In both cases, the microbiological criteria specifically apply to RTE foods and take into consideration if growth of Listeria in the food may occur or not. However, in both approaches the main focus is on controlling L. monocytogenes during processing of RTE foods and in the production environment. The Eu- ropean Regulation on microbiological criteria (Regulation (EC) no 2073/2005) is embedded in the so called ‘hygiene package’ of legislations, and in the Codex Ali- mentarius microbiological criteria are presented in an Annex of the ‘Guidelines on the Application of General Principles of Food Hygiene to the Control of Listeria monocytogenes in Ready-to-Eat foods’ (CAC/GL 61-2007), only. In the European Community, food business operators have an obligation to demonstrate compliance with microbiological criteria and thereby enable verification of their GMP and HACCP systems. The approach of using risk-based microbiological criteria for L. monocytogenes as management option for controlling Listeria in foods and thus to reduce the likelihood of listeriosis in vulnerable populations will be critically discussed.

* Plenary Supported by FCT

REFERENCE USDA regulatory approach and considerations for the control D / PL of Listeria monocytogenes 11 Engeljohn, 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 the control programs for ready-to-eat meat and poultry will be presented. Use of risk assessments to inform the verification and enforcement strategy will be highlighted. Lessons learned regarding the implementation of a “zero tolerance” approach will be discussed, along with future plans for ensuring that progress towards good control are not negated in further processing operations.

* Plenary Supported by FLAD

27 AREA // D // Strategies for prevention and control of Listeria monocytogenes PLENARYLECTURES // PL/ 10–12

REFERENCE / Listeria monocytogenes, an emergent pathogen D PL in Chile and Latin America 12 Hormazábal, J. C.* Public Health Institute of Chile Food borne diseases are a major health burden in Latin America. In Chile Listeria monocytogenes is a pathogen under laboratory surveillance since 2004. All the isolates from human cases must be confirmed at the National Reference Laboratory at the Public Health Institute of Chile. Until 2008 L. monocytogenes was an infrequent pathogen, approximately 45 clinical cases per year were reported in all the country. At the ending of 2008 an unusual increase of listeriosis was detected in the Metropolitan region of Chile. In this scenario, the Reference Laboratory included for first time molecular tools for the detection of related cases. PFGE including PulseNet International protocols, was a powerful upgrade for the surveillance system. In parallel The Ministry of health, in absence of a specific regulatory framework for L. monocytogenes in food industry, made important changes in sanitary food regulations, including specific microbiological criteria for Listeria in food and environment. The integration of surveillance data, clinical, environmental and food industry isolates, allowed the creation of a single database, including molecular typing information. This dynamic system allowed the detection of the first Chilean outbreak of L. monocytogenes, and made possible the link of clinical cases to potential sources, including the environment, raw or processed food products. After few weeks of an intensive investigation the outbreak source was confirmed (goat and brie cheese). The inclusion of molecular tools in surveillance provides valuable information for the early detection of listeriosis outbreaks. PulseNet Latin America Network gives an important support for the construction of national and regional databases for the detection of local outbreaks and virulent clones with potential international spread. In Latin America Listeria keeps as an infrequent and unknown pathogen, major efforts are necessary specially in health staff and general population education including a sensitive surveillance system and a strong regulatory framework in food industry.

* Plenary Supported by FCT

28 AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control PLENARYLECTURES // PL/ 13–14

REFERENCE How can the social sciences help us understand the prevalence / of listeriosis in the UK? E PL Wadge, A.* 13 Chief Scientist, Food Standards Agency, UK There has been a marked change in the epidemiology of listeriosis in humans in the UK over the past decade. A doubling of reported cases has been seen in England and Wales since 2001 and this has occurred largely in people aged 60 years and over and presenting with bacteraemia rather than central nervous system infection. The incidence of listeriosis among other age ranges has remained unchanged and the incidence in pregnant women has been stable since the early 1990s. This presentation will explore the possible reasons for the increase in the over 60s focussing on several strands of work that are being undertaken to address this. Firstly in 2007 the UK Advisory Committee on the Microbiological Safety of Food (ACMSF) considered that the change in the epidemiology seen in the UK was most likely linked to social factors rather than changes occurring in the microorganism and referred the issue of listeriosis in the elderly to its ad hoc group on vulnerable groups for further consideration. The presentation will highlight some of the group’s findings which are available in a report (www.food.gov.uk/multimedia/pdfs/committee/acmsflisteria.pdf). Amongst their recommendations they recognised the importance of food consumption and food handling behaviours in the over 60s including those in vulnerable groups. One of the report’s recommendations was for this to be considered by the Food Standards Agency’s (FSA) Social Science Research Committee (SSRC). The presentation will highlight findings from the SSRC’s report which showed that very little 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 although the reason for this difference is unclear. Specific differences reported included poor refrigeration and defrosting practices; differences in cooling, storage and reheating of leftovers; variable adherence to ‘use-by’ and ‘best-before’ dates; and differences in personal and domestic hygiene such as hand-washing and cleaning of kitchen surfaces. The report noted that there is relatively little evidence regarding the current levels of food hygiene knowledge among those aged 60 years and over and no information on whether the level of knowledge differs with generations or has changed as people age. Emphasising the need for correct storage and handling of food in the home particularly by those over 60s was a key theme of Food Safety week in the UK in 2009 and the presentation will illustrate the approach that was taken by the FSA in this campaign. The presentation will conclude by highlighting some of the gaps in our knowledge and the SSRC recommendations concerning research. * Plenary Supported by FCT

REFERENCE Consumer perceptions, behaviour and microbial food safety; / implications for Listeria control E PL Frewer, L. J.* 14 Wageningen University, The Netherlands In order to understand fully the problem of food safety linked to the occurrence of listeriosis, it is important to consider how consumers respond to food safety issues, in terms of their psychology and how this determines their behaviour, for example in relation to food preparation behaviour. An important research objective relates to consumer activities after food purchase. Risk perception determines how consumers react to different types of risks. Very generally, risks which are perceived to be unnatural in origin and involuntarily imposed on the individual who is exposed to them are perceived to be more 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 consumer concern. In addition, consumers tend to exhibit an “optimistic bias” in relation to microbiological food risks, which means that food safety information tends to be perceived as applying to more vulnerable, consumers in the population. Research suggests that consumers are reasonably knowledgeable about safe food preparation practices, but that this knowledge is not always applied in practice. Food preparation tends to be a habitual behaviour, which is difficult to change. Introducing food safety messages during food preparation (for example, in recipe development) tends to activate existing food safety knowledge, as does the inclusion of materials designed to elicit affective responses to food safety issues, for example disgust. Some groups within the population are more vulnerable than others, and targeting risk communication messages to the needs of these groups is particularly relevant. It is argued that, whilst consumer observation studies, combined with modelling of critical control points in food preparation, might indicate the riskiest behaviours in terms of Listeria, this information should be combined with activation of general food safety knowledge if an effective approach to reducing the incidence of food borne disease is to be developed. * Plenary Supported by FCT

ORAL PRESENTATIONS

AREA // A // Biology of Listeria monocytogenes ORALPRESENTATIONS // O / 01–13

The cold shock associated proteins (Csps) promote tolerance of different REFERENCE A / O environmental stresses and host cell invasion of Listeria monocytogenes Tasara, T.1, Klumpp, J.2, Loessner, M. J.2 and Stephan, R.1 01 1. Institute of Food Safety, University of Zurich, Switzerland 2. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland The food-borne pathogen Listeria monocytogenes has to withstand various stress conditions in order to survive and proliferate on foods and within different types of host cells. The bacterial cold shock family proteins (Csps) consist of small, highly conserved nucleic acid-binding proteins, and are assumed to have an influence on the expression of various microbial genes. In addition to cold adaptation functions, some prokaryotic Csp proteins are also involved in promotion of other cell processes during normal bacterial growth, as well as in adaptation to nutrient starvation and stationary growth phase stresses. The possible functional contribution of L. monocytogenes CspA, CspB and CspD proteins were investigated under food-related environmental stress, and during host cell invasion. We show that the three L. monocytogenes Csp components, although highly homologous, provide distinct cellular functions during cold, osmotic and oxidative stress adaptation as well as host cell invasion processes. All three csp genes are constitutively expressed but are dispensable for viability and growth of this organism at optimal temperature. However, a hierarchy in Csp functional importance during cold (CspA>CspD>CspB) and osmotic (CspD>CspA/CspB) stress adaptation is observed. With respect to double (DcspBD) and triple (DcspABD) csp deletion mutants, we also observe reduced survival of oxidative stress and a reduced ability to invade Caco-2 and J744A.1 murine macrophages cells. Overall, our data indicate important functional roles of L. monocytogenes Csp proteins in promotion of cellular functions which facilitate environmental stress resistance and host cell invasion processes of this food-borne pathogen.

Different levels of flagellin detected during growth REFERENCE A / O of Listeria monocytogenes strains at low temperature Cabrita, P.1,2,3*, Batista1, S., Moes, S.4, Jenö, P.4, Trigo, M. J.3, Boavida Ferreira, R.1,2 02 and 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, Portugal 3. Instituto Nacional dos Recursos Biológicos, IP, Oeiras, Portugal 4. 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 face since refrigeration is present throughout the food chain. This study aimed to investigate whether unique or common proteins are up- or down-regulated under nutritional stress conditions and low temperature. To achieve this, a simplified methodology to analyse the extracellular protein profiles of four L. monocytogenes strains was used. These strains, belonging to four different serovars, were selected according to differences in virulence. Cultures were incubated in minimal medium (Modified Welshimer Broth) at 10°C. Proteins present in the supernatants of the cell cultures, in late exponential phase, were precipitated and separated by SDS- PAGE, using equivalent amounts of total secreted proteins. For each bacterial strain, at least three independent culture assays were set. The most abundant polypeptide bands and those suggesting the widest range in differential expression among strains were identified by ESI LC / MS-MS. The p60 virulence protein, one of the major proteins previously detected at 37°C, was still detected in relatively high amounts at 10°C in all strains. The virulence proteins listeriolysin O and internalin C expressed at 37°C, were not detected at 10°C in all strains, confirming previous findings. In serovar 1/2a strain, flagellin was the major protein identified. Two other strains, from the rare serovares 4c and 4d/4e, showed lower levels 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 steel surfaces. Serogroup 1/2a has been reported as prevalent among food isolates. Therefore, the different levels of flagellin detected may be related with the ability of some strains to colonize food contact surfaces at refrigeration temperatures. This hypothesis will be further investigated.

* Participation Supported by IUFoST

33 AREA // A // Biology of Listeria monocytogenes ORALPRESENTATIONS // O / 01–13

REFERENCE Phenotypic and corresponding transcriptomic responses of Listeria A / O monocytogenes strains in the presence of unprotonated organic acids 03 Lee Chang, K. J., Pinfold, T., Koshy, A. and Bowman, J. P. University of Tasmania, Australia Sodium diacetate-resistant L. monocytogenes food and clinical isolates delineated through a culture-based screening process were found to possess significantly better survival levels following challenge to pH2.4 acid challenges compared to various reference strains. Sodium diacetate was found to directly and substantially improve survival and was synergistic with acid tolerance resultant from the shift to the stationary growth phase. The resistant strains had comparatively lower intracellular levels of acetate and K+ ions that corresponded to lower transmembrane delta pH values. Another observed feature was that when grown in the presence of sodium diacatete cell wall stability was enhanced as revealed by lysis assays, utilising bead-beating and mutanolysin, suggesting acetate induces various cell wall modifications that may also influence diffusion of unprotonated organic acid into cells. Transcriptomic analysis of pH5.0-habituated sodium diacetate-resistant strain FW04/0025 compared with reference strain EGD that were grown with 21mM (8mM unprotonated) sodium diacetate at pH 5.0 indicated substantial variation in genetic responses with EGD much more reactionary to the presence of mineral acid. These differences were reflected in regulon-level gene expression trends with EGD strongly activating and repressing the SigB- and CodY regulons, respectively, while in FW04/0025 the response of these regulons were muted. The transcriptome of FW04/0025 only becomes more congruent with that of EGD when in the presence of sodium diacetate, though several distinct genetic expression differences still occur. Gene expression trends deriving from exposure to sodium diacetate suggest extensive cell wall and membrane modification, alterations to branched-chain amino acid/fatty acid metabolism/biosynthesis, induction of an SOS-like DNA repair and thioredoxin-mediated antioxidant responses occurs, though strain-level specific responses are quite divergent. Corresponding proteomic analyses are underway to further characterize responses to food pre- servative organic acids by L. monocytogenes.

REFERENCE Internalin profiling, multilocus sequence typing A / O and virulence assesments suggest evolutionary history 04 of the Listeria monocytogenes-Listeria innocua clade Chen, J. and Fang, W. Zhejiang University, China The morphological, ecological, biochemical and genetic resemblance, and the clear difference of virulence between L. monocytogenes and L. innocua make this bacterial clade attractive as models to examine the evolution of pathogenicity. This study was attempted to examine the population structure of L. monocytogenes and L. innocua, and further to investigate the microevolution in this clade via profiling of 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 in vivo virulence assessments. Results indicate that L. monocytogenes comprises three recognized lineages I, II and III, including a set of lineage III strains displaying strong phospholipase activity and subdued virulence. While resembling L. monocytogenes in having a nearly identical Listeria pathogenicity island I, and inlA and inlB, 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 young species descending from L. monocytogenes, which comprises four subgroups: two major subgroups I and II, and one atypical subgroup IV exhibiting the least genetic distance to L. monocytogenes. All L. innocua strains lack 17 virulence genes found in L. monocytogenes, except for subgroup IV strains harboring inlJ, and are nonpathogenic to mice. As shown by the estimation of the time to the most recent common ancestor, L. monocytogene lineages I and II appeared at approximately the same time, and this is also the case with L. innocua subgroups I and II. The nonpathogenic L. monocytogenes strains and L. innocua subgroups IV constitute the possible evolutionary intermediates between L. monocytogenes and L. innocua. The evolutionary history in the L. monocytogenes-L. innocua clade represents a rare example of evolution towards reduced virulence of pathogens.

34 AREA // A // Biology of Listeria monocytogenes ORALPRESENTATIONS // O / 01–13

REFERENCE The SOS response of Listeria monocytogenes is involved / in stress resistance, mutagenesis, and biofilm formation A O van der Veen, S.1,2 and Abee, T.1,2 05 1. Top Institute Food and Nutrition (TIFN), Wageningen, The Netherlands 2. Laboratory of Food Microbiology, Wageningen University and Research Centre, Wageningen, The Netherlands The food-borne pathogen Listeria monocytogenes is widely distributed in the environment. As a consequence, raw materials used by the food industry could introduce L. monocytogenes to food processing facilities. L. monocytogenes has evolved various strategies and networks to survive and adapt to changing conditions e.g. during food processing. One of the stress response mechanisms we recently identified in L. monocytogenes is the SOS response. The SOS response is a conserved inducible pathway that is involved in DNA repair and restart of stalled replication forks. We identified the SOS response regulon of L. monocytogenes and showed that it is important for stress resistance and adaptive mutagenesis. In the present study, we investigated the role of the SOS response in L. monocytogenes biofilm formation. L. monocytogenes static biofilms on polystyrene and glass consists of a homogeneous layer, while on stainless steel L. monocytogenes biofilms consist of single attached cells or microcolonies. Static biofilms contain the small rod-shaped morphology, which is very similar to the morphology of planktonic cells. However, L. monocytogenes continuous flow biofilms consist of ball-shaped microcolonies, which are surrounded by a dense network of knitted chains composed of elongated cells. We showed that continuous flow biofilm formation and not static biofilm formation is dependent on the SOS response. Using Q-PCR analysis, promoter reporters, and SOS response mutants, we showed that the SOS response is activated during knitted-chain biofilm formation and that deletion of its regulon member yneA, which is involved in cell elongation during SOS response activation, results in diminished biofilm formation in continuous flow conditions. Furthermore, we demonstrated that activation of the SOS response during continuous flow biofilm formation induced mutagenesis: wild-type biofilms showed considerably higher rifampicin resistant fractions than ∆recA biofilms or wild-type planktonic cultures. Our results show that the SOS response of L. monocytogenes is important for stress resistance, adaptive mutagenesis, and continuous flow biofilm formation, and may therefore contribute to the survival and persistence of this pathogen in food processing environments.

Clonal diversity of Listeria monocytogenes, a worldwide perspective REFERENCE A / O Chenal-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 06 1. Institut Pasteur, National Reference Center and WHO Collaborating Centre for LISTERIA, Paris, France 2. Institut Pasteur, Genotyping of Pathogens and Public Health Platform, Paris, France Listeria monocytogenes is a foodborne pathogen that can cause listeriosis, a severe invasive disease in human with a high fatality rate. L. monocytogenes is widespread in nature. Molecular typing methods have grouped L. monocytogenes into two major genetic lineages (lineages I and II) and an additional minor lineage (lineage III). They differ according to virulence and ecological origin. We have developed a DNA sequencing-based subtyping method, multilocus sequence typing (MLST) to examine the epidemiology and population genetics of L. monocytogenes. In the present study, we have undertaken the first spatiotemporal analysis of L. monocytogenes biodiversity by sequencing internal portions of seven housekeeping genes in 300 strains isolated from the six continents. We selected a set of 300 unrelated strains of L. monocytogenes collected from 41 countries and 6 continents isolated between 1935 and 2009 from different sources. MLST data based on seven housekeeping genes (3,288 nucleotides) were obtained as described previously (Ragon et al., PLoS Pathogens (2008)). The results show a pattern of biodiversity similar to the one we had initially reported in France. Indeed, the population structure is similar to that obtained on 360 strains isolated principally from France by Ragon et al. Sequences and allelic profiles are available on the Internet-accessible database (www.pasteur.fr/mlst). L. monocytogenes appears to exhibit a homogeneous clonal diversity across continents, indicating a high dispersal rate at a global scale.

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REFERENCE Life without a cell wall: Listeria monocytogenes L-form cells feature A / O a unique mode of division 07 Briers, 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 maintain or build a rigid peptidoglycan envelope. Although L-forms had been studied for decades and many reports exist on their morphological, serological and biochemical properties, very little was known about the basic cell biology and molecular mechanisms underlying the transformation process. Of particular interest is the question how L-form bacteria are able to proliferate in the absence of a mature cell wall. We have investigated the biology of stable, non-reverting Listeria monocytogenes L-form cells (native and GFP labelled). Transmission electron microscopy demonstrated that L-form cells are devoid of the typical thick Listeria type 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 peptidoglycan-bound proteins such as Internalin A, whereas membrane-anchored proteins such as Internalin B are still present. Monitoring L-form growth by time- lapse confocal laser scanning microscopy revealed the development and maturation of vesicles within maternal L-form cells. We propose a novel model for growth and division of L-form bacteria, which may explain their ability to multiply in the absence 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 functions 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 into macrophages are not killed but seem able to survive for prolonged periods of at least 48 hours. In conclusion, we show that L. monocytogenes L-forms (i) can arise and survive in the environment, (ii) are able to multiply and divide, and (iii) show intracellular survival in macrophages.

REFERENCE A / O Pangenomic analysis of Listeria monocytogenes 08 Deng, 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, USA 2. Center for Bioinformatics and Computational Biology, University of Maryland, College Park, USA 3. National Center for Food Safety and Technology, Food and Drug Administration, Summit, USA Listeria monocytogenes is well known for its adaptability to diverse environment and host niches and its high fatality rate among infected immunocompromised populations. Three genetic lineages have been identified in this species. Strains of genetic lineages I and II account for >90% of human infections in the United States, whereas strains from genetic lineage III are rarely implicated in human infections for unclear reasons. Here we compare the genomes of 26 L. monocytogenes strains representing the three lineages based on both in silico comparative genomic analysis and high-density, pan-genomic DNA array hybridizations. We uncover 86 genes and 8 small regulatory RNAs that likely make L. monocytogenes lineages differ in carbohydrate utilization and stress resistance during their residence in natural habitats and passage through the host gastrointestinal tract. We also identify 2,330 to 2,456 core genes in the listerial pan-genome that define this species and assess the impact of lysogenic bacteriophages on genomic diversifi- cation. Phylogenomic reconstructions based on 3,560 homologous groups suggest a polyphyletic population infrastructure and gradual loss of genes as this saprophytic species diversified into a rare and probably defective lineage.

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REFERENCE Evidence for an antiporter-independent glutamate decarboxylase / (GAD) system in Listeria monocytogenes: Influence of growth media A O on GAD system activity 09 Karatzas, K.-A., Brennan, O., Heavin, S. and O’Byrne, C. P. SFI, Ireland The GAD system plays an important role in survival of Listeria monocytogenes under acidic conditions (eg. acidic foods) and in virulence (eg. survival in stomach). The GAD system imports extracellular L-glutamate (Glu)e through an antiporter, converts it to γ-aminobutyric acid (GABA) resulting in the consumption of an intracellular proton and thus the increase of the intracellular pH. We show for the first time that (Glu)e added in Defined Medium (DM) is not used by the GAD system and therefore it cannot increase the ability of this bacterium to grow in mild acidic conditions, neither does it enhance acid resistance at lethal pH values as it does in BHI. The rate of GABA export was monitored in BHI at various pH values showing that it initiates at pH4.6 and the rate increases as pH values decrease. We also demonstrate that there are activators of the antiporter-based GAD system in BHI, while its inactivity in DM is not due to the presence of inhibitors in this medium. Activation was at the transcription level with the expression of gadD2T2 being more than 100-fold higher in BHI than in DM where levels of gadD2 were undetectable. Furthermore we demonstrated for first time that in both acidified DM and BHI, L. monocytogenes accumulates high levels of intracellular GABA (GABA)i (>42mM) and despite the slower rate of accumulation in DM the final levels were identical in both media. Since DM does not contain any (Glu)e we have shown for first time that this bacterium converts (Glu)i to (GABA)i, which is not exported and thus is stored intracellularly. This suggests an alternative mechanism of acid resistance based on the GAD system that circumvents the antiporter-based mechanism. We suggest that the (GABA)i accumulation might buffer the intracellular pH and thereby contribute in survival under extreme acidic conditions.

Role of Listeria monocytogenes tyrosine phosphatases in conferring REFERENCE A / O listeriophage resistance Paz, R.-N. 1, Eugster, M. R.2, Zeiman, E.1, Loessner, M. J.2 and Calendar, R.3 10 1. Hadassah Hebrew University Medical Center, Israel 2. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland 3. Department of Molecular and Cell Biology, University of California, Berkeley, USA Protein tyrosine phosphatase (PTP)-like proteins exist in many bacteria and are segregated into 2 major groups: Low molecular weight, and conventional. These PTP are suggested to be involved in many aspects of bacterial physiology including stress response, DNA binding proteins, virulence and capsule/cell wall production. By annotation Listeria monocytogenes (LM) possesses 2 potential low molecular weight and 2 conventional PTPs. Although no tyrosine kinases were identified yet in LM, using Immunoprecipitation (IP) on total cell lysate and MS plus MS/MS on the IP products we have identified at least 10 tyrosine phosphorylated proteins. These proteins vary in their physiological function and were associated with carbohydrate metabolism, DNA and RNA binding, processing and transcription and transport. Using LM WT strain 10403S, we have created an in-frame deletion mutant lacking all 4 PTPs, as well as 4 additional complemented strains harboring each of the PTPs. No major physiological differences were observed between the WT and the mutant lacking all 4 PTPs. However, the deletion mutant strain was found to be resistant to listeriophages A511 and P35, and sensitive to other listeriophages such as U153 and A118. This phage resistance was attributed to reduced attachment to the cell wall. Additionally, the mutant lacking all PTPs was found to lack N-acetylglucosamine in its teichoic acid. Phage sensitivity was rescued in a complemented strain harboring a low molecular weight PTP (LMO2540). We also found that attachment of the phages is partially restored by the same PTP and by one with conventional weight PTP (LMO1800). Thus, it seems that PTPs probably affect many processes in LM, but mostly resistance to listeriophages.

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REFERENCE Thiolomics – the thiol: disulfide redox metabolism A / O of Listeria monocytogenes 11 Ondrusch, 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, Germany 2. Dept. of Microbiology , University of Mysore, India 3. Dept. of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel The thiol: disulfide redox metabolism (TDRM), found in all living cells, constitutes a multi-component network. It counteracts oxidative stress, serves to maintain the proper intracellular redox potential, assists in protein folding, is essential for the formation of deoxyribonucleotides for DNA synthesis and helps to repair damaged proteins. The best-characterized biological thiols are the tripeptide glutathione (GSH) and the small proteins of the thioredoxin (Trx) and glutaredoxin (Grx) family. Oxidized GSH and Trx are recycled by cognate reductases (GSH reductase – Gor/GshR; thioredoxin reductase – TrxB). A considerable number of genes/gene products putatively involved in the TDRM of L. monocytogenes EGD- e has been identified in the genome sequence. It comprises the unique fused gene gshF (GSH synthetase), grx, two putative gor/gshR, six members of the thioredoxin family, trxB, class I and class III ribonucleotide reductases (nrdAB and nrdDG), gpo/gpx, prx, tpx, ohrA/R (detoxification of organic hydroperoxides), msrA (methionine sulfoxide reductase ) and several regulators, e.g. perR (peroxide regulon), spx & rex (redox-sensing regulators), fur, zur & mntR (metal uptake), nrdR and the redox-sensitive chaperone hsp33. We investigated the regulation and function of these genes/gene products in the TDRM in particular and in the physiology of L. monocytogenes in general, also with respect to infectivity and virulence, by the following approach: i) construction of selected mutants, ii) study of their multi- plication in vitro and in vivo, and iii) genome-wide transcription profiling of wild type 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 the glutathione/glutaredoxin system showed extensive changes in their transcription profiles. Affected were virulence genes, genes for regulators, transporters, stress response factors and also for metabolic enzymes. These results emphasize the central role of the TDRM, their impact on cellular processes and pathogen-host interaction will be discussed.

REFERENCE RNA-structures acting at a distance A / O Johansson, J. 12 Umeå University, Sweden Riboswitches are RNA-elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that an S-adenosylmethionine (SAM) riboswitch, SreA, in Listeria monocytogenes can also function in trans, and act as a non-coding RNA. We show that SreA controls expression of the virulence regulator PrfA by binding to the 5´-untranslated region of its mRNA in a temperature-dependent manner. Absence of the SAM riboswitch increases the level of PrfA. Thus, the impact of the SAM riboswitch on PrfA highlights a link between virulence and the nutritional status of the bacterium. Together, our results describe a novel role for riboswitches and a new class of regulatory non-coding RNAs in bacteria.

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Deep RNA sequencing of Listeria monocytogenes reveals overlapping REFERENCE A / O and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs 13 Oliver, 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, USA 2. University of Sydney, Australia 3. 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 to enable development of more effective control strategies. The stationary phase stress 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 stationary phase cells of L. monocytogenes 10403S and an otherwise isogenic ∆sigB B mutant, which does not express the alternative sigma factor σ , a major regulator of genes contributing to stress response, including stresses encountered upon entry into stationary phase. Overall, 83% of all L. monocytogenes genes were transcribed in stationary phase cells; 42% of currently annotated L. monocytogenes genes showed medium to high transcript levels under these conditions. A total of 96 genes had significantly higher transcript levels in 10403S than in ∆sigB, indi- B cating σ -dependent transcription of these genes. RNA-Seq analyses indicate that a total of 67 noncoding RNA molecules (ncRNAs) are transcribed in stationary phase L. monocytogenes, including 7 previously unrecognized putative ncRNAs. Application of a dynamically trained Hidden Markov Model, in combination with B B RNA-Seq data, identified 65 putative σ promoters upstream of 82 of the 96 σ - B dependent genes and upstream of the one σ -dependent ncRNA. The RNA-Seq data also enabled annotation of putative operons as well as visualization of 5’- and 3’-UTR regions. The results from these studies provide powerful evidence that RNA-Seq data combined with appropriate bioinformatics tools allow quantitative characterization of L. monocytogenes transcriptomes, thus providing exciting new strategies for exploring transcriptional regulatory networks.

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REFERENCE / Probiotics reduce Listeria monocytogenes-induced tissue invasion and B O stillbirths in pregnant guinea pigs 14 Smith, 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 miscarriage or stillbirth, premature delivery, or infection of the newborn. Recently we have published a risk assessment based on dose response data from nonhuman pri- 7 mates and guinea pigs. Both animal models estimate LD50s of approximately 10 L. 6 monocytogenes cfu similar to the FAO/ WHO estimated human LD50 of 1.9 x 10 cfu based on outbreak data. The similarities between the dose response curves and LD50s suggest these animal models are appropriate for testing therapies and preventive strategies applicable to humans. Recently, probiotics have been proposed to protect hosts from pathogens introduced to the system through ingestion. Our objective was to determine the efficacy of probiotics in yogurt in pre- venting L. monocytogenes invasion and stillbirths. Using our pregnant guinea pig model for listeriosis, 5 ml of yogurt containing Lactobacillus and Bifidobacterium was administered orally to pregnant guinea pigs on gestation days (gd) 32–36. On gd35, guinea pigs were orally fed L. monocytogenes (109 cfu) in sterilized whipping cream 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 isolated 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 with 108 L. monocytogenes cfu have stillbirths compared to 14% when treated with yogurt and L. monocytogenes. These results present opportunities to develop treatment and preventive therapies for listeriosis. In summary, consumption of yogurt containing Bifidobacterium and Lactobacillus reduced the invasion and number of stillbirths after L. monocytogenes exposure in pregnant guinea pigs.

REFERENCE Listeriolysin O favors Listeria monocytogenes growth in co-culture with B / O the ciliate Tetrahymena pyriformis 15 Ermolaeva, S. and Pushkareva, V. Gamaleya Institute of Epidemiology and Microbiology, Russian Federation Listeria monocytogenes was isolated from soil, water, sewage and sludge. We explored 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 pyriformis. Axenic T. pyriformis and the following bacterial strains were used: wild type L. monocytogenes strains EGDe, VIMVR081, VIMVW039, VIMHA034, VIMVF870, EGDe derivative EGDe::Dhly and wild type L. innocua strain NCTC11288. Experiments were performed in LB broth at 28°C. Bacteria were counted bacteriologically or by qPCR. T. pyriformis trophozoites and cysts were counted 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. monocytogenes culture or with T. pyriformis cysts infected L. monocytogenes. After 7 days of co-culturing, wild type L. monocytogenes strains reduced trophozoite and increased cyst concentrations up to 21.1 and 6.1 times, respectively. EGDe::Dhly failed to cause mortality among protozoa and to trigger protozoan encystment. In concordance, LLO deficiency deteriorated L. monocytogenes growth in the presence of T. pyriformis. Replenishment of the hly gene in the mutant strain restored toxicity towards protozoa. L. innocua transformed with the LLO-expressing plasmid caused extensive mortality and encystment in ciliates. L. monocytogenes EGDe entrapped in cysts caused infection in guinea pigs upon ocular and oral infection. The L. monocytogenes virulence factor LLO promotes bacterial survival and is responsible for L. monocytogenes toxicity for protozoa and induction of protozoan encystment. Therefore, LLO activity might support bacterial survival in the natural habitat outside of a host.

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Atopy is a risk factor for listeriosis REFERENCE B / O Kawamoto, K., Matsubara, S., Da Silva, M. and Makino, S.-I. 16 Obihiro Univ. Agri. Vet. Med., Japan Listeria monocytogenes is a Gram-positive bacterium that causes meningitis, bacteremia, and febrile gastroenteritis. The outcome of listeriosis is dependent on host factors such as age, pregnancy, and HIV infection, which influence host im- munocompetency. A Th-1 type cytokine IFN-gamma; plays an important role in the innate immune response against intracellular bacterial pathogens. In contrast, Th2-biased immune responses often associate with most atopic diseases. In this study, we examined whether atopy affected the immune response to L. monocytogenes infection. NC/Nga is a model mouse for human atopic dermatitis, which has a genetic predisposition to develop atopic skin lesion. We compared the susceptibility to listeriosis of NC/Nga mice with BALB/c and C57BL/6 mice. The course of infection was characterized by monitoring survival of mice, and determination of bacterial numbers in the organs. NC/Nga mice were highly susceptible to L. monocytogenes infection: the 50% lethal dose was significantly lower and the number of bacteria in livers, spleens and brains were higher in NC/Nga than those for other inbred mice. The increased permeability of blood-brain barrier was observed in NC/Nga brains at day 3 post infection, but not in BALB/c and C57BL/6. As compared to BALB/c and C57BL/6 mice, plasma IFN-gamma-levels of NC/Nga were comparative. However, markedly increased IL-10 levels were detected in NC/Nga plasma. Pretreatment with neutralizing antibodies to IL-10 partially protected mice but retarded the clearance of bacteria. Yet the molecular mechanisms by which the infection of L. monocytogenes induces overproduction of IL-10 in NC/Nga mice remain unknown, our results suggest that the differential cytokine production may at least partially underlie the higher susceptibility to L. monocytogenes 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-bacterial REFERENCE B / O vectors to target the vasculature of progressive tumors Paterson, Y., Seavey, M. M., Maciag, P. C. and Sewell, D. 17 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 immune system for the immunotherapy of cancer and as prophylactic vaccines for infectious disease. Given the antigenic instability of tumor cells, we recently turned our attention to the use of immunotherapy to destroy cells actively involved in forming new blood vessels that support the growth and spread of cancer. We constructed Lm expression systems that would target two central cell types involved in angiogenesis – endothelial cells and pericytes. Two proteins are highly expressed on each cell during active angiogenesis – Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) (endothelial cells) and High Molecular Weight Melanoma Associated Antigen (HMWMAA) (pericytes). We selected fragments from each molecule that included peptides predicted to bind to the HLA-A2 molecule. We fused the genes encoding these fragments to the gene encoding the first 420 residues of Listeriolysin-O (LLO) and used Lm to deliver these fusion proteins. Even though the immune system should be tolerant to these self-molecules, both vaccines elicited potent anti-tumor CTL responses. Lm-LLO-VEGFR2 was able to reduce tumor microvascular density (MVD), cause regression of established breast tumors and protect against tumor re-challenge 100+ days post last immunization. The Lm-LLO-HMWMAA vaccine also dramatically reduced MVD, 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 the tumor-associated antigen Her-2/neu. Neither immunotherapeutic interfered with the generation of normal vasculature in wound healing or gestation. We are currently testing these vaccines to impact the progression and metastasis of advanced breast cancer, we hypothesize that reduced angiogenesis will prevent the formation of distal metastases blunting cancer spread and improving overall survival.

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REFERENCE The intracellular carbon metabolism of Listeria monocytogenes B / O in comparison to that of other intracellular bacterial pathogens 18 replicating in mammalian host cells Gotz, A.1, Eylert, E.2, Stoll, R.1, Eisenreich, W.2, and Goebel, W.3 1. Biocenter-Microbiology, University of Würzburg, Germany 2. Institute of Biochemistry, LMU München, Germany 3. Max-von-Pettenkofer Institute, LMU München, Germany Intracellular bacterial pathogens replicate either in the cytosol (e.g. Listeria monocytogenes, Shigella spp. and the closely related enteroinvasive Escherichia coli [EIEC]) or in specialized phagosomal compartments (e.g. Salmonella enterica Serovar Typhimurium) of the infected host cells. Our knowledge on the metabolic adaptation processes between intracellular pathogens and their host cells, allowing their efficient intracellular growth, and on the influence of the intracellular bacterial metabolism on the expression of those virulence genes, that are decisive for the intracellular life cycle of the intracellular pathogens, is still rather limited. We have carried out initial studies concerning these important questions with the above mentioned intracellular bacteria. For this goal we constructed mutants impaired in the uptake and/or catabolism of specific carbon sources and studied by NMR- or MS-based 13C-isotopologue profiling their intracellular carbon metabolism in comparison to the isogenic wild-type strains after infection of suitable mammalian host cells. The results obtained show that the intracellular carbon metabolism of L. monocytogenes differs significantly from that of the two other pathogens. Whereas glucose, but not glucose-6-P is the major carbon substrate for 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 transporters in L. monocytogenes. However, the intracellular C-metabolism of all three pathogens in mammalian host cells is surprisingly flexible. Mutants defective in the uptake of the preferential carbon source switch readily to alternative carbon sources and the lower energy supply of the utilized secondary carbon sources seems to be compensated by an increased uptake of anabolic monomers from the host cells. As shown for L. monocytogenes the intracellular carbon metabolism appears to be adapted to optimal expression of the virulence genes that are essential for the intracellular life style.

REFERENCE / LIMP2 links late phagosomal trafficking with the onset B O of the innate immune response to Listeria monocytogenes: 19 a role in macrophage activation Fernandez-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 de Valdecilla – IFIMAV, Spain The innate immune response to Listeria monocytogenes depends on phagosomal bacterial degradation by macrophages. Here, we describe the role of LIMP2, a lysosomal type III transmembrane glycoprotein and scavenger-like protein, in Listeria phagocytosis. We show here that LIMP2 is not involved in bacterial recognition at the cell surface but participates in the degradation of Listeria within phagosomes. LIMP2 appears linked to Rab5a activation and controls the late-endosomal/lysosomal fusion machinery. Importantly, LIMP2 deficient mice display a macrophage-related defect in innate immunity. They produce less acute-phase pro-inflammatory cytokines/chemokines, MCP-1, TNF-α and IL-6, but normal levels of IL-12, IL-10 and IFN-γ and a 20-fold increase in susceptibility to Listeria infection. This macrophage dysfunction results in a low listericidal potential, impaired phago-lysosome transformation into antigen-processing compartments and uncontrolled LM cytosolic growth that fails to induce normal levels of acute- phase pro-inflammatory cytokines. Therefore, the role of LIMP2 appears to be connected to the activation of Listeria-primed macrophages through internal signals linking the regulation of late trafficking with the onset of the innate Listeria immune response.

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The tetraspanin CD81 is required for entry of Listeria monocytogenes REFERENCE B / O in mammalian cells Tham, T. N.1,2,3, Gouin, E.1,2,3, Rubinstein, E.4,5, Boucheix, C.4,5, Cossart, P.1,2,3 20 and Pizarro-Cerdá, J.1,2,3 1. Institut Pasteur, Unité des Interactions Bactéries-Cellules, Département de Biologie Cellulaire et Infection, Paris, France 2. INSERM, U604, Paris, France 3. INRA, USC2020, Paris, France 4. Institut André Lwoff, Université Paris-Sud, Villejuif, France 5. INSERM, U602, Villejuif, France Listeria monocytogenes is a facultative intracellular bacterial pathogen that invades epithelial cells by subverting signaling cascades associated with its two main cellular receptors, E-cadherin and Met. We recently identified the type II phos- phatidylinositol 4-kinases (PI4KIIs) α and β as required for bacterial entry downstream of Met. In this work we investigated whether tetraspanins CD9, CD63 and CD81, 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 expressed and detected at the cellular surface and also within intracellular compartments, particularly in the case of CD63. In resting cells, colocalization between these tetraspanins and the PI4KIIα is only detectable in restricted areas of the perinu- clear region. Upon infection with Listeria, endogenous CD9, CD63 and CD81 were recruited at the bacterial entry site but did not colocalize strictly with endogenous PI4KIIα. Live cell imaging confirmed that tetraspanins and the PI4KIIα do not follow the same recruitment dynamics to the Listeria entry site. Depletion of CD9, CD63 and CD81 levels by small interfering RNA (siRNA) demonstrated that CD81 is required for bacterial internalization, identifying for the first time a role for 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 entry site but not that of the Met receptor, suggesting that this tetraspanin could act as a membrane organizer required for the integrity of signaling events occurring at Listeria entry sites.

Listeria innate immune evasion by peptidoglycan modification REFERENCE B / O Aubry, C. 21 Department of Cellular Biology and Interaction, Institut Pasteur, France Peptidoglycan (PG) plays an important role in host-pathogen interaction because PG is the site of anchoring of virulence factors and an important target for the innate immune system. As reported previously, inactivation of pgdA, encoding a PG N-deacetylase in Listeria monocytogenes, revealed a key role of PG modification in virulence as survival of the mutant was severely impaired in mice (Boneca et al., 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 by macrophages. Interestingly, inactivation of the deacetylase induced a strong secretion of IFN beta by infected macrophages, through a surprisingly and unchar- acterized TLR2-dependent pathway. We have now shown that TLR9 but not TLR4 contribute to this IFN beta secretion. The key adaptors and the new signaling pathway leading to IFN beta production are under investigation. We found that L. monocytogenes PG is also modified by a putative O-acetyltransferase (OatA) essential for virulence. A Listeria oatA mutant shows an increased sensitivity to hen egg lysozyme, bacteriocin and cell wall targeting antibiotics in vitro. Its growth is impaired in macrophages, possibly contributing to immune escape. Modification of PG is a highly efficient mechanism used by pathogenic Listeria to evade innate host defenses.

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REFERENCE Constitutive activation of the central virulence transcriptional B / O regulator PrfA enhances Listeria monocytogenes pathogenesis but 22 reduces bacterial fitness outside of the host Freitag, N. E.1 and Bruno, J. C.2 1. Department of Microbiology & Immunology, University of Illinois at Chicago, USA 2. Department of Global Health, University of Washington, USA Listeria monocytogenes survives as a saprophyte in soil and decaying vegetation while maintaining an ability to invade mammalian cells and cause serious disease. Survival and replication of L. monocytogenes within mammalian hosts requires the regulated synthesis and expression of multiple gene products that enable host cell invasion, bacterial replication within the cytosol, and spread to adjacent cells. The transcriptional regulator PrfA controls the expression of multiple bacterial virulence factors and is required to facilitate the L. monocytogenes transition from saprophyte to pathogen. PrfA has been shown to exist in both low and high activity states, with the transition to high activity occurring within the cytosol of host cells. A number of mutations within prfA have been described (prfA* mutations) that serve to lock the protein into a high activity state. We examined the consequences of constitutive PrfA activation on L. monocytogenes physiology and pathogenesis by assessing the fitness of prfA* strains in a variety of in vitro and in vivo conditions. prfA* strains exhibit a competitive advantage over wild strains in animal models of infection, with ten-fold higher numbers of prfA* bacteria recovered from the livers and spleens of infected mice. However, despite apparently normal growth in broth culture, the prfA* mutants exhibited a fitness defect when grown in the presence of wild type bacteria, and this fitness defect was exacerbated when mixed cultures were placed under various stress conditions. Strains containing prfA* mutations were also defective in their ability to adapt to a long term survival phase following prolonged growth in broth culture. Taken together, these results indicate that L. monocytogenes must maintain a critical balance of PrfA activity to promote bacterial fitness both inside and outside of infected host cells.

REFERENCE The lvfH gene of Listeria monocytogenes encodes a novel virulence factor B / O highly activated during infection 23 Carvalho, 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 can induce potentially lethal listeriosis in immunocompromised human hosts. As a facultative intracellular pathogen, it is able to invade and replicate inside different eukaryotic cell types and, by cell-to-cell spread, disseminate infection. Such processes are highly dependent upon expression of specialized proteins which act as virulence factors on certain steps of the bacterial infectious cycle. Our recent studies on the L. monocytogenes EGD-e transcription profile in infected mice revealed genes that were highly activated throughout the infection timeline and could be involved in virulence. Among these is lvfH, a serotype 1/2a-specific gene that encodes a protein putatively involved in the L-rhamnose biosynthesis pathway, a mechanism that provides substrate for the decoration of cell wall teichoic acids. In order to confirm and characterize the role of this gene in L. monocytogenes virulence, we generated an lvfH deletion mutant and investigated the influence of this mutation in the Listeria infectious process. In vitro assays showed that the mutant was unaffected in its host cell membrane-adhering properties, but was significantly impaired in its ability to invade different mammalian cell lines. Organs of mice intravenously infected with the lvfH mutant displayed a significant decrease in bacterial load, as compared to animals infected with wild type bacteria. This phenotype is unrelated with an intrinsic growth defect of the mutant, as it presented growth profiles similar to the wild type strain in broth and within murine macrophages. Animal organs infected with an lvfH-complemented strain showed wild type-like infection levels, confirming the specific requirement of LvfH for full virulence in this model. Functional characterization of LvfH and analysis of the relation between teichoic acid rhamnosylation and virulence could reveal a new role for teichoic acid decoration in L. monocytogenes pathogenesis.

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Human listeriosis due to Listeria ivanovii REFERENCE C / O Guillet, 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.1 24 and Lecuit, M.1,2,4 1. Necker-Enfants Malades hospital, Paris Descartes University, Paris, France 2. Institut Pasteur, NRC and WHO-CC for Listeria, Paris, France 3. University of Edinburgh, Scotland, UK 4. 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), considered a ruminant-specific pathogen. We describe here a case of gastroenteritis and bacteremia due to Lii in a kidney transplant recipient. A 55-year-old man was referred to our hospital with a three-week history of non-bloody diarrhoea, vomiting, dehydratation, and low-grade fever. He underwent a renal transplantation for chronic renal failure and was chronically infected with hepatitis C virus. Stool and blood cultures allowed the isolation of Lii. Intravenous amoxicillin (6g/day) and gentamicin (6 mg/kg/day) treatment was associated with resolution of symptoms. The 4 Lii isolates from this patient had indistinguishable ApaI and SmaI PFGE patterns, phenotypic characters and classical resistance for antibiotics. These human isolates were indistinguishable from prototypic ruminant strains based on (i) the activation status of the central virulence gene regulator PrfA, (ii) the presence of the L. ivanovii-specific pathogenicity island LIPI-2 by PCR mapping, and (iii) invasion assays using Madin-Darby Bovine Kidney cells and huma, HeLa cells. So, Lii is pathogenic for humans. As for Lm, Lii route of infection is foodborne. A review of the literature identified 8 cases, mostly in immunosuppressed patients. The rarity of human Lii infections probably reflects low exposure given the rare occurrence of this species in nature compared to Lm.

REFERENCE Foodborne listeriosis in India: An update C / O Barbuddhe, 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 25 1. ICAR Research Complex for Goa, India 2. Division of Veterinary Public Health, Indian Veterinary Research Institute 3. Nagpur Veterinary College, India 4. Institute of Medical Microbiology, Justus Liebig University, Giessen, Germany Listeria monocytogenes is a foodborne pathogen that can cause serious invasive illness, mainly in certain well-defined high-risk groups, including elderly and immunocompromised patients, pregnant women, newborns and infants. In India, the pathogen has been isolated from humans, animals, a variety of foods including milk and milk products, meat and meat products and vegetables. In India, studies on molecular epidemiological aspects of L. monocytogenes are largely lacking. Therefore, we do not know the genetic variability of strains isolated and their epidemic potential. The genetic diversity of the Listeria strains from various sources namely, milk and milk products (112), fish and seafood (47), wild life (7), poultry meat (19), meat and processed meats (37), animal clinical cases (41) and human clinical cases (21) isolated/collected from different places in the country have been characterized biochemically and with in vitro assays before attempting for serotyping and virulence gene profiles. Out of the strains characterized phenotypically, 195 strains have been serotyped using multiplex PCR and pulsed field gel electrophoresis. The predominant serotype among Indian Listeria isolates is L. monocytogenes 4b. Significant variation among the isolates recovered from different sources has been observed as evident by PFGE analysis. As many as 34 different PFGE profiles have been observed. An electronic database for the characterized strains has been created. This is an interactive web based database so that the data can be exchanged between laboratories electronically. The molecular characterization of Listeria isolates from India would help in better understanding of the sources of infection and their risk assessment, routes of transmission of the infective agent, influencing factors, clinical forms and virulence characteristic of the 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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REFERENCE Human listeriosis and co-morbidities in England, 1999 to 2008: C / O quantifying the risk 26 Mook, 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 epidemiology of listeriosis in England and Wales changed between 2001 and 2008, with more patients aged ≥ 60 years presenting with bacteraemia. We examined the risks in this age group, and quantified the role of co-morbidities for listeriosis in all age groups. Case-patients were resident in England between 1999 and 2008 and were reported to an enhanced national surveillance scheme by hospital microbiologists using a clinical case questionnaire. We coded co-morbidities of non pregnancy-related cases of L. monocytogenes infection according to ICD-10. These data 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 received a clinical questionnaire for 81% of cases (N=1141). Eighty-two percent (N=934) had one or more underlying medical conditions and we recorded 1239 ICD-10 codes on co-morbidities from these 934 cases. The ≥ 60 years age group comprised 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]), malignancies 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 & other parts of CNS (66.3 [35.3-113.3]). We have highlighted several underlying conditions not previously thought to be strongly associated with listeriosis. The extent to which these co-morbidities are correlated with each other requires further investigation to enable much better, targeted prevention.

REFERENCE Risk factors for death in Listeria monocytogenes infection, England and C / O Wales, 1990 to 2008 27 Mook, P., Grant, K. and Gillespie, I. Health Protection Agency, UK Listeriosis, caused by the Gram positive bacterium Listeria monocytogenes, is a rare but severe food borne disease. The elderly, pregnant and the seriously ill are most often affected, with high mortality rates in all patient groups. The epidemiology of listeriosis in England & Wales has changed in recent years, with an average of 179 cases reported annually from 2000 to 2008 compared with 110 cases on average between 1990 and 1999. Much of the increase has occurred in patients > 59 years presenting with bacteraemia but without central nervous system involvement. To examine factors influencing L. monocytogenes mortality, non-pregnancy associated cases resident in England & Wales reported to national surveillance between 1990 and 2008 (N=1832) were analysed further. The overall mortality rate for this period was 39% (N=719). Univariable and subsequent multivariable analyses were performed and the role of key factors (season, age, underlying conditions and treatment) in this outcome investigated. Our initial findings suggest that, for a disease which disproportionately affects the elderly and the infirm, mortality is greatest in older patients and in those with underlying conditions. Un- derlying conditions themselves (particularly alcoholism and malignancies of the lymphatic/haematopoietic system and the breast) appear to be more important than the treatment they were receiving for an underlying condition, even when the effect of age is considered. Given that listeriosis is largely a food-borne disease, and therefore preventable, it would seem appropriate to actively target high risk groups with specific advice on what foods to avoid in order to minimise the risk of listeriosis.

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A discrete event model to track Listeria monocytogenes REFERENCE C / O in the retail environment Pouillot, R.1 and Gallagher, D.2 28 1. CFSAN/FDA, USA 2. Va Tech, USA A recent comparative Listeria monocytogenes (Lm) risk assessment predicts that of the listeriosis cases attributed to deli meat, a majority are associated with deli meat sliced and packaged at retail. There is a need to: 1) identify potential sources and practices that contribute to Lm contamination of food at the retail level; and 2) identify retail practices that would reduce or eliminate Lm contamination of ready-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 at retail, a dynamic discrete-event model that tracks Lm cells at various locations over time in a retail setting was developed. This model considers i) the transfer of bacteria from some defined compartment to others (unsliced products, sliced products, food contact surfaces, non food contact surfaces, slicer, gloves, hands, etc.); ii) the bacterial inactivation through cleaning and disinfection; and iii) the bacterial growth according to the physic and chemical environment (temperature, pH, water activity, presence of growth inhibitors). Sequences of events were derived using specifically acquired data from an observational study of food handling practices in retail deli departments. Additional data on the transmission of Lm in the retail environment is being collected through laboratory studies. More- over, this risk assessment is designed to evaluate the relative effectiveness of retail Lm control measures along with other risk management scenarios. The major uncertainty is in the potential existence of niches and their interaction with the retail environment. This presentation will describe this model, provide an illustration using current data and highlight the data gaps that mainly influence the outputs.

Clinical and epidemiological aspects of listeriosis in Israel REFERENCE C / O Hershko-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 29 1. Meir medical center, Kfar-Saba, Israel 2. Hadassah Hebrew University Medical Center, Israel 3. Israel Ministry of Health, Central Laboratories 4. Tel-Aviv Souraski medical Center, Tel Aviv, Israel 5. Rambam medical center, Haifa, Israel Listeria monocytogenes (LM) is a ubiquitous foodborne pathogen. Invasive infections are rare, mainly affecting elderly people, the immunocompromised and neonates. In pregnancy it causes fetal loss, preterm delivery and neonatal morbidity. Although the incidence of listeriosis is low compared with other en- teropathogens, it carries a high mortality. Our aim was to characterize LM morbidity, risk factors and molecular epidemiology in Israel to improve understanding of the disease burden in Israel and assist public health policy-making. We performed a nationwide retrospective study of all LM cases during 1998–2007. Cases were actively sought in the records of all hospital-based clinical microbiology laboratories, the national reference LM laboratory and LM cases reported to the dis- trict physicians. 481 cases were identified, of which 166 were pregnancy-associated. The yearly incidence peaked in 2006 and 2007 at 9.6 cases/million, which is almost 5 times than the current rate in the USA. The incidence in pregnancy varied between 5 and 25 cases/100,000 pregnancies/year. Annual rates above 15 cases/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 survival improved as pregnancy age advanced (P<0.05). LM associated late abortion occurred in 26.6% and preterm labor in 46.8%. A single maternal death was recorded. The case fatality in non-pregnancy cases was 18%. Additionally a gradual increase 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 associated cases were caused by a single clone. In Israel, Listeria monocytogenes is an important foodborne pathogen causing appreciable morbidity and mortality. Fur- ther studies of the sources, molecular epidemiology and specific virulence determinants should be undertaken.

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REFERENCE Human isolates of Listeria monocytogenes during C / O half a century in Sweden 30 Lopez-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 invasive 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 predominant serovar in human invasive listeriosis in Sweden. During the 1980s, some listeriosis outbreaks caused by L. monocytogenes serovar 4b, belonging to a special PFGE type, were diagnosed in the USA and Europe and were due to consumption of dairy products. The predominant strain in listeriosis cases in Sweden during the same time belonged to this type. The hypothesis is that a majority of human listeriosis serovar 4b strains in Sweden came from soft cheeses imported from Mediterranean countries. The hunt for L. monocytogenes serovar 4b in food production plants is intensive in EU and USA and during recent years, cheeses have had higher microbiological quality due to certified dairy farms and increased microbiological control. This may be the reason for the decrease of serovar 4b cases of listeriosis in Sweden. In 1996, serovar 1/2a became the major serovar in listeriosis cases in Sweden. The consumption of gravad and cold-smoked salmons has increased in Sweden, and in a recent study, 12.9% of ready-to-eat vacuum-packed gravad salmon and 28.0% of cold-smoked salmons harboured L. monocytogenes. The maximum number of L. monocytogenes was 1500cfu/g product. The most common PFGE types found in human cases of listeriosis in Sweden today are also the types frequently encountered in vacuum-packed cold-smoked and gravad salmon/rainbow trout.

REFERENCE Estimated incubation periods for listeriosis vary according to clinical C / O form of disease 31 Goulet, 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 can be calculated precisely when a patient has had a single exposure to a confirmed source of contamination. This information can be collected during the investigation of point-source food borne outbreaks. Our study aimed to estimate the listeriosis incubation period using available outbreak investigation data. Cases of invasive listeriosis from confirmed food borne point-source listeriosis outbreaks with precisely documented incubation periods and clinical forms of infection were identified during the period 1985–2008 by literature review and from non-published French outbreaks. Data from 9 published papers and from 6 French outbreaks were analysed. A precise incubation period was documented for 28 cases: 13 with central nervous system (CNS) involvement (10 French outbreak and 3 published cases), 15 pregnancy-associated cases (12 French outbreak and 3 published cases). A confirmed food vehicle was identified for all outbreaks (rice salad, ice- cream, cheese, meat-based products). The overall median incubation period was 17 days (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 involvement (median: 10 days (range: 2–19 days)). Insufficient data did not allow for analysis of the incubation period of the bacteraemic form of infection. Our results suggest that the incubation period for listeriosis varies according to the clinical form of disease. A longer incubation period was observed for pregnancy-associated cases than for cases with CNS involvement. This information could have implications for the investigation of food borne listeriosis outbreaks as the incubation period is used to determine the time period for which a food history is collected. Adapting this time period according to the clinical form of infection could facilitate a more precise identification of food products likely to be the source of contamination.

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Listeria monocytogenes in food and animals REFERENCE C / O in the European Union in 2008 da Silva Felício, M. T., Rizzi, V., Boelaert, F. and Makela, P. 32 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 resistance and food-borne outbreaks. These data are reported to the European Food Safety Authority (EFSA) who publishes the Community Summary Report. In 2008, a large number of investigations (128,000) concerning ready-to-eat (RTE) foodstuffs were reported by 26 MSs. The food categories most often covered were RTE meat products, cheeses and fishery products. L. monocytogenes was seldom detected above the legal safety limit of 100 cfu/g from RTE foods and findings over this limit were most often reported from fishery products, cheeses, meat products and sandwiches at levels of 0.2–0.5% in the EU. L. monocytogenes was isolated from both cheeses made from raw or low-heat-treated milk and pasteurised milk as well as from soft/semi-soft and hard cheeses. L. monocytogenes was most often detected in soft and semi-soft cheeses made from pasteurised milk. L. monocytogenes was also reported, generally at a relatively low prevalence, from various animal species in 2008, demonstrating that animals act as one reservoir of Listeria bacteria although they rarely serve as a direct source of human infections. The highest 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 carried in MSs to ensure compliance with L. monocytogenes criteria. Compared to previous years the quality of the data received improved as regards reporting of the stage of sampling and the use of appropriate test methods that has eased the assessment of compliance with Listeria criteria at Community level.

Listeria monocytogenes infection in the over 60s in England REFERENCE C / O between 2005 and 2008: a retrospective case-control study utilising market research panel data 33 Gillespie, 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 investigation of factors which might alter people’s risk of infection is important to inform on prevention and control. The incidence of listeriosis in England has dou- bled since 2001, with a largely unexplained increase in people aged ≥ 60 years presenting with bacteraemia without central nervous system involvement. Stan- dardised epidemiological data has been sought on cases of listeriosis reported in England since 2005, but the value of the data accrued is limited without some knowledge of exposure prevalence in the population at risk of listeriosis. The exposures of listeriosis cases aged ≥ 60 years reported in England from 2005–2008 were compared to those of market research panel members representing the same population and time period. Exposures were grouped to facilitate comparison. Odds ratios and 95% confidence intervals were calculated. Cases were more likely than panel members to report cold cooked meats (beef and pork, but not chicken), smoked fish (specifically salmon) and shellfish (prawns), dairy products (milk and certain cheeses) and mixed salads. They were less likely to report the consumption of sandwiches and fresh vegetables. To our knowledge this is the first time that market research data has been applied to infectious disease epidemiology in this way. The diversity of high-risk food exposures reflects the ubiquity of the microorganism in the environment and/or the susceptibility of those at risk, and suggests that a wider variety of foods can give rise to listeriosis. Food safety advice on avoiding listeriosis should be adapted accordingly.

49 AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis ORALPRESENTATIONS // O / 24–36

REFERENCE Better and faster typing, MLVA – shall we play together? C / O 34 Larsson, J. T.1, Roussel, S.2 and Moller Nielsen, E.1 1. Statens Serum Institut, Copenhagen, Denmark 2. Agence Française de sécurité sanitaire des aliments, Maisons-Alfort, France In 2007 and 2008 there were four papers published on the subject of MLVA (Multi Locus VNTR [Variable Number of Tandem Repeats] Analysis) in L. monocytogenes. During the same period a protocol with an extensive bioinformatic approach was developed at SSI. The yet unpublished SSI MLVA protocol was created after analysis of 20 genomes. Ten VNTRs were chosen on the basis of both nucleotide and amino acid sequences analysis. The VNTRs have repeat lengths ranging from 6 to 15 basepairs. Degenerate primers were designed to match the diversity in the genomes, taking good care not to include any gaps (which were present in several loci/genomes). Finally three multiplex PCR groups were created and the VNTRs were analysed by electrophoresis. The SSI method has been evaluated with the last years Danish human listeriosis cases and the North American ILSI Listeria collection. Results indicate a very close match to two-enzyme PFGE and an even better separation of isolates. These results in concert with the reduced time and cost for using MLVA for typing started off an incentive for unification and stan- dardisation of MLVA in L. monocytogenes. In a recently started cross laboratory study, a comparison of the protocol published by Kate Sperry (2007) and the protocol developed at SSI is under way. The methods are evaluated together with traditional typing methods such as PFGE and agglutination serotyping. The MLVA protocols are tested using capillary and agarose gel electrophoresis. Strain collec- tions includes; a selection of the WHO subtyping study from 1986 and a selection from 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 the two assays. The results from this cooperation will lead to a recommendation on a MLVA protocol for use in Denmark and France.

REFERENCE comK prophage junction fragments in Listeria monocytogenes contain C / O SNPs that differentiate subclones of ECII and ECIII that are unique to 35 individual 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, especially those associated with ready-to-eat meat and poultry products. Both of these epidemic clones have backbone genomes that are highly conserved, but contain a hypervariable 40 Kb comK prophage. All isolates analyzed in the present study that were suspected of being ECII or ECIII were first confirmed as such by multiplex PCR and multi-virulence-locus sequence typing (Chen and Knabel, 2007) and included those from the 1998–1999 hot dog and 2002 turkey deli outbreaks in the U.S., those from multiple states that were generated as part of USDA FSIS's meat and poultry plant surveillance program, and those that were previously isolated from two turkey processing plants in two different states (Eifert et al. 2005). Upstream and downstream prophage junction fragments in all ECII and ECIII 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 ECIII isolates from a sporadic case in 1988 and from an outbreak in 2000 due to turkey deli meat in the U.S. were also amplified and sequenced. Cluster analysis based on junction fragment sequences revealed the presence of five subclones of ECII, the ECIII subclone and the Canadian 2008 subclone, which were unique to individual meat and poultry processing plants. However, the ECII subclone that caused the 2002 outbreak was found in three different processing plants, two of which were originally associated with this outbreak. We speculate that evolution of these subclones may be due to extensive recombination and subsequent niche-specific adaptation acting on the comK prophage, which may represent a “Rapid Adapta- tion Island” in Listeria monocytogenes. Genes that were unique to the comK prophage were identified as putative “Adaptons” and their possible roles in colonization, transmission between and within meat and poultry plants, and subsequent contamination of foods and humans will be discussed.

50 AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis ORALPRESENTATIONS // O / 24–36

Genetic diversity of Listeria monocytogenes measured by multiple-locus REFERENCE C / O variable-number tandem repeat analysis (MLVA) Hyytia-Trees, E., Sabol, A., Graves, L. and Ribot, E. 36 Centers for Disease Control and Prevention, USA Listeria monocytogenes is a genetically and phenotypically diverse organism, encompassing at least three genetically distinct lineages that appear to differ in their likelihood of causing human and animal disease. Classification of isolates into these three lineages has been confirmed by different subtyping methods, and also correlates with serotype classification. In the present study, 250 epidemiologically 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 characterized using multiple-locus variable-number tandem repeat analysis (MLVA). The isolate set also included one isolate from each of ten well characterized outbreaks. The data was analyzed in BioNumerics software and a dendrogram was constructed by UPGMA clustering using the categorical coefficient. A minimal spanning tree was also created using the Manhattan coefficient. In the minimum spanning tree, a clonal complex was defined as a group of related patterns that differed from each other at a single locus by one or two repeats. A total of 139 unique patterns were detected among the 250 isolates. Thirty one MLVA patterns were associated with more than one isolate, with the most common pattern including 16 isolates with isolation years varying from 1983 to 2007. With very few exceptions, the isolates clustered according to their lineage. Nineteen clonal complexes were detected, 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 outbreak related isolates were located in three different clonal complexes that included nine or more isolates with multiple isolation years and origins in different continents. In conclusion, the MLVA scheme used in the present study is in ac- cordance with the currently used lineage classification and indicated the presence of successful epidemic clones.

51 AREA // D // Strategies for prevention and control of Listeria monocytogenes ORALPRESENTATIONS // O / 37–46

REFERENCE Time Temperature Indicators can be used as an effective Risk D / O Management Tool for Listeria monocytogenes in Ready-To-Eat foods 37 Koutsoumanis, 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 RTE foods. In a previous study we presented a microbial Time Temperature Indicator (TTI) system based on the growth and metabolic activity of a Lactobacillus sakei strain. In the latter system, an irreversible color change of a chemical chromatic indicator (from red to yellow) progressively occurs due to the pH decline as a result of L. sakei growth and metabolism in a selected medium. In this study we show that both the L. sakei growth and colour change of the microbial TTI system exhibit similar kinetic responses with the growth of L. monocytogenes. With an appropriate 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) can be adjusted in order to indicate a certain level of L. monocytogenes growth in the food during distribution and storage. Thus, the use of the microbial TTI can assure a maximum limit in the growth of the pathogen from production to consumption time by informing the consumers when this limit is exceeded in a product unit. The latter limit, which we call Growth Tolerance Criterion (GTC), can be considered as a performance criterion for the growth of L. monocytogenes during distribution and storage. The applicability of the microbial TTI in reducing consumer exposure to L. monocytogenes from the consumption of pate was evaluated in a simulation study. The TTI was appropriately adjusted to provide a GTC=3 logs CFU/g. The concentration of the pathogen and the colour of the TTI at the time of consumption were estimated with a probabilistic approach using Monte Carlo simulation. The simulation results showed that the application of the TTI resulted in a significant reduction of consumer exposure to L. monocytogenes. Assuming that packages in which the TTI end point has been reached before consumption are discarded, the predicted percentage of consumed products contaminated with L. monocytogenes concentrations above 103 CFU/g was reduced from 5% to 0.2% with the use of TTI.

REFERENCE Safety of salad leaves and herbs D / O 38 Garland, C. D.1 and Clark, A.2 1. FWE Health, North Hobart, Tasmania, Australia 2. 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 in a commercial facility in Tasmania, Australia. Production has increased from 27 tonnes in 1995 to 2000 tonnes in 2009, with cultivation now undertaken at 3 field sites and processing in a centralised factory certified to third party audited food safety standards. The range of consumers is wide, including typical high-risk groups. More than 1200 samples of RTE products have been tested since 1995 and currently 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 in field conditions are implemented for: lettuce transplants; cultivation soil; catch- ment animals and wild life; irrigation water; harvesting; acceptance/rejection criteria for raw ingredients; potable water; transport of harvest to factory. CPs/CCPs in the factory relate to: restricted access; strict separation of work spaces into low- medium-high risk zones; temperature control; reduction of microbial loads; product packing; staff training; sensory monitoring of product; physical and microbiological 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 microbiological and physical CPs/CCPs relating to: animal access to the field, soil addi- tives and composts; surfaces, equipment, potable water, irrigation water, raw ingredients and RTE products; factory temperature control (ambient, water, product); removal of organisms by washing, centrifugation and infrared drying, and disinfection by chlorination (with auto adjustment of free available chlorine and pH); transport.

52 AREA // D // Strategies for prevention and control of Listeria monocytogenes ORALPRESENTATIONS // O / 37–46

Environmental factors affect adhesion of Listeria monocytogenes REFERENCE D / O to inert surfaces through flagellum expression Tresse, O. 39 INRA, France Listeria monocytogenes could adhere to inert surfaces and form biofilms. Biofilms of L. monocytogenes constitute eventually a reservoir for cross- or re-contaminations of food products in food-processing plants. Biofilm initiation includes an irreversible attachment of cells (adhesion) to inert surfaces. Adhesion is therefore crucial for the biofilm development and subsequently for dissemination and persistence of food pathogens. Adhesion assays from four to 101 L. monocytogenes strains 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 concentrations and temperatures. In parallel, flagella were observed after cultivation in the different environmental conditions using optical microscopy. Three naturally afla- gellated strains, five environmental flaA mutant strains and one flagellum-paralyzed 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 equal to6,at8°Cand20°Corat0%and6%NaClthanatpH5,at37°Corat11%NaCl. Correlatively, flagella were not detected at pH 5, at 37°C and at 11% NaCl. This result indicates that the temperature is not the only environmental factor that could regulate flagellum expression in L. monocytogenes. NaCl concentrations and pHs closed to non-growing conditions inhibit flagellum expression. Naturally afla- gellated strains and ∆flaA mutants adhered at the same level of strains cultivated in flagellum inhibiting conditions. In addition, ∆flaA mutants adhered equally while adhesion capability of the respective parental strains varied according to the strain. Furthermore, adhesion was also affected in the flagellum-paralyzed strain indicating that motility plays an important role in adhesion capability. In conclusion, conditions of food conservation have an effect on adhesion capability of L. monocytogenes strains through flagellum regulation and flagella are responsible for adhesion variation among strains.

Shelf-life laboratory durability and challenge studies for REFERENCE D / O Listeria monocytogenes in ready-to-eat foods: a presentation of the European technical guidance document intended for laboratories 40 Beaufort, 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 refrigeration temperatures and may be present in ready-to-eat (RTE) foods. In annex I of regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs, a specific attention to food safety criteria for L. monocytogenes in RTE foods is given and annex II of this regulation specifies that food business operators (FBO’s) shall conduct, as necessary, studies to evaluate the growth of L. monocytogenes that may be present in the product during the shelf-life under reasonably foreseeable storage conditions. But this annex does not describe the procedure to conduct such shelf-life studies to ensure that the criterion of 100 L. monocytogenes/g is met over the entire intended shelf-life of the product. The European technical guidance document, intended for laboratories conducting shelf-life studies for L. monocytogenes in RTE foods 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 microbiological procedures for determining growth of L. monocytogenes using challenge tests and durability studies. Challenge tests, defined as tests providing information on the behaviour of L. monocytogenes artificially inoculated in the food, can be performed with two different objectives: (i)Classify whether a food does or does not support growth of L. monocytogenes; (ii) Quantify the growth of L. monocytogenes by assessing either the growth potential (δ) or the maximum growth rate (∝max). Durability studies allow an evaluation of the growth of L. monocytogenes in a naturally contaminated food during its storage according to reasonably foreseeable conditions. This technical document, prepared by the EU Community Refer- ence Laboratory for L. monocytogenes in collaboration with a working group consisting 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.

53 AREA // D // Strategies for prevention and control of Listeria monocytogenes ORALPRESENTATIONS // O / 37–46

REFERENCE Successful strategies against Listeria monocytogenes in Switzerland D / O 41 Imhof, R. Agroscope Liebefeld-Posieux Research Station ALP, Switzerland In the aftermath of the massive Listeriosis outbreak in 1987 due to a soft cheese made out of raw milk, the Swiss government decreed the creation of appropriate means 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 detection of Listeria and to develop a nationwide Listeria monitoring programme (LMP) in cooperation with the Swiss dairy industry. LMP covers about 70 to 80% of the cheese production of Switzerland. The aims are to detect contamination in cheese production sites and ripening centres as early as possible, to stop the spreading of Listeria by means of appropriate measures and above all to prevent contaminated 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 are not open to the public, ALP closely works together with the concerned public authorities in cases of enterprises with problems. ALP also created a special task force: 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 participation in the process of complete redevelopments. Following defined proceedings the ALP team helps in cooperation with the enterprises own emergency team to find individually designed solutions. Periodical controls in the following years proof the success and the sustainability of interventions and worked out security concepts. The history of outbreaks and the European RASFF notifications of the last 15 years concerning Switzerland manifest the success of this specific approach. Despite many ingenious efforts and product developments, there exists – to our knowledge after 20 years of specific experience – no single measure or method to solve a problem within the dairy food chain caused by L. monocytogenes. But observance of the principles of Good Manufacturing Practice, a specifically designed concept of security – which has to be lived by staff and personnel – besides to periodical controls guarantee that every production site is capable to produce and distribute safe and healthy dairy products.

REFERENCE Absence of Listeria monocytogenes growth during raw milk D / O cheesemaking: a modelling approach 42 Jordan, K.1, Schvartzman, S.1,2, Butler, F.2 and Tenenhaus-Aziza, F.3 1. Teagasc, Moorepark Food Research Centre, Ferrmoy, Cork, Ireland 2. Biosystems Engineering, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Ireland 3. CNEIL, National Interprofessionnal Center for Dairy Economy, Paris, France The presence of Listeria monocytogenes in certain foods and the risk that this poses to public health and food quality is still a problem. Currently, the field of food microbiology focuses on obtaining data on the behaviour of microorganisms in food, but the responses obtained provide little insight into the relationship between physiological processes and growth or survival. This link can be made through mathematical models. In a simple form, a mathematical model is a simple mathematical description of a process. The application of mathematical models to food microbiology has been developed in recent years and now constitutes a new discipline named as Predictive Microbiology. However, most predictive models are based on laboratory experiments in microbiological media under static conditions. As such models tend to be inaccurate, we have undertaken our experiments in a food system under dynamic conditions. Cheese was made with raw and pasteurised milk deliberately contaminated with L. monocytogenes. Listeria was monitored through the manufacture and ripening period of the cheese. The results showed that L. monocytogenes did not grow during manufacture of raw milk cheese, but did grow during manufacture of pasteurised milk cheese. The data obtained for growth, survival and inactivation was modelled. The application of models that can explain the behaviour of Listeria in cheese and the further predictions that can be obtained from these models are useful for the improvement of ongoing research on biotraceability and for the better understanding of the general behaviour of these microorganisms under dynamic conditions, such as in dairy products.

54 AREA // D // Strategies for prevention and control of Listeria monocytogenes ORALPRESENTATIONS // O / 37–46

A predictive model to set high pressure processing criteria for REFERENCE D / O Listeria monocytogenes inactivation on dry-cured ham Bover-Cid, S.1, Belletti, N.2, Garriga, M.1 and Aymerich, T.1 43 1. IRTA. Food Technology, Spain 2. Università di Bologna, Italy The aim of the work was to validate and use a predictive mathematical model for 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 developed from the inactivation data obtained after processing sliced dry-cured ham, spiked with L. monocytogenes, at different HHP conditions according to a central composite design: at 347–852MPa; for 138 to 945 seconds; at 7.6 to 24.4°C. The best fitting and most significant polynomial equation indicated that pressure and time were the most important factors determining the inactivation extent. By contrast, temperature showed no significant influence within the assayed range. The statistically significant quadratic terms of pressure and time indicate the little effect observed below 450MPa. Similarly, holding time longer than 10min did not result in a meaningful reduction of L. monocytogenes counts. The mathematical model was validated with data obtained from further experiments within the assayed experimental domain, including data from international scientific literature. The accuracy and bias factor were within the proposed ac- ceptable values demonstrating the suitability of the model for predictive purposes. As a practical application of the validated model, the mathematical equation was used to predict the HHP process conditions needed to ensure the safety performance criteria established by different authors and organisations, in terms of logarithmic reductions of L. monocytogenes loads. The general performance criteria of 6Log reduction, as equivalent to pasteurization, suggested by the Food and Drug Administration would be achieved with treatments above 600MPa, for instance applying 807MPa for 5min or 746MPa for 10min. More product-specific performance criteria proposed, i.e. 4D and 2.39D, would be achieved with 5min treatments at 703MPa and 613MPa, respectively.

REFERENCE Application of a validated predictive model to prevent growth / of Listeria monocytogenes in ready-to-eat foods – importance D O for product development and risk management 44 Mejlholm, O. and Dalgaard, P. Seafood & Predictive Microbiology, Technical University of Denmark Predictive microbiology is a most active research area within food microbiology and many successfully validated models are today available and useful for assessment and management of microbial food safety and quality. Recently, an extensive growth and growth boundary model for Listeria monocytogenes was developed and successfully validated for ready-to-eat meat, seafood, poultry and non-fermented dairy products. This model is flexible and includes the effect of 12 environmental parameters (temperature, NaCl/aw, pH, smoke intensity, nitrite, CO2, acetic acid, benzoic acid, citric acid, diacetate, lactic acid and sorbic acid) as well as their interactive effects. It can predict the growth boundary and thereby combinations of product characteristics and storage conditions that prevent growth of L. monocytogenes and this is most important in relation to EU regulations and codex guidelines. Nevertheless, growth boundary predictions alone are insufficient for formulation of safe foods because the likely variability in product characteristics and storage conditions must be taken into account. For this, stochastic models can be 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 alternative that allow variability in product characteristics and storage conditions to be taken into account when conditions that prevent growth of L. monocytogenes are predicted. This approach relies on validated cardinal parameter growth models. The effect of interaction between environmental parameters is used to predict both 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 interfaces can be predicted in a sufficient distance from the growth boundary to account for variability in the environmental conditions.

55 AREA // D // Strategies for prevention and control of Listeria monocytogenes ORALPRESENTATIONS // O / 37–46

REFERENCE / Application of predictive microbiology to control the growth D O of Listeria monocytogenes – dairy products as an example 45 Lobacz, A. Faculty of Food Sciences, University of Warmia and Mazury in Olsztyn, Poland Predictive microbiology is a relatively new scientific discipline which is used for modelling behaviour of microorganisms, mainly food borne pathogens, as a response to environment. It is based on assumption that the reaction of bacteria on particular environment factors is reproducible, and on the basis of researches and observations made in past, it is possible to predict the microbiological behaviour in defined environment. Many mathematical models, with regard to Listeria monocytogenes, have been generated, which describe survival, inactivation or growth of this food borne pathogen as a response to environmental parameters. Models are generated usually in microbiological liquid media, although more often data are obtained in food products. In the following paper usefulness of predictive models describing the growth of L. monocytogenes is presented. Application of tertiary models – ComBase Predictor (www.combase.cc) and Pathogen Modeling Program (www.ars.usda.gov) is also discussed. Moreover, microbiological experiment was performed in order to evaluate behaviour of L. monocytogenes in Camembert type cheese during ripening and storage at following temperatures: 3, 6, 9, 12, 15 and 21°C. Primary and secondary models were generated and their performance was checked by mathematical and graphical validation. Predictive microbiology is a new tool to microbiological risk assessment. As a subdiscipline of food microbiology, it is an intensively developing field. Gener- ated and validated predictive models become more accurate and more often used in food industry in order to eliminate food borne outbreaks.

REFERENCE Characterization of Food Alert for Listeria monocytogenes in France in D / O 2008 46 Leclercq, 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 and WHO-CC for Listeria, Paris, France 2. Direction Générale de l’Alimentation, Ministère de l’Alimentation, de l’Agriculture et de la Pêche, France 3. Institut de Veille Sanitaire (InVS), France 4. 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 health and consumers, the European regulation EC 178/2002 indicates that food business operators shall notify all foodstuffs not in compliance with the food safety criteria for Listeria monocytogenes (Lm) described in regulation EC 2073/2005. A food alert is established when a foodstuff presenting a risk is on the market, or when rapid action (withdrawal or recall) is required. In the present study, our aim was to characterize all these French food alerts (FAs) for Lm in 2008. In case of FA, competent authorities asked the food business operators or/and inspection services to send Lm food strains from own-checks and official control to the NRC for Listeria to obtain their genoserotype and combined AscI/ApaI patterns by PFGE. Weekly, comparison of microbiological characteristics of food and human strains were performed on a period of 6 months and were sent to involved competent authorities and French Institute of Public Health Surveillance (InVS) with the aim of possible complementary investigation, if necessary. In 2008, 280 FAs for Lm have been registered, mainly at the retail level (184), from own-checks (215) and official control (65). Among these 280 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 (40 FAs). 179 FAs were only followed by food strains invoice [Range: 1-52 strains/FA; 100 FAs with a single strain associated], representing 673 food strains. Genoserotypes of food strains were at 55% IIa, 21% IVb, 19% IIb and 4% IIc. Combined AscI/ApaI patterns of strains varied from 1 to 27 per FA [137 showed single combined AscI/ApaI pattern]. 60% of combined AscI/ApaI patterns for FAs food strains have been also observed for human strains in 2008. 84 FAs have food strains found microbiologically similar to human strains but no FA was linked with human cases in 2008. Work is in progress for comparison of these data to 2007 and 2009 data. Even if high food contamination were sometimes observed, FAs were not at the origin or followed by human cases in 2008. In addition with the measures performed by the food business operators in the context with their sanitary control plan, food management measures linked to FAs may have contributed in the French situation of no Lm outbreak since 2003.

56 AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control ORALPRESENTATIONS // O / 47–49

REFERENCE Efforts to update and perform health education on listeriosis / in Los Angeles County, California, by the County of Los Angeles E O Department of Public Health 47 Guevara, R. E.* County of Los Angeles Department of Public Health, USA Listeriosis became a relatively well-known disease in Los Angeles County after the large southern California listeriosis outbreak in 1985. However, 20 years later, the tasks of keeping the general population and medical community properly educated have not been easy with public health priorities shifting across topics such as bioter- rorism preparedness, MRSA, West Nile Virus, pandemic influenza, and most recently H1N1 influenza. In 2005, health education efforts on listeriosis were specific to only certain high-risk groups and needed a broader audience. Understanding the County of Los Angeles Department of Public Health’s (DPH) health education mission to en- compass the general community, a multi-disciplinary team composed of epidemiolo- gists, public health nurses, and health educators produced new listeriosis education materials. Public health messages were developed by those who conducted case investigations by interviewing patients, relatives, and providers of listeriosis cases, and by those who performed epidemiologic surveillance. A standard of attractiveness was set before recruiting health educators to help design the new education materials. Finding various images and pictures, health educators drafted different designs for posters, flyers, and brochures. Public health nurses formed focus groups to help re- fine health education messages, particularly for materials translated into Spanish. No systematic measurement for success of the health education materials was established; however, the materials continue to be used by DPH as various health care providers and organizations request them, and even when H1N1 influenza became the focus of public health in April 2009, listeriosis was the most common search term in the DPH website. This presentation describes the approach the DPH used to more effectively communicate the risks of listeriosis not just to those who are vulnerable to the disease, but also to those who might know or even take care of them.

* Participation Supported by IUFoST

Awareness of listeriosis among Portuguese pregnant women REFERENCE E / O Mateus, T.1,3, Maia, R. L.2 and Teixeira, P.1 48 1. CBQF/Escola Superior de Biotecnologia – Universidade Católica Portuguesa, Porto, Portugal 2. CECLICO/Centro de Estudos Culturais, da Linguagem e do Comportamento Faculdade de Ciências Humanas e Sociais – Universidade Fernando Pessoa, Porto, Portugal 3. Escola Universitária Vasco da Gama, Coimbra, Portugal The role of Listeria monocytogenes as a foodborne pathogen was definitively recognized during the 1980s. This recognition was the consequence of a number of epidemic human outbreaks due to the consumption of contaminated foods, in Canada, the US and Europe. Listeriosis is especially severe in immunocompromised individuals such as pregnant women. The disease has a low incidence, although this is undeniably increasing, and a high fatality rate amongst those infected. In pregnant women listeriosis may cause miscarriage, foetal death or neonatal morbidity in the form of septicaemia and meningitis. Improved education concerning the disease, its transmission and prevention measures for immunocompromised individuals and pregnant women has been identified as a pressing need. The aim of this study was to evaluate the awareness, in terms of food safety and listeriosis, of pregnant women in the last trimester of pregnancy or with babies up to 3 months old. For this purpose 956 women were ques- tioned as to their knowledge and practices relating to what foods to avoid, good hygiene and cooking practices. Also of interest were the sources of information on Liste- ria spp. and listeriosis, and from whom and how women would like to receive such information. Two hundred and twenty nine women said they had not avoided any kind of food, whilst 306 said they had not made any particular changes to their food safety related habits when they prepared and cooked meals. Four hundred and seventy seven women considered that they had received enough information about nutrition during pregnancy, and that this information had been acquired from their doctor, who was also the person considered the most competent to give this information. A hundred and seventeen women said they had heard about listeriosis, although only 58 of these had been able to describe listeriosis symptoms. Women showed plenty of interest in new information about listeriosis, and they would like doctors to provide it, as well as by brochures and in the pregnancy advice book provided by the State Medical Service. It appears that plan is required, to raise awareness amongst health professionals for the need for food safety education for pregnant women.

57 AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control ORALPRESENTATIONS // O / 47–49

REFERENCE The development and progress of a risk-based strategy E / O for the control of Listeria monocytogenes in New Zealand 49 Castle, M. and Crerar, S. New Zealand Food Safety Authority The New Zealand Food Safety Authority (NZFSA) has developed a risk-based strategy for the control of Listeria monocytogenes. One of the objectives of this strategy is ‘no increase in the reported incidence of foodborne listeriosis over the five year period 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 support industry and NZFSA make risk based decision; (iii) the application of management controls for industry using a risk-based approach; (iv) the implementation of the principles in the Codex Guidelines on the Application of General Principles of Food Hygiene to the Control of Listeria monocytogenes; (v) risk communication for vulnerable consumers and food businesses. The strategy also recognises that there is the potential for the increased incidence of listeriosis cases due to the impact 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 the New Zealand food industry and consumers to enhance the control of L. monocytogenes. The L. monocytogenes strategy was launched in 2008 and the initial response to the risk management tools developed will be explored including uptake and response from industry.

POSTER PRESENTATIONS AREA // A // Biology of Listeria monocytogenes POSTERPRESENTATIONS // P / 00–46

REFERENCE / Compartmentalization of IFN-gamma and IL-6 receptors signalling A P in Listeria monocytogenes phagosomes: immune vesicles 00 Ramos-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 phagosomes. Here, we describe a novel listericidal Stat1 mediated signalling pathway involving IFN-γ and IL-6. This pathway is compartmentalized into IFN-γ/IL-6 phago-receptosomes, innate immunity vesicles that link LM destruction with specific immunity and IL-6 regulation. The Stat1-mediated signal triggered within this compartment, links Rab5a with the activation of the lysosomal enzyme effec- tor, cathepsin-D, and with the induction of oxidative mechanisms such as the phox regulators, p67phox/Rac2-GTP, and the iNOS. Three downstream events link these listericidal IFN-γ/IL-6 phago-receptosomes with specific immunity: (i) a Stat1- independent transformation into MIIC-like vesicles regulated by Rab5a-GTP that allows the acquisition of αβ SDS-stable MHC-II dimers, cathepsin-D, Lamp-2 and Limp-2. (ii) A Stat1-dependent degradation of the Listeria immune-dominant antigen, listeriolysin O by cathepsin-D. (iii) A downstream Stat1 signal to regulate IL- 6 production and drive a Th-1 cell-mediated immunity.

AREA // A // Biology of Listeria monocytogenes POSTERPRESENTATIONS // P / 00–46

REFERENCE Antimicrobial susceptibility among Listeria monocytogenes isolates / from non human sources in France over a ten year period A P Granier, S. A.1, Moubarek, C.2, Colaneri, C.1, Roussel, S.1, Courvalin, P.2 and Brisabois, A.1 01 1. AFSSA, France 2. Institut Pasteur, France Listeriosis is public health concern since 1980’s. This food-borne infectious disease has very high rates of hospitalization and fatality. It usually requires antimicrobial treatment. Recommendations are: Penicillin G or ampicillin combined or not with aminoglycosides. Antimicrobial resistance has increased among Gram positive bacteria during the last past years but very few data are available for Listeria monocytogenes, especially from non-human sources. In order to determine the frequency of resistance within the species L. monocytogenes, a collection of 205 food and environmental isolates, randomly selected among a collection of 10 year period (1996–2006) was tested for antimicrobial susceptibility. This collection of non epidemiologically related isolates included major serotypes (1/2a,1/2b,1/2c and 4b strains) encountered, each of them presenting a unique PFGE profile. The phenotype 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 four strains displayed an original phenotype: two isolates (serotype 4b) were resistant to erythromycin, two isolates (serotype 1/2a) were resistant to tetracycline, one of the two last isolate was additionally resistant to trimethoprim. PCR analysis detected the presence of erm(B) gene in the 2 erythromycin resistant strains, tet(M) in the tetracycline resistant strains and dfr(D) was identified in the unique trimethoprim resistant strain. Interestingly, int gene, involved in the mobility of conjugative transposons, was found in the strains harboring tet(M) gene. The present results do not 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 resistance situation in L. monocytogenes doesn’t shift.

REFERENCE Five homologous small RNAs are involved in the response A / P of Listeria monocytogenes to cell wall acting antibiotics 02 Kiil Nielsen, P. and Kallipolitis, B. Department of Biochemistry and Molecular Biology, Denmark Small RNAs (sRNAs) are important regulatory elements involved in a variety of cellular responses, including stress tolerance and virulence. In Listeria monocytogenes five homologous sRNAs, named LhrC1-5, were identified recently by co- immunoprecipitation with the RNA chaperone protein Hfq. The objective of the present study is to improve our understanding of the role(s) of LhrC1-5 in L. monocytogenes. Our study involves the identification of factors that influence the expression of LhrC1-5 and investigations of genes affected by LhrC1-5. A range of different environmental conditions and regulatory mutant strains were employed to identify factors that affect the expression of LhrC1-5. The presence and stability of the RNA transcripts were determined by Northern blotting. Furthermore, expression of lhrC1-5 was followed by construction of promoter-lacZ fusions for all five sRNA-encoding genes. These studies revealed a differentiated transcription of LhrC1-5 in response to the presence of cell wall acting antimicrobial agents, such as ethanol and β-lactam antibiotics. Cell wall acting agents have previously been shown to activate the two-component systems LisRK and CesRK in L. monocytogenes. Our experiments revealed that transcription of lhrC1-5 is completely dependent on the presence of a functional LisRK system, whereas the CesRK system appears to be important for a differentiated transcription of lhrC1-5 in response to the above-mentioned cell wall acting agents. Finally, a mutant strain lacking lhrC1-5 was constructed and characterized, and genes regulated by LhrC1- 5 were investigated by comparing the global gene expression of a wild type strain vs. a ∆lhrC1-5 mutant strain. In summary, this study shows that L. monocytogenes responds to cell wall acting agents through a complex gene regulatory network which includes five small non-coding RNAs.

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REFERENCE Phenotypic analysis of selected listerial secretion mutants A / P 03 Halbedel, S.*, Galander, S. and Flieger, A. Robert Koch Institute, Germany To be secreted into any extracellular compartment, proteins need to be translocated across the cytoplasmic membrane first. Protein translocation is an energy driven process and mediated by different sophisticated nano-machines that link energy consumption to substrate translocation. Bulk protein translocation in the Firmicutes is mediated by the Sec translocon together with the YidC proteins that have 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 systems have been described that transport certain subsets of translocation substrates only, such as the Tat system, or that are supposed to be involved in protein secretion since they seem to form proteinaceous membrane pores. Among the latter is the Bacillus subtilis SpoIIIAH protein that recently was shown to be the mother cell-specific part of a channel connecting the mother cell with the prespore compartment in sporulating B. subtilis cells. A homologue of this gene can be found in L. monocytogenes as well (lmo0791), whereas the prespore-specific component (spoIIQ) and the other seven genes of the B. subtilis spoIIIA operon seem to be absent from the listerial chromosome. L. monocytogenes SpoIIIAH consists of an N-terminal transmembrane helix and a putative extracellular domain at the C- terminus that shares similarity with the ring forming proteins of the YscJ/FliF family associated with type III protein secretion. We therefore hypothesized that SpoIIIAH might be involved in secretion of proteins or other biomolecules into the extracellular space in L. monocytogenes. To test this hypothesis and to describe the impact of YidC homologues on protein secretion we generated strains with deletions of the spoIIIAH, yidC or yqjG genes. Here, we will present data describing the phenotypic and proteomic analysis of these mutants.

* Participation Supported by IUFoST

REFERENCE A / P Distribution of serotypes and pulsotypes of Listeria monocytogenes in pig farms (France 2008) 04 Boscher, E. AFSSA, France This work was undertaken to estimate the prevalence of L. monocytogenes and, distribution 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 from sows in 73 pig farms localized in Brittany, France. Detection of L. monocytogenes was carried out according to the ISO 11290-1/A1 method. Isolates were serotyped and typed by PFGE. We found that 46.6% of the farms (34/73) had at least one positive 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 of the 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 4b and 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 in several farms; as example, genotypes G18, G12 and G10 were found in 4, 5 and 5 farms respectively. This study provided recent valuable information on the occurrence of L. monocytogenes in French pig farms. This microorganism is prevalent in the sows in farrow-to-finish pig farms (46.6%). Our work highlighted for some farms several serotypes and genotypes suggesting several sources of contamination. Furthermore, the serotypes found in this study are identical to those usually involved in human listeriosis in Europe (Goulet et al. 2008).

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REFERENCE Comparative phylogenomics of Listeria monocytogenes reveals / an adaptation profile A P Silveira Nalério, E.1, Padilha Silva, W.1, Stabler, R.2 and Wren, B. W.2 05 1. Universidade Federal de Pelotas, Brazil 2. London School of Hygiene and Tropical Medicine, UK Listeria monocytogenes is the causative agent of listeriosis which may cause a range of diseases from gastroenteritis to death. In fact, disease outcome can be related to strain serotype/lineage thus molecular analyses has demonstrated that L. monocytogenes is a highly diverse species which can be grouped into three lineages. Whole-genome microarray can be employed to study phylogenetic relationships among Listeria strains either species or serotype level, in addition to demonstrate differences on their virulence potential and/or environmental adaptation. The aim of this study was the whole genome comparison of L. monocytogenes strains from different origins. Ninety-nine L. monocytogenes strains from different geographical origins (Brazil, Denmark, Austria, Ireland, USA and unknown), including clinical strains (humans and animals), food and food industries strains were analyzed. DNA from all strains were competitively hybridized on a L. monocytogenes DNA 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 performed as previously described by Stabler et al. (2006). Comparative phylogenomics clustered the L. monocytogenes strains into two central clades which is representative of the two main lineages of this species. In addition each of these clades was divided into two further subclades. Clade formation was independent of the geographical origin of strains with the exception of the clade containing persistent strains (strains that persist in food-processing environment), where none of the Brazilian strains were present. It was found 18 specific genes for lineage I strains (1/2a and 1/2c serotypes). These genes are related to carbohydrate metabolism, two component regulatory system, ABC transporter complex and bvrB and bvrC genes. Significantly all persistent strains clustered together in the same lineage I clade. We achieved a set of unique genes belonging exclusively to L. monocytogenes persistent strains pointing to be responsible for its adaptation profile. The genes are involved in stress resistance and are related to carbohydrate transport and metabolism, environmental information processing, signal transduction mechanisms, cell surface protein, amino acid transport and metabolism, nucleotide transport and metabolism, translation, cell wall biogenesis, replication, recombination and repair, transport of small molecules similar to ABC transporter, metabolism of lipids and unknown function. Interestingly from 14 virulence listed genes most of them were present in all studied L. monocytogenes strains with exception of inlE and inlG genes. These findings indicate that genetic variability of L. monocytogenes strains point to niche adaptation instead virulence differentiation despite of different origins. Persistent strains clustered suggesting genetic origin to survival in this environment.

Serotyping and PFGE patterns of Listeria monocytogenes isolated REFERENCE A / P from poultry meat Vasantrao Kurkure, N.1, Kalorey, D. R.1, Rodrigues, J.2, Gunjal, P.1 and Barbuddhe, S. B.2 06 1. Nagpur Veterinary College, Maharashtra Animal & Fishery Sciences University, India 2. ICAR Research Complex for Goa, India Listeria monocytogenes is a gram positive, facultative, intracellular bacteria which is ubiquitous in nature. Poultry meat and products frequently have been implicated in outbreaks of food borne illness. In India there is practice to sell freshly slaughtered poultry meat. A total of 119 poultry meat samples were collected from poultry meat shops. These samples were processed for isolation of Listeria spp. by adopting standard protocol. Out of 47 Listeria spp. recovered 17 isolates were confirmed as L. monocytogenes by biochemical and in vitro pathogenicity test. The L. monocytogens isolates were subjected to multiplex PCR based serotyping and PFGE analysis. All the isolates were grouped as 4b, 4d and 4e serotypes. By PFGE analysis four Asc I profiles were observed. Thirteen isolates were observed to be of similar profile indicating homogenecity among the isolates recovered from poultry meat. Present findings indicated the need of improvement in hygienic practices while slaughtering and sell of poultry meat as L. monocytogenes is of zoonotic pathogen.

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REFERENCE Genotypic characterization of Listeria monocytogenes isolated A / P from fresh leafy vegetables 07 Warke, 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, India 2. ICAR Research Complex for Goa, India Listeria monocytogenes is a food borne pathogen responsible for listeriosis, an illness characterized by meningitis, encephalitis, abortion and septicemia in human beings. A total of 417 samples comprising eleven different types of fresh leafy vegetables 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 samples were processed for isolation of L. monocytogenes by two step enrichment followed by plating on selective media. Confirmation of the isolates was on basis of biochemical characters, haemolysis on blood agar and Christie, Atkins, Munch Pe- tersen test (CAMP). The isolates were also tested for the virulence associated genes 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), coriander (3), radish (4), amaranthus (2) and mint leaves (2). All the genes were detected in seven strains, four genes (hlyA, actA iap and prfA) in three strains. The hlyA, actA and iap in six strains. Three genes (hlyA, iap and prfA) were detected in five strains. The hlyA and iap genes were detected in ten strains. Fourteen isolates were subjected to multiplex PCR serotyping and all belong to 4b, 4d and 4e serogroup. Contamination of vegetables with L.monocytogenes is a cause of concern as most of the vegetables are consumed raw as salad. Thus, there is increased risk for transmission of listeriosis. Therefore proper washing and cleaning of fresh leafy vegetables is recommended at harvesting and consumption.

REFERENCE Comprehensive appraisal of the exoproteome of Listeria monocytogenes A / P by genomic and proteomic analyses 08 Desvaux, 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 environment and are relevant to the bacterial lifestyle. By definition, secreted proteins are proteins actively transported via secretion systems but they can have radically different final destinations. In monoderm (Gram-positive) bacteria, such secreted proteins can (i) anchor to the cytoplasmic membrane, (ii) associate with the cell wall, or (iii) be released into the extracellular milieu and are the called exoproteins. The subset of proteins localized in the extracellular milieu constitutes the extracellular proteome, also called exoproteome. Listeria monocytogenes is a bacterial species both pathogenic and saprophytic, which exhibits a highly variable level of virulence from one strain to another and such biodiversity might express within the exoproteome. The present investigation aimed at developping of a generic genomic strategy to predict exoproteins in monoderm bacteria as well as determining those differentially expressed within a panel of representative strains. Genomic analysis was performed following a novel rational bioinformatic approach to predict exoproteins, which expression was determined by proteomic analyses following 2-DE and identification by MALDI-TOF MS. The rational bioinformatic approach integrates the structural properties of the proteins potentially secreted as well as the respective secretion systems involved. Comparison of theoretical and experimental 2-DE maps provided a comprehensive picture of the exoproteome. Exoproteins were further discriminated between those found in all or only in a subset of L. monocytogenes strains. While the core exoproteome included proteins related to virulence and cell wall biogenesis, variation in the protein members of these categories constituted the variant exoproteome. The novel and rational in silico strategy here developed to predict exoproteins is adaptable and fully applicable to other monoderm bacteria. This proteomic analysis resulted in the first definition of the core and variant exoproteomes of this species and provides auxiliary insight in bacterial physiology.

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REFERENCE Investigating differences in lineages of Listeria monocytogenes / using comparative genomics A P McIlwham, S., Farber, J. and Pagotto, F. 09 Bureau of Microbial Hazards, Canada Listeria monocytogenes causes foodborne listeriosis and is ubiquitous in the environment. Strains are often classified into lineages I (food); II (human illness); and III (food-production). It is not clearly understood how lineages develop a tropism for a particular niche. Using comparative genomics, we attempted to identify unique genetic attributes within lineages that contribute to the tropism of the pathogen. A mixed genome array, created from a genomic library of 15 L. monocytogenes isolates, was used to compare the genomes of different strains. An isogenic mutant for a glycoside hydrolase family 65 (GH65) was created based on microarray screening. Forty-five strains from different sources were studied in Mueller- Hinton broth with ampicillin, vancomycin or acriflavine. Additionally, the growth rates of eleven strains in Listeria Enrichment Broth (LEB) were compared. A GH65 gene was found to be present in lineage II isolates but absent in lineage I isolates. A GH65 isogenic deletion mutant and other lineage I isolates showed a reduced ability to proliferate in the presence of vancomycin and ampicillin. When growth rates were compared in LEB, the GH65 deletion mutant behaved similarly to lineage I strains. The GH65 gene, present in lineage II strains, may provide a competitive advantage in the host environment. The difference in growth rate of the lineage I and II strains in LEB may provide insight as to why certain serotypes are more readily isolated from food commodities.

Autolysis in Listeria monocytogenes – a proteomic approach REFERENCE A / P Pinto, E.1, Marques, N.2, Andrew, P. W.3 and Faleiro, M. L.1 10 1. Instituto de Biotecnologia e Bioengenharia, Centro de Biomedecina Molecular e Estrutural, Universidade do Algarve, Portugal 2. BioFig, Universidade do Algarve, Portugal 3. Department of Infection, Immunity and Inflammation, University of Leicester, UK Autolysins play a role in many cellular processes including cell growth and division, cell wall turnover, motility and also pathogenicity. Several growth conditions may interfere with cell wall synthesis, leading to unrestricted activity of the autolysins which can degraded the cell wall to levels that compromise the cell integrity. During our work differences between Listeria monocytogenes strains in the tendency to undergo autolysis was found. The objective of the present study was to test the hypothesis that as cells enter stationary phase the pattern of protein 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 were collected immediately before autolysis. The samples were subjected to a proteome analysis. Remarkably at favourable autolysis condition the autolytic strain expressed 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 a negative impact on cell wall synthesis. In both proteomes of the non-autolytic strain EGD and the autolytic strain C897 in non favourable autolyis conditions revealed a higher expression of proteins involved in the energy generation, amino acid and fatty acid metabolism. In the secretome of C897 the p60 protein was over expressed at 30°C but no significant differences were found between the strain C897 and EGD. However p60 levels were higher in the autolytic strain intracellular proteome when grown at 20°C. The levels of the autolysin p45 were higher in the secretome of C897 at 30°C and significant differences between the two strains were found.

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REFERENCE Role of flhA, cheR and motA in growth of Listeria monocytogenes A / P at low temperature 11 Mattila, M., Lindström, M., Somervuo, P. and Korkeala, H. University of Helsinki, Finland Temperature-dependent induction of flagella is a well-characterized phenomenon in L. monocytogenes. At 37°C L. monocytogenes is virtually non-motile and the motility 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 during growth at low temperatures is still unclear. To study this relationship, we prepared knock-out mutants of three motility and chemotaxis genes, flhA, cheR, and motA on L. monocytogenes strain EGD-e. We compared the expression levels of these genes in cultures grown at 3, 25 and 37°C by using qRT-PCR. The role of these genes in the cold adaptation of L. monocytogenes was also studied with growth curve analysis, motility assays and electron microscopy. The relative expression of flhA, cheR and motA in EGD-e cells grown at 3°C was significantly (p<0.01) increased compared 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 cultures grown at 3°C both the growth rates and maximum optical densities at 600 nm were significantly (p<0.001) lower for the mutant strains ∆flha, ∆cheR and ∆motA compared to the wild-type strain. At 37 and 25°C we did not detect any significant differences between the wild-type strain and the mutants. The motility assays showed that ∆flhA and ∆motA strains were completely non-motile at all three temperatures whereas ∆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. The results suggest that flhA, cheR, and motA have a role in the cold tolerance of L. monocytogenes strain EGD-e, and that flagellum production and chemotaxis are linked to cold adaptation of L. monocytogenes.

REFERENCE The effect of acetic acid (at pH 5.5) or benzoic acid (at neutral pH) A / P on lipid composition and fluidity of Listeria monocytogenes membrane 12 Ioannis, 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 or the 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 protective role upon (acidic) stress decreasing the membrane fluidity and thus obstructing the entrance of the stressful compounds. The aim of our work was to investigate the modification of L. monocytogenes membrane lipids molecules (neutral, NL or polar, PL lipids) and also their FA composition in response to low (acetic acid) or neutral pH (benzoic acid) and their consequences on membrane fluidity. L. monocytogenes (DP-L1044, D. Portnoy, University of Pennsylvania) total lipids,TL extracted from stationary phase cells cultured (1L BHI broth, 30°C) with low initial pH (5.5) by acetic acid, LmAA (optical density OD600=0.210±0.031, 72h, n=12) or with neutral pH by benzoic acid (1g/L), LmBA (OD600=0.682±0.013, 10h, n=9) and compared with those of optimal conditions cells, Lmcontrol (OD600=0.816±0.032, 10h, n=8). The results revealed: i) Only acetic acid enhances the antimicrobial activity. ii) The sums of 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) of each TL acidic adapted culture (increase of high-melting FA). iii) Singularly acetic acid adaptation in L. monocytogenes was correlated with a higher content (49.3% among TL) of neutral lipids class than those of Lmcontrol. Moreover, we correlated the lipid changes with thermodynamic analysis (Differential Scanning Calorimetry, DSC) of lipids in order to estimate experimentally the initial hypothesis, that both adaptation mechanisms (the high-melting point FA increase and the NL accumulation) would promote the decrease of membrane fluidity. The results revealed that in each culture LmBA and LmAA TL was increased the phase transition temperature,Tc (5.3°C and 3°C) and enthalpy difference, ∆Η (10.2% and 53.8% respectively) than Lmcontrol. The thermodynamic findings confirmed the above hypothesis of decreasing fluidity. The high ∆Η value of LmAA (high energy amount to change its lipids) is interpreted by NL accumulation.

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REFERENCE Elucidation of the responses to weak acids in the human pathogen / Listeria monocytogenes using gene microarrays A P O’Byrne, C.1, Heavin, S.1 and Morrissey, J.2 13 1. NUI Galway, Ireland 2. University College Cork, Ireland Contamination of processed food with Listeria monocytogenes is a major human health risk. Food preservation regimes often include the use of weak organic acids to reduce microbial growth. Although the response of L. monocytogenes to strong acids has been investigated extensively, there little is known about how L. monocytogenes responds to weak acids, particularly at the molecular level. The objective of this study was to investigate the molecular response of L. monocytogenes to five food-grade organic acids, acetic, lactic, sorbic, citric and benzoic acid. Careful growth experiments were carried out to determine the inhibitory concentrations of each of these acids on the growth of L. monocytogenes. Microarray and quantitative RT-PCR experiments were then performed to elucidate the molecular responses of the organism to weak acids. In total 222 genes were found to be differentially expressed in response to at least one of the weak acids. The temporal response of 9 of these genes to sudden exposure to lactic acid or citric acid was studied using RT-PCR. These data highlighted the distinct responses observed to the 2 weak acids and identified genes that showed strong unique transcriptional responses (both up-regulation and down-regulation) to each. At a global level there was surprisingly little overlap between genes that responded to each acid. For example, the 7 genes differentially expressed in the presence of acetic acid were not influenced by any of the other acids. Together the data suggest that each organic acid influences the cell in a very specific way, a finding that suggests that each acid has a different inhibitory mode of action.

REFERENCE The immunogenic surface protein IspC acts as an / N-Acetylglucosaminidase in Listeria monocytogenes serotype 4b A P Ronholm, J.* 14 University of Ottawa, Canada IspC is a surface protein isolated from Listeria monocytogenes serotype 4b which acts as an autolysin and virulence factor. Several autolysins including P60, Ami and Auto have been isolated from various L. monocytogenes serotypes but the role for their apparent functional overlap remains unknown. Further characterizing these autolysins may help to gain insights into their roles in Listeria biology. The novel autolysin IspC remains understudied. A panel of 15 monoclonal antibodies (mAbs) directed against the surface of L. monocytogenes serotype 4b were generated, characterized as recognizing IspC and will facilitate further study of this protein. Based on serological assays with these mAbs we have determined that IspC expression is limited to certain L. monocytogenes serotypes including 4a, 4ab, 4b, and 4e, although, other serotypes have genes highly homologous to IspC. Sequence homology 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 acetylation would affect IspC mediated PG hydrolysis. PgdA, the only one of three PG deacetylases predicted to localize to the cell surface, is required for de-acetylation of PG, which is assembled fully acetylated. To determine how acetylation affects IspC PG hydrolysis, a pgdA deletion mutant was created. The mutant PG (fully acetylated) was much more susceptible to hydrolysis by IspC than PG from the wild-type, as assessed by re-naturing SDS-PAGE. Currently we are making a pgdA complement to restore PG deacetylation in the deletion mutant. Further investigation is being done on IspC hydrolysis of PG from the deletion mutant using mass spectrometry. This will provide insight into the effects of the pgdA deletion on PG structure and IspC PG hydrolase activity.

* Participation Supported by IUFoST.

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REFERENCE Listeria monocytogenes EGD chitinolytic activity is regulated by A / P carbohydrates but also by the virulence regulatory gene, PrfA 15 Halberg 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 major component of fungal cell walls and insect and crustacean exoskeletons and is one of the most abundant polymers in marine and terrestrial environments. Chitin hydrolysis by Listeria monocytogenes depends on two chitinase encoding genes chiA (lmo1883) and chiB (lmo105) and the aim of this study was to investigate their regulation. The production of the chitinases is substrate regulated and subjected to catabolite control. Thus, chitin induces expression of both chitinases however chiA but not chiB is furthermore induced by the monomer GlcNAc. In growth medium supplemented with chitin and glucose the expression of both chitinases is repressed. Expression of chiA and chiB is growth phase dependent with higher expression in late exponential growth phase compared to early exponential phase. Chitinases expressed by bacterial pathogens have proven to be important not only for nutrient acquisition 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 significantly reduced in ∆prfA mutant cells compared to the wild type cells. In agreement with this result northern blot analysis showed that the amounts of chiA and chiB transcripts were significantly lower upon induction by chitin in the ∆prfA mutant compared with the wild type. Furthermore, in contrast to the wild type, no chiA transcript could be detected in the mutant lacking PrfA during growth in medium supplemented with GlcNAc. Regulation of chitinolytic activity in L. monocytogenes is complex and the results obtained in this and other studies indicate that the biological role of this activity may not be limited to the external environment.

REFERENCE Infectious dose curves for guinea pigs challenged with a Listeria A / P monocytogenes epidemic clone strain and a strain carrying a naturally- 16 occurring virulence-attenuating mutation in inlA show a significant shift in median infectious dose Nightingale, 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, USA 2. Grocery Manufacturers Association, USA 3. U.S. Food and Drug Administration, USA 4. Health Canada 5. Exponent, USA 6. Cornell University, USA Listeria monocytogenes contains two subpopulations, including (i) epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide 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 associated with human disease (approx. 5%). The virulence factor Internalin-A (InlA; encoded by inlA) binds certain isoforms of the cellular receptor E-cadherin to facilitate crossing of the intestinal barrier by L. monocytogenes. The current FDA/FSIS/CDC L. monocytogenes risk assessment was developed with dose response data from murine challenge experiments, a model that fails to probe InlA mediated virulence due to the inability of InlA to bind the murine isoform of E-cadherin. 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 a listeriosis outbreak or (ii) a strain carrying the most common inlA PMSC mutation. Dose-response curves for tissue infectivity were constructed with either a log-logistic, beta poisson, or exponential (for spleen data) fit to the raw individual and combined organ data. The log logistic and beta poisson models based on combined organ data showed an approx. 1.3 log10 CFU increase in the median infectious dose for the strain carrying a PMSC in inlA relative to the EC strain. Inclusion of strain effect significantly improved the ability of the model to explain the observed data, supporting a significant difference in tissue infectivity between EC and PMSC strains. L. monocytogenes strains thus show notable differences in infectious dose required to establish an infection. Results from this work support the dose-response relationship 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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REFERENCE MudPIT based proteomic analysis of alkaline adapted, environmentally / persistent Listeria monocytogenes strains A P Nilsson, R. E., Ross, T. and Bowman, J. P. 17 University of Tasmania, Australia Soluble protein expression profiles of environmentally persistent and sporadic Listeria monocytogenes strains in mid-exponential and stationary growth states at pH 7.3 and adapted to pH 8.5 were compared using multidimensional protein identification technology (MudPIT). Searches employed the X!Tandem algorithm and an in house data validation criteria was applied using the Trans Proteomic Pipeline (Version 3.4) and comparison against a decoy database. Qualitative and relative abundances were compared through functional ontology (TIGR) and determination of spectral abundances. A total of 1661 proteins were identified following integration analysis. Significant differences in protein expression were observed for both growth state and culture condition. Increased presence and abundance of proteins associated with the cell wall, transport and binding, amino acid/cofactor biosynthesis and protein stabilization were identified. The study findings suggest alkaline adapted L. monocytogenes strains may use cytoplasmic acidification by surrogate proton sources (both endogenous and exogenous) as a means of persisting in high pH environments. Furthermore, proteins associated with stabilization of cellular processes and cell wall modification may be aiding in minimizing cellular damage under these conditions. This trend was identified in both growth states; however it was more pronounced in exponential phase. No significant difference was observed between the L. monocytogenes strains studied. The results of this work support the notion that exposure to conditions favouring alkaline homeostasis may be an important contributor to the development of persistent L. monocytogenes, independent of strain.

RpoN, the alternative sigma factor, is associated with the growth phase REFERENCE A / P transition and pathogenesis in Listeria monocytogenes Okada, Y., Suzuki, H., Monden, S., Igimi, S. and Okada, N. 18 Ministry of Health, Labour and Welfare, Japan Listeria monocytogenes has 5 types of sigma factor. RpoN, an alternative sigma factor, is involved in the bacteriocin-resistance and the osmotolerance in this bacterium, however, its’ function remains almost unknown. We found that the rpoN deletion mutant showed higher growth than its parental strain at the stationary phase. To identify the RpoN regulated genes which are associated with the growth phase transition, DNA microarray analysis was performed. From L. monocytogenes strain EGD and its rpoN in-frame deletion mutant, total RNA samples were isolated at early exponential and stationary phase. The gene expression profiles in both strains at each growth phase were compared, and in the results, especially, 27 genes associated with oxidoreductase showed the different patterns of expression between two strains. So we examined the survival of these strains after exposure to the sub-lethal concentration of hydrogen peroxide. Both strains showed the similar levels of tolerance against 100mM hydrogen peroxide at stationary phase. How- ever, the mutant was significantly tolerant to hydrogen peroxide than EGD at the mid exponential phase. From the result of electrophoresis, the chromosomal DNA was degraded after exposure to hydrogen peroxide only in EGD, but not in the mutant. Next, we examined the pathogenicity of the rpoN mutant. The mutant showed the higher growth in J774 cells and the peritoneal adherent cells from BALB/c mice. In spleen and liver of mouse, the mutant was able to grow at the similar level of EGD, however, the growth was slowly. These results suggest that the rpoN mutant is able to grow higher than the parental strain at the stationary phase because of the increased DNA stability against the active oxygen accumulated in the bacterial cells as a result of the respiration during the exponential growth, and RpoN is associated with the pathogenicity of L. monocytogenes.

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REFERENCE Cellular lipid fatty acid pattern differences between reference and A / P ice-cream isolate of Listeria monocytogenes as response to cold stress 19 Anita, 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 its ability to grow at low temperatures, allowing refrigeration at 5°C to act as an effective enrichment for the organism. There is little information about its ability to withstand 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 in the cold (5°C) contain significantly less a-17:0 than those grown at higher temperatures. The purpose was to investigate the effect of ice-cream storage in L.monocytogenes FA profile, after inoculation of pilot ice-cream with reference strain DP-L1040 (D. Portnoy, University of Pennsylvania). The inoculation was performed with reference strain Lmref, (BHI, 30°C, 8h, OD600=0.8) in order to yield 6.30 log cfu/g ice-cream. Cells of L. monocytogenes isolated from inoculated ice-cream, (Lmice), after 6 month of storage and cells of Lmref were grown in BHI broth, 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 analyzed. Our results show that significant differences exist between the FA profile of Lmref30°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 in cells of ice-cream isolate. There was also an increase of unsaturated FA. The Lmice5°C showed an a-15:0/a-17:0 ratio of 11.2, while Lmref5°C was characterized by 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 reference strain. These results showed that L. monocytogenes strain origin needs to be considered when physiological studies are performed with this bacterium. The food matrix and environmental conditions can influence the FA pattern and L. monocytogenes survival. A greater understanding of cold adaptation mechanism may offer methods for controlling L. monocytogenes in chilled and frozen foods.

REFERENCE Role of the dihydroxyacetone metabolism in the resistance of Listeria A / P innocua to pediocin 20 Milohanic, E. INRA, France Listeria monocytogenes is a Gram-positive bacterium, the causative agent of listeriosis, 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. We were interested in the dha system of Listeria innocua that involves the general PTS enzymes, the main transport and phosphorylation system of carbohydrates, which also occurs in L. monocytogenes. It catalyses the intracellular PEP-dependent phosphorylation of dihydroxyacetone (Dha). In silico studies allowed us to hypothesize that the last gene of the dha system, pedB, could encode resistance to pediocin, a bacteriocin produced by the lactic acid bacterium Pediococcus acidilactici. We sought to understand why this gene is associated with the dha system. We first confirmed the operon organization of this system. Quantitative PCR experiments revealed 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 of this operon. We also showed that the wild-type strain of L. innocua is sensitive to pediocin while the dhaR mutant is resistant. The pedB mutant is under investigation. In addition, we purified the five proteins involved in Dha phosphorylation and could reconstitute in vitro these metabolic steps. Interestingly the dha operon encodes many enzymes involved in the pentose phosphate pathway especially the Tpi-like protein. These results would suggest a link between Dha metabolism, pentose phosphate pathway and virulence in L. monocytogenes.

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REFERENCE Glucose transport system in Listeria monocytogenes and their impact / on virulence gene expression A P Moussan Ake, F. 21 INRA, France In Listeria monocytogenes at least two phosphoenolpyruvate (PEP): carbohydrate phosphotransferase systems (PTS) of the mannose class and also two non-PTS permeases can transport glucose. Mutants lacking EIIAB (Lmo0096) or EIIC (Lmo0097) of the glucose/mannose PTS grew slightly slower on minimal medium containing up to 10 mM glucose and consumed glucose 3-times slower than the wild-type strain. A lmo0784 mutant (EIIA, mannose type PTS) exhibited an extended lag phase before it started growing on glucose and glucose consumption was slowed 3.5-fold. All PTS transporters are inactive in a ptsI mutant, which nevertheless grew at high glucose concentrations (>15mM) after an extended lag phase. Two GlcU-like non-PTS glucose transporters (Lmo0169 and Lmo0176) probably catalyze glucose uptake in the DptsI mutant. Expression of either one of them in an Escherichia coli DptsHIcrr mutant restored glucose utilization. The efficient utilization of glucose affects the PrfA-dependent expression of L. monocytogenes virulence genes, including hly. Nevertheless, inactivation of the glcU genes in a strain carrying a Phly-gus fusion had no effect on PrfA activity. However, deletion 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 activity in the EIIABGlc/Man mutant was also elevated when it was grown on solid minimal medium containing 5 or 10mM glucose. A similar behaviour was observed for the lmo0784 mutant, which in addition exhibits very low man operon expression. The highest gus expression was observed in the DptsI mutant grown at glucose concentrations between 25 and 50mM.

Antimicrobial susceptibilities of Listeria monocytogenes isolated REFERENCE A / P in Japan Monden, S., Okutani, A., Suzuki, H., Asakura, H., Nakama, A., Igimi, S., Okada, Y. 22 and Maruyama, T. Japan Antibiotics, such as ampicillin and co-trimoxazole, are usually used as the first choice for treatment of listeriosis. However, the antimicrobial resistant strains of Listeria monocytogenes are often isolated in many countries. In this study, in vitro antimicrobial susceptibility of 232 L. monocytogenes strains (101 isolates from patients of listeriosis and 131 isolates from food and food-related environment) obtained in Japan from 1974 to 2008 were examined their susceptibilities against 6 kinds of antibiotics by plate dilution method. According to the breakpoint from the guideline of National Comittee for Chemical Laboratory Standard, all strains tested here were susceptible to gentamicin and kanamycin, and all clinical isolates showed susceptibility to ampicillin and erythromycin. One strain from beef was resistant to ampicillin, and the another one from pork showed resistance to 3 kinds of drug, chloramphenicol, erythromycin and enrofloxacin. 56.4% of clinical isolates and 37.8% of food and environmental isolates were resistant to enrofloxacin, and the rest of isolates showed intermediate phenotype to this antibiotics. The rates of enrofloxacin-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 this study, most of clinical isolates were susceptible to antibiotics except enrofloxacin. On the other hand, some strains from food and environment showed resistance or intermediate to ampicillin, chloramphenicol or erythromycin. Furthermore, all strains were more than intermediate to enrofloxacin, a kind of quinolone that used for treatment of animal infection in Japan. These results suggest that the origin of listerial isolates in involved in their antibiotic susceptibility.

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REFERENCE Influence of sub-lethal concentrations of disinfectants A / P on Listeria monocytogenes adhesion and invasion in Caco-2 cells 23 Gaedt Kastbjerg, V.1, Halberg Larsen, M.2, Ingmer, H.2 and Gram, L.1 1. National Institute of Food Research, Technical University of Denmark 2. 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. monocytogenes is not always eliminated by the disinfection process. One reason could be that the bacteria are only exposed to sub-lethal concentrations of the disinfectant. We have recently shown that sub-lethal concentrations of disinfectants used in the food industry affect virulence gene expression on transcript level in L. monocytogenes, and the effect depend on the active components of the disinfectants. The aim of the present study was to determine if disinfectants used routinely in the food industry affect the virulence of this pathogen studied in a cell-model. Two different disinfectants were used. Compound 1 contains peracetic acid and hydrogen peroxide as the active ingredients and reduces the expression of virulence genes in L. monocytogenes whereas Compound 2 contains quaternary ammonium compounds (QAC) and induces virulence gene expression. L. monocytogenes EGD was exposed to the two disinfectants for one hour in a non-inhibiting and a sub-lethal concentration, and subsequently the bacterial cells were added to a monolayer of Caco-2 cells. Bacterial adhesion and invasion were monitored. Exposure of L. monocytogenes to the sub-lethal concentration (0.0031%) of the QAC disinfectant significantly increased adhesion to Caco-2 cells as compared to bacteria exposed to water (control) and resulted in a slightly higher invasion. No effect was observed of the non-inhibiting concentration of the QAC disinfectant (0.0016%). In contrast, the adhesion was unaffected and the invasion slightly decreased after exposure to the non-inhibiting concentration of 0.125% of the peracetic disinfectant as compared to bacteria exposed to water. On-going studies will evaluate how long term exposure to disinfectants will affect adhesion and invasion to Caco-2 cells and these data will also be discussed.

REFERENCE The SOS response in Listeria monocytogenes – a stress A / P survival mechanism 24 Kiil 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 variety of bacteria. The SOS response involves two central regulatory components, RecA and LexA, which coordinate the expression of target genes leading to arrest of cell division, DNA repair and mutagenesis. Historically, the SOS response is linked to the presence of single stranded DNA and stalled replication forks induced by UV ra- diation. However several other stress conditions, which are not expected to cause massive DNA damage, have been shown to induce SOS response e.g. mild heat treatment, antibiotics, ethanol and salt stress. The SOS response of the food borne pathogen Listeria monocytogenes is presently not well understood. We find it likely that the SOS response is important for the growth and survival of this pathogen in contaminated foods and during infection. It is therefore of great interest to investigate this response in L. monocytogenes into more details. To this end, we focused our studies on the auto regulated repressor protein LexA, the genes targeted by this regulator, and the environmental signals leading to activation of the SOS response in L. monocytogenes. LexA regulated gene expression is analyzed by comparing expression profiles of WT L. monocytogenes with ∆lexA and lexA* strains expressing a non functional and a constitutive active LexA, respectively. In order to further elucidate the regulatory mechanisms and timing of the SOS response, we construct promoter-reporter gene fusions, which report the size and duration of activation of LexA controlled protein expression. As reporter genes we employ both lacZ and degradation tagged gfp. We thus expect to be able to monitor time resolved changes in SOS gene expression and thus come closer to the apparently versatile role of this stress response mechanism in Listeria monocytogenes.

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REFERENCE Acid shock triggers heavy metal detoxification / in Listeria monocytogenes A P Müller, S., Neuhaus, K. and Scherer, S. 25 Technische Universität München, Germany L. monocytogenes is a food borne pathogen ubiquitous in the environment. The bacterium 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 associated with food production. In this work, the acid shock response at pH 5 for 15min, 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 microarrays. Besides transcriptional induction of well characterized genes which are involved in acid tolerance, an up regulation was also found for genes whose products are potentially participating in heavy metal detoxification, such as ion export proteins (heavy metal exporting ATPase: lmo0641, lmo1852-1854; cation efflux protein: lmo2231, lmo2231) or heavy metal reduction proteins (arsenate reductase: lmo2230). Furthermore, it was demonstrated that, after induction of the acid tolerance response (60 min, pH 6) L. monocytogenes exhibits a higher resistance against various heavy metal ions, e.g. against CdII, HgII, and NiII; and especially against CuII and AsV. Acidic pH appears to serve as a trigger not only for the induction of genes involved in acid tolerance itself, but also for the induction of genes involved in heavy metal resistance. Since metals become more bioaccessible at low pH, especially in soil, acid solubilized heavy metals might reach noxious doses in certain environments and, therefore, detoxification genes may be up regulated. Ongoing analysis using deletion mutants will help to elucidate the role of detoxification under acidic growth conditions.

Listeria monocytogenes mutants defective in growth at 5°C REFERENCE A / P and in high salt environment Burall, L., Laksanalamai, P. and Datta, A. 26 US Food and Drug Administration, USA Listeria monocytogenes can survive and grow in refrigerated temperature and in the presence of high salt. In an effort to better understand the associated mechanisms, a library of 5280 transposon mutants of LS411, an isolate from the Jalisco cheese 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 altered growth profiles have been identified. 63-C8 showed a significant reduction in growth in BHI at 5°C. Additionally, the mutant had reduced growth in the presence of 9% but not 7% NaCl. Sequencing located the transposon between two divergently transcribed genes, iap and one encoding a putative SecA subunit. qPCR revealed 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 subunit gene. 36-F11 showed no growth reduction at 5°C but had attenuated growth in the presence of 7% NaCl. The salt attenuation was exacerbated at 9% NaCl, which resulted 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 increased avidity in the bacterial associations within the clumps. Sequencing of the insertion region of 36-F11 identified the transposon between two divergently transcribed genes. The insertion site is 36bp upstream of a gene encoding a glycine betaine/L-proline ABC transporter, which is down-regulated in the mutant while the divergently transcribed putative mechanosensitive ion channel appeared slightly upregulated. Further studies are being conducted to identify the mechanisms underlying these phenotypes.

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REFERENCE Revival of 5,000 Listeria strains from Seeliger’s historical collection A / P with a semi-automated microbiological pipeline 27 Haase, J.1, Hof, H.2 and Achtman, M.1 1. Science Foundation Ireland 2. University of Heidelberg, Mannheim, Germany Prof. Seeliger from the University of Würzburg, Germany, collected more than 7,000 Listeria strains, globally, between the 1920s and the 1980s from humans, animals, food and the environment. Within this project, this collection, the “Special Listeria Culture Collection” (SLCC), has been recultured and frozen at -80°C. Strain information has been transcribed from the original notebooks and is now stored in an SQL-based database (Bionumerics, Applied Maths) and the physical strain locations are stored in a LIM system (ItemTracker). A large collection such as this required the set up of a comprehensive “strain pipeline”. This ensures correct cataloguing, efficient storage and retrieval of cultures. This was achieved via custom scripts (Python) that allow communication and archiving of data obtained from the initial culturing to isolation of DNA and downstream phylogenetic analyses. The collection now provides an exceptional opportunity to investigate the population structure and the evolutionary history of L. monocytogenes.

REFERENCE A / P Two point mutations are responsible for the lack of glycosidic substition in cell wall teichoic acids in Listeria monocytogenes serovar “7” 28 Eugster, M. R.1, Huwiler, S.2, Morax, L.1 and Loessner, M. J.1 1. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland 2. Institute of Microbiology, ETH Zurich, Switzerland The currently defined six Listeria species are further differentiated into at least 15 distinct serovars based on serological reactions with specific antisera directed against O and, to a lesser extent, H antigens. Some L. monocytogenes strains (such as WSLC 1034) have previously been assigned to serovar “7”. These cells contain ribitol phosphate wall teichoic acids (WTA) similar to serovars 1/2 and 3, but without glycosyl substition. Purified WTA polymers and associated sugars were analyzed by a novel technique using electrospray ionization tandem mass spectrometry (ESI-MS/MS). In addition, WTA carbohydrates were identified using fluorescently labeled N-acetylglucosamine (GlcNAc) binding proteins (CBD-P35) and Listeria phage A118 which requires rhamnose-substituted WTAs for adsorption. We found that the absence of rhamnose and GlcNAc in WTAs of strain 1034 is based 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 acid decoration with GlcNAc. The second mutation is a single base deletion and produces a frameshift at position 165 in gene lmo1083, whose product is involved in rhamnose biosynthesis, resulting in a truncated enzyme and lack of rhamnose in WTA. Deletion mutants constructed in a serovar 1/2a background (EGDe Dlmo2550 Dlmo1083) resulted in loss of the rhamnose and GlcNAc WTA components. Complementation with wt lmo2550 and lmo1083 in the EGDe mutant, in serovar “7” strain 1034, and in other mutants featuring a lack of sugar substitution not only restored the rhamnose and GlcNAc content in EGDe, but also produced a serovar 1/2 phenotype in the “serovar 7” strain and other mutants lacking GlcNAs from their WTA. These findings suggest that Listeria serovar “7” strains (at least 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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REFERENCE High-throughput genome sequencing of two Listeria monocytogenes / clinical isolates during a large foodborne outbreak A P Gilmour, M.1, Graham, M.1, Van Domselaar, G.1, Tyler, S.1, Kent, H.1, Trout-Yakel, K.1, 29 Larios, O.1, Allen, V.2, Lee, B.3, Nadon, C.1 and Kearney, A.1 1. Public Health Agency of Canada 2. Ontario Agency for Health Protection and Promotion, Canada 3. Canadian Food Inspection Agency, Canada A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis (PFGE) revealed two similar but distinct AscI PFGE patterns. Whole genome sequencing of two L. monocytogenes isolates was completed using the Roche GS FLX™ platform to rapidly determine the genome sequence of the primary outbreak strain and to investigate the genetic diversity associated with a change of a single restriction enzyme fragment observed by PFGE. The chromo- somes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs) and three indels, including a 33kbp prophage that accounted for the difference in PFGE pattern. The distribution of these traits was assessed within clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes. High- throughput genome sequencing therefore provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. These data allowed us to determine evolutionary lineages and unequivocally define the full breadth of genetic variation between two subtype variants identified by the internationally standardized PulseNet PFGE typing method. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.

Expression of antimicrobial activity in food and clinical REFERENCE A / P Listeria monocytogenes isolates Barbosa, J., Ferreira, V., Borges, S., Azevedo, I., Magalhães, R., Santos, I, Almeida, G. 30 and Teixeira, P. Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Portugal Listeria monocytogenes is a Gram-positive facultative intracellular pathogen capable of causing fatal infections in risk groups, such as pregnant women, elderly and immunocompromised individuals. Little information is available on the antimicrobial susceptibility of L. monocytogenes, particularly of strains isolated from food and production environments. Minimum inhibitory concentrations (MIC’s) of ampicillin, penicillin G, chloramphenicol, erythromycin, tetracycline and vancomycin 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% were intermediary and resistant, respectively, to erythromycin. Only one isolate demonstrated a resistance profile to tetracycline, an antibiotic largely used in veterinary practice, 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 as a drug of second choice in cases of ampicillin allergy. Although in vitro antibiotic susceptibility testing does not always reflect the in vivo situation, results demonstrated that some of the isolates investigated are resistant to antibiotics of clinical importance commonly used to treat listeriosis. Listeriosis is usually an easily treat- able disease and no reports have been presented on antibiotic resistant isolates from 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, it contains very valuable and important data. Only by keeping an on-going track of the resistance patterns can these be monitored for unexpected changes.

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REFERENCE The Listeria monocytogenes sigma B and sigma H regulons overlap, but A / P only sigma B appears to be important for survival of acid, alkaline and 31 oxidative stress Chaturongakul, S.1, Raengpradub, S.2, Wiedmann, M.3 and Boor, K. J.3 1. Mahidol University, Thailand 2. Silliker, Inc., USA 3. Cornell University, USA Previous transcriptomic analyses in Listeria monocytogenes using whole genome microarrays and Hidden Markov Model (HMM) based promoter searches identified B many genes that were controlled by more than one transcriptional regulator (e.g., σ , H L σ , σ , PrfA, HrcA or CtsR). The regulons of the two stationary phase sigma factors, B H σ and σ shared the largest number of genes. To further investigate the regulatory B H features shared between σ and σ , whole genome microarrays were performed and transcript profiles for ∆sigB, ∆sigH, and ∆sigB∆sigH strains were compared to that of the parent strain 10403S. In the present study, using cut-off criteria of a 1.5 fold dif- B H ference at p<0.05, we identified 301 σ -dependent genes, 82 s -dependent genes, and a total of 61 genes with expression profiles that suggest regulatory interactions B H between σ and σ . The 61 co-regulated genes grouped into 4 different quality threshold (QT) clusters: Cluster 1 is comprised of 19 genes that are positively regu- B H lated by both σ and σ (e.g., mRNA levels for cspD, encoding a cold shock protein and for rpoD, encoding an RNA polymerase sigma factor, were significantly lower in the ∆sigB∆sigH strain than in the other strains), Cluster 2 contains 17 genes that are B H negatively regulated by both σ and σ (e.g., mRNA levels for genes encoding ribosomal proteins (rpsR and rplJ), RNA polymerase 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 ∆sigH strains, but with transcript levels that are the same in both the ∆sigB∆sigH and wild type strain (e.g., ATP-dependent Clp protease clpP), and Cluster 4 has 14 genes that are 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 rpsD and rplK, which encode ribosomal proteins). In addition to transcript profiling, 10403S and the 3 otherwise isogenic mutant strains were also phenotypically characterized. After exposure to acid (1 h at final pH of 2.5), alkaline (1 h at final pH of 12), or oxidative stresses (15min at 13mM cumene hydroperoxide final concentration), the phenotype of the ∆sigB∆sigH strain was identical to that of the ∆sigB strain, i.e., with cell numbers that were significantly reduced (CFU/ml) when compared to those of B the parent strain. This latter finding further verifies the central role of σ in stress re- H sponse, and suggests that σ is less important for survival of these stresses.

REFERENCE / Virulence gene expression in Listeria monocytogenes strains isolated A P from different sources 32 Alessandria, V., Rantsiou, K. and Cocolin, L. University of Turin, Italy Listeria monocytogenes is an important food-borne pathogen that has the capacity to cause severe infections. Ubiquitous microorganism, it is commonly isolated from foods of animal origin, mainly meat and milk products. However, due to its capacity to develop at refrigeration temperatures, human listeriosis outbreaks are most often associated with ready-to-eat food products that are consumed without prior cooking. The incidence of the disease depends on different factors including the infective dose and the immunity conditions of the host. The present work focuses on the expression analysis 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, SCOTT A), 7 isolated from food matrices (4 of which isolated from meat products and three from diary products) and 1 isolated from humans. In the first step, the expression analysis 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 different strains. The combined use of reverse transcription and quantitative PCR (qRT- PCR) was used to evaluate gene expression. As a second step, we wanted to evaluate the 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 differences emerged for the different genes, without showing any significant association between the expression of the genes and the origin of the different strains.

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REFERENCE Differentiation of Listeria monocytogenes, Listeria innocua and / Listeria marthii, a novel Listeria species isolated from the natural A P environment, Finger Lakes National Forest 33 Graves, 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, USA 2. Cornell University, USA 3. 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 phenotypically similar to Listeria spp. were isolated from soil, standing water, and flow- ing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined the same species by whole genome DNA-DNA hybridization studies, but sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to form a separate species. 16S ribosomal RNA sequence analysis confirmed their close phylogenetic relatedness to L. monocytogenes and 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 prs showed 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°C with 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 DNA sequences was between 7.5% and 9.5%. In reactions at 70°C, FSL S4-120T DNA showed an average of 45% relatedness to the three L. monocytogenes strains and one L. innocua strain (range, 30–53%). The isolates yielded positive reactions in the 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 a homologue of the L. monocytogenes virulence gene island. A new species L. marthii is 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 lmo0028 REFERENCE A / P and lmo1638 genes. Yurov, D.*, Varfolomeev, A., Kaminskaya, A. and Ermolaeva, S. 34 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 and lmo1638 that encode L,D-carboxypeptidases. The lmo0028 promoter has a recognition site for the virulence regulator PrfA. Mutant L. monocytogenes strains GIM0028 and GIM1638 were obtained by site-specific insertions into the chromosome of the wild type EGDe strain. Morphology was characterized with light microscopy. Septation was studied with fluorescent vancomycin. Genome copy numbers were determined with quantitative PCR. L. monocytogenes virulence was studied on BALB/c mice and human colon carcinoma HT29 cells. The mutation in lmo0028 caused dispersion in cell lengths: the length/width ratio ranged between 1.5 and 15 for GIM0028 and 1 and 4.4 for EGDe. The insertion in lmo1638 caused shortening in cells. The average length/width ratios were 3.75, 1.2 and 3.0 for GIM0028, GIM1638 and EGDe, respectively. The longish GIM0028 cells were defected in septum formation, GIM1638 were not. GIM0028 demonstrated two fold decreasing in invasion efficiency. Doubling times in HT29 cells determined by plating were 51 minutes and 70 minutes for EGDe and GIM0028, respectively. However, evaluation of intracellular bacterial genome numbers with qPCR did not reveal a difference between the strains. GIM0028 bacteria increased their length upon intracellular growth: average length/width ratio was about 4.8 for invading bacteria and 9.2 after 6 h of intracellular growth while for the wild type bacteria it was 3.5 (p<0.005). GIM0028 demonstrated reduced virulence for BALB/c mice 6 4 with LD50 of 1x10 and 1.5x10 CFU/mouse for GIM008 and EGDe strains, respectively. The Lmo0028 L,D-carboxypeptidase is involved in septum formation while Lmo1638 seems to be involved in cell elongation. A mutation in lmo0028 decreases L. monocytogenes virulence.

* Participation Supported by IUFoST.

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REFERENCE Antimicrobial susceptibilities of Listeria monocytogenes isolated A / P from retail beef, pork and poultry in Japan 35 Ida, 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 the most important link between food-producing animals and humans. The aim of this study is evaluate antimicrobial susceptibilities of Listeria monocytogenes isolated 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 were included in the study. The minimum inhibitory concentrations of antimicrobial agents were determined by the agar dilution method according to the guidelines of Clinical 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 as quality control strains. All isolates were susceptible to antimicrobials except for oxytetracycline. Three strains, isolated from domestic pork, domestic poultry and beef imported from Australia were resistant (>32µg/ml). All of them were isolated in 2001 and belonging to serotype 1/2c. This study showed that antimicrobial resistance is not highly prevalent in L. monocytogenes in meat in Japan.

REFERENCE Genetic basis of two low pathogenic L. monocytogenes strains A / P with apparent phospholipase C activity 36 Jiang, 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 C activity were characterized for their genetic basis and pathogenecity. Sequencing analysis 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 of the enzyme at the N-terminus. The strains were of serovar 4a, had low pathogenecity 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 mutation of these sites to the plcB version of strain 10403S abolished the enzyme activity, increased virulence by one log (from 8.12 to 7.07), but did not have significant effect on plaquing. This abolishment did not seem to be related to presence of G145S in the central regulator prfA, which was found to induce overexpression of virulence genes independent of environmental conditions in other strains. However, this does not mean that prfA was not involved in regulation of its expression because transposon mutagenesis identified two mutant strains lacking apparent phospholipase activity due to disruption of the prfA by insertions at nt364 and nt828. There are 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 examine if apparent activity of the novel phospholipase in these strains results from easy access to zinc metalloprotease encoded by mpl for maturation, or if amino acid substitutions of T165A and K197N other than G145S in prfA also contribute to regulation of downstream virulence genes. Using one of the strains as a model organism, we are testing how phospholipase C and listerolysin interact in determining its pathogenecity and if there are other genes involved in their low pathogenecity by genome wide scanning using Solexa sequencing technology.

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REFERENCE Molecular genotyping and antimicrobial resistance / of Listeria monocytogenes from foods and the environment A P Parisi, A.1, Miccolupo, A.1, Fraccalvieri, R.1, Latorre, L.1, Normanno, G.2 and Santagada, G.1 37 1. Experimental Zooprophylactic Institute Apulia and Basilicata, Italy 2. University of Bari, Faculty of Veterinary Medicine, Italy Listeria monocytogenes is generally susceptible to a wide range of antibiotics, however an increasing number of strains resistant to one or more antibiotics have been reported. Objectives of the present study were to evaluate molecular genotyping and antimicrobial susceptibility in L. monocytogenes isolates from foods and the environment. 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 21 antimicrobials was performed using Gram-positive GPN4F kit (TREK Diagnostic System). 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 to six 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 which represented by a single isolate. ST9 (21%), ST121 (12%), ST3 (5%) and ST199 (5%) were the most prevalent STs. Moreover MLST allowed a good discrimination of the two main genetic lineage of L. monocytogenes. Our results confirm the low prevalence of food and environmental isolates resistant to antimicrobials. Inter- estingly, a good correlation between resistance to oxacillin and genetic lineage I was observed (Yates corrected =35,46; p<0.005); this is in agreement with the clonal organization of L. monocytogenes population. The severity of L. monocytogenes infections and the reported increasing of antimicrobial resistance make necessary the continuous monitoring of this trend although the recorded data do not appear to be alarming. The comparison of phenotypic and genetic characters allow to validate the evolutionary models speculated for this microbial species.

Virulence transcriptome analysis of Listeria monocytogenes REFERENCE A / P by application of microarrays in vitro and in situ Rantsiou, K., Alessandria, V. and Cocolin, L. 38 University of Turin, Italy With the complete genome sequence available for Listeria monocytogenes as well as other Listeria spp., it is nowadays possible to use tools that allow global analysis of the biology of this pathogenic microorganism. Microarrays give the possibility to study concurrently a large number of genes and deduce information regarding their expression. This approach accelerates significantly the rhythm by which new information is generated. In this way, the effect of different conditions on the expression of a series of genes and as a consequence on the physiology of a microorganism can be investigated. Within the 6th FP project Pathogen Combat, it was developed a microarray including 72 genes of L. monocytogenes, encoding for virulence, adhesion and stress response genes. In this study we sought to apply this array to study the effect of different environmental conditions on the expression of virulence genes. First, different strains of L. monocytogenes were grown in vitro, at different temperatures, pH and salt concentrations. Then, L. monocytogenes was artificially inoculated in different food matrices, to simulate the conditions of ‘virulence’ of this microorganism at time of consumption. Global analysis of the expression of virulence genes in different L. monocytogenes strains will lead to a better undestanding of the physiology of this microorganism and eventually to the prediction of its potential to cause disease to humans.

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REFERENCE Effects of growth conditions on surface properties of Listeria; A / P a proposed role for AI-2 39 Wong, 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 environments due to its ability to colonize surfaces and then become persistent by adapting to low nutrient and low temperature conditions. We have been investigating the 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) and also the D10 medium supplemented with duck meat extract. Growth in the BHI medium was fastest, but significant increases in growth were seen when D10 was supplemented with 10% duck meat extract. One feature of cells that become persistent in factory environments is their ability to form biofilms that are resistant to cleaning and disinfection. We have found that the surface hydrophobicity (measured using Microbial Attachment to Solvents (MATS) assays) alters significantly when the organism is grown under low nutrient conditions, with cells becoming more hydrophobic. Also these cells have a greater tendency to flocculate in liquid culture. This affect is seen when cells are grown above 30°C and therefore is not related to the synthesis of flagellae. The ability to form biofilms in Listeria is known to be affected by the synthesis of a functional autoinducer 2 (AI-2)-like signal, and that an intact luxS gene is associated with inhibition of attachment and biofilm formation. Using a Vibrio harveyi reporter strain we have found that when cells are grown in minimal media, the amount of detectable AI-2 is reduced. As AI-2 has a role in the down-regulation of components required for attachment and biofilm formation, this result is consistent with our observation of changes in the surface properties of Listeria under low nutrient conditions that are more likely to promote surface attachment.

* Participation Supported by IUFoST.

REFERENCE A / P Stress behaviour of a Listeria monocytogenes 568 Lmo1634 transposon mutant 40 Truelstrup-Hansen, L.1, Holman, D. B.1 and Ells, T. C.2 1. Dalhousie University, Canada 2. Agriculture and Agri-Food Canada Listeria monocytogenes is an important psychrotrophic foodborne pathogenic bacterium with tendency to persist in food processing plants. Heat treatment is routinely used as a means to control pathogenic bacteria in food products. Therefore, the thermotolerance of L. monocytogenes and mechanisms responsible are of interest. Previous research using transposon mutagenesis yielded several heat resistant mutants, including one, named 1B4, that was mapped to a putative alcohol/ac- etaldehyde dehydrogenase gene, lmo1634 or adh, also identified as Listeria adhesion protein. The objective of this study was to investigate the function of this gene in relation to a variety of environment stresses and in biofilm formation. The transposon 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) Lm568 2 strain. Attachment of 1B4 (7.06log10 CFU/cm ) to stainless steel coupons was not 2 significantly (p>0.05) different from the WT Lm568 (6.91log10 CFU/cm ). Neither 1B4 nor Lm568 exhibited greater survival after desiccation (43% RH) at 20°C for 7 days. The alcohol dehydrogenase activity of Lm568 (0.197U/mg) was found to be significantly (p>0.05) greater than in 1B4 (0.0458U/mg). 1B4 was complemented in trans with the WT lmo1634 gene and the successful transcription of lmo1634 in the complemented strain was confirmed by RT-PCR. In addition, no polar effect on the genes immediately downstream of lmo1634 in 1B4, i.e., lmo1635, lmo1636, or lmo1637, was observed. The trans complementation of 1B4, however, did not completely restore the WT phenotype. Attempts to create a deletion mutant in lmo1634 ultimately proved unsuccessful. Despite these technical issues, it is clear that a mutation in lmo1634 yields a multi-stress resistant phenotype.

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Investigation of the conditions that trigger the activation of the alter- REFERENCE A / P native sigma factor σB in Listeria monocytogenes Utratna, M., Shaw, I. and O’Byrne, C. 41 National University of Ireland, Galway, Ireland Listeria monocytogenes has evolved multiple strategies to overcome diverse stress conditions and it is able to occupy extreme environmental niches. As a facultative intracellular foodborne pathogen L. monocytogenes successfully invades host cells, surviving in gastric tract before the final colonization of the target organs. The al- B ternative sigma factor (σ ) plays an essential role in the stress tolerance and viru- B lence of L. monocytogenes. Many components of the σ regulon have been identi- B fied but the mechanisms that regulate σ are still unknown. The current model B for σ regulation, based on work done in Bacillus subtilis, suggests that the activ- B B ity of σ is independent of the levels of σ in the cell. The expression of OpuCA has B been shown to be controlled by σ . Therefore the levels of this protein can act as an B indirect reporter of the activity of σ in L. monocytogenes. Polyclonal antibodies against OpuCA were developed in chickens and used in Western blotting. Based B on this reporter system it was shown that σ is more active in stationary phase B when compared to exponential phase (OD600=0.6). σ activity under conditions encountered in the human gut was also determined. Levels of OpuCA increase gradually in the range of salt up to 0.9M NaCl and when the pH is reduced from pH B 7.2 to pH 5.0. Higher σ activity was also detected under limited oxygen conditions and at 4°C. However, no change in the levels of OpuCA was observed in the presence of low concentrations of bile salts up to 5mM. Additionally an attempt B was made to develop a transcriptional reporter fusion system by cloning the σ B promoter from strongly σ -dependent gene, lmo2230, into the reporter vector pTCV-lac allowing the measurement of β-galasctosidase activity under various conditions. This genetic system will also be a useful tool in monitoring the activity B of σ in L. monocytogenes.

REFERENCE Characterization of Listeria monocytogenes 1/2a, 1/2b, 1/2c and 4b A / P by Amplified Fragment Length Polymorphism and evaluation of their geographical distributions in Portugal 42 Maia, 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, Portugal 2. Escola Superior Agrária, Instituto Politécnico de Castelo Branco, Portugal 3. Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Portugal Listeriosis is a bacterial infection caused by the ingestion of foods contaminated with Listeria monocytogenes, that can affect humans, especially the group that includes pregnant women, newborn infants, the elderly and immunocompromised individuals. AFLP molecular subtyping, using EcoRI restriction enzyme, was used to do the differentiation between strains isolated from different Denomination of Protected Origin regions of cheese production in Portugal and as well from clinical specimens. A set of 159 strains belonging to serotypes 1/2a, 1/2b, 1/2c and 4b isolated from samples of Serra da Estrela ewe’s cheese, São Jorge cow´s cheese, Serpa ewe´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 was possible to predict the serovar from the AFLP pattern found in most of the isolates. In the opposite direction, each one of the serovars was associated to several AFLP molecular types, which demonstrates the grater discriminatory capacity of the AFLP subtyping. Similarities between profiles, based on band positions, were derived from the Dice correlation coefficient (SD) with a maximum position tol- eranceof1%,3%or4%. Listeria monocytogenes strains were clustered by the technique of the UPGMA and a dendrogram was constructed to reflect the genetic distance between them.

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REFERENCE Global analysis of the Listeria monocytogenes surface proteins A / P of the LPXTG family 43 Botello-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 Superior de Investigaciones Científicas (CSIC). Darwin 3, Madrid, Spain 2. Unidad de Proteómica, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain 3. Departamento de Biología Molecular, Universidad Autónoma de Madrid. Campus de Cantoblanco, Madrid, Spain Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that causes serious systemic diseases in humans and animals. Comparative genomics revealed the presence in the Listeria genus of a large number of genes encoding surface proteins containing an LPXTG sorting motif. This signature makes these proteins recognized by sortases, the enzymatic machinery that anchors proteins covalently 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 relevance of surface proteins for communication with the environment and the host, the biological role of most members of this family remains unknown in L. monocytogenes. In a collaborative effort with other European groups that contributed to generate mutants defective in specific LPXTG proteins, we accomplished a comprehensive proteomic analysis in peptidoglycan material purified from each of these mutants. A total of 30 LPXTG mutants have been analysed to date. Pro- teomic analysis was also performed in peptidoglycan purified from intracellular wild-type bacteria upon infection of epithelial cells. A major finding of this global approach was the cross-regulation existing among certain LPXTG proteins. This phenomenon involved in most cases the down-regulation of concrete LPXTG proteins in response to the lack of a specific LPXTG protein. Interestingly, the inverse phenomenon, the up-regulation of a LPXTG protein in response to a deficiency in another LPXTG protein, was also observed. These proteomic data were confirmed 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.

REFERENCE Antimicrobial susceptibility of Listeria monocytogenes strains A / P derived from food and food-processing bakery plant 44 Eusébio, 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 bakery industry, from food and food-processing environment, to 11 antibiotics was determined by the standard agar dilution methodology. The tested antibiotics were: ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, nitrofurantoin, penicillin, rifampicin, streptomycin, tetracycline and vancomycin; minimal inhibitory concentrations values were used to classify the strains into sensitive, moderately resistance and resistant. All the tested isolates were found to be susceptible to ampicillin, chloramphenicol, gentamicin, nitrofurantoin, penicillin, rifampicin, tetracycline streptomycin and vancomycin. In the case of erythromycin, 54 isolates (32%) were susceptible, 68 (41%) displayed moderately resistance and 45 (27%) were resistance. Concerning to ciprofloxacin, a moderate resistance was observed in 12 strains (7%) against 155 strains (93%) that were susceptible. Generally, this study showed that L. monocytogenes strains are susceptible to the antibiotics commonly used in the treatment of listeriosis. Concerning that antibiotic resistance in some L. monocytogenes strains has already been described, a con- tinued study of emerging antimicrobial resistance is important to guarantee an effective treatment of human listeriosis.

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REFERENCE A physiological study to purpose a new formal method / to obtain L. monocytogenes cells adapted to BAC A P Saá Ibusquiza, P., Cabo, M. L., Herrera, J. J. R., Vázquez, D., Carrera, S. and Eiriz, E. 45 Instituto de Investigaciones Marinas (Marine Research Institute), CSIC, Vigo, Galicia, España Maximum adaptation potential of Listeria monocytogenes (L. monocytogenes) cells to benzalkonium chloride (BAC) was classically checked by successive exposition of stationary cells to increasing sublethal BAC concentrations. However, this method is time consuming and do not ensure we reach the maximum level of adaptation of the strain. So, in the present work we develop a new method to obtain BAC-adapted cells of L. monocytogenes that is based in only one exposition of L. monocytogenes exponential-phase cells to sublethal concentrations of BAC. Moreover, in order to predict the optimal conditions for achieving the maximum level of adaptation, a factorial design to formalize the effects of the initial concentration of cells and the concentration of BAC during the exposition was carried out. Obtained empirical model demonstrated a positive effect of inoculum and a positive interaction between both variables. However, a negative first order effect for the BAC indicated the convenience of using moderately high concentrations of it during the expositions. As a consequence of applying this procedure, adaptation of L. monocytogenes 5873 to BAC was increased aproximately 4 times respecting to the wild type strain in only 36h. Finally, comparison between the expression profiles in the wild and the highest BAC-adapted variant was also carried out.

REFERENCE Characterization of a RNA-helicase in the human pathogen / Listeria monocytogenes A P Netterling, S. and Johansson, J. 46 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 as being able to grow in a span from -1 to 45°C. Listeriosis can cause severe symptoms such as meningitis, with high lethality rate. The ability to grow at low temperatures is one of the most interesting aspects of this pathogen. We have looked at RNA helicases; proteins that are able to unwind short dsRNA structures in an ATP- dependent manner. Most mRNAs develop complex secondary and tertiary structures during the maturation process, these structures needs to be unwound before translation initiation. The stability of these complex structures is influenced by temperature, often becoming more rigid at lower temperatures thereby hin- dering e.g. translation, resulting in undesired cold-stabilized RNA structures. RNA helicases may function by relaxing local RNA-RNA interactions or by dissociat- ing RNA from proteins. In L. monocytogenes RNA helicases have earlier been shown to be up-regulated during growth in a cold environment. RNA-helicases have 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. monocytogenes. This DExH-box RNA helicase deletion strain displayed an inability to grow at 4°C and had an impaired growth at temperatures below 37°C. Motility was also affected in the deletion strain implying that it cannot spread in food sources as well as the wild-type strain. All phenotypes were rescued in a complemented strain. Our results indicate that RNA helicases is of high importance to the bacterium for growth at lower temperatures, and provides a link between RNA helicases and motility.

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REFERENCE A Listeria monocytogenes strain is still virulent despite non-functional B / P major virulence genes: Optical Mapping shows a potential mechanism 47 Roche, 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, Agents transmissibles 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, France and CHRU, Tours, France L. monocytogenes is a pathogenic species, but some strains exhibit low virulence. In previous studies, 24 naturally occurring low-virulence L. monocytogenes strains were 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 phenotypic characteristics, these low-virulence strains have been assigned by cluster analysis to one of four groups. The 11 strains belonging to Group I exhibit a mutated PrfA whereas five out of the six strains belonging to Group III have causal mutations in plcA, inlA and inlB genes. New strains exhibiting the same PFGE specific profile than the low-virulence Group III strains have been identified and characterized. All were low-virulence strains exhibiting the same mutations characteristic of the Group III strains, beside the A23 strain which has been characterized as a virulent strain in the mouse model. Interestingly, this strain exhibited 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 inactive InlB, PI-PLC and PC-PLC proteins and to a lack of InlA expression which are known to be important for virulence. Despite these mutations in these major virulence 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 two Group III strains in comparison with the EGDe genome showed specific fragments inserted in the genome of the Group III strains. Identification of the genes modified by the insertion could explain the virulence of the A23 strain compared to the Group III strains. Phylogenetic analysis of these strains could be fruitful to understand the evolution of virulence trait as well as plasticity of virulence genes.

REFERENCE Protein expression of lineage I, II, and III Listeria monocytogenes B / P strains in murine macrophages 48 Donaldson, 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 from genetic lineages I, II, and III (serovar 1/2a strain EGD, serovar 4b strain F2365, and serovar 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 but could not establish prolonged infection within the macrophages. By contrast, strains EGD and F2365 proliferated within macrophages for at least 7 hr. We further characterized this interesting phenotypic difference by determining the protein expression profiles of these strains at 0 hr, 3 hr, and 5 hr post-infection using two dimensional liquid chromatography coupled with electrospray ionization tandem mass spectrometry. We found that metabolic and cell wall associated proteins were expressed by all three strains at 3 hr post-infection. However, increased expression of stress response and DNA repair proteins was detected in the lineage I and II strains at 5 hr post-infection. These proteins were not significantly increased in the lineage III strain at this time point. By comparing the protein expression patterns of these three L. monocytogenes strains during intracellular growth in macrophages, we were able to detect biological differences that may determine the ability of L. monocytogenes to survive and persist in macrophages.

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REFERENCE Prevalence of L. monocytogenes in bovine mastitic milk samples: / Possible source of food borne infection B P Kalorey, D. R.1, Warke, S.1, Kurkure, N. V.1 and Barbuddhe, S. B.2 49 1. Nagpur Veterinary College, India 2. ICAR Research Complex for Goa, India Listeria monocytogenes is a food borne pathogen responsible for listeriosis, disease characterized by meningitis, encephalitis, reproductive disorders and septicemia in human beings. One of the major source of Listeria is milk. A study was conducted to know the prevalence of Listeria monocytogenes in mastitic milk from central India. Quarter milk samples (n=1221) were collected from dairy cows and screened for sub clinical mastitis by California mastitis test (CMT). CMT positive samples were examined for the presence of L. monocytogenes following two step enrichment and plating on selective agar. Confirmation of isolates was based on biochemical tests, haemolysis on blood agar and CAMP test. The L. monocytogenes confirmed isolates were subjected to PCR assay for detection of virulence marker genes (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 prfA genes were detected in 33 strains. The hlyA and actA were in seven strains. Eleven strains harbored hlyA, actA and iap genes. All isolated strains were subjected to serotyping 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. Detection of 4b serogroup is of highly significance owing to its association with food borne listeriosis outbreaks.

Agr-dependent peptide sensing in L. monocytogenes – Effects on biofilm REFERENCE B / P formation, virulence and global gene expression Waidmann, M. S.1*, Monk, I. R.2, Auchter, M.1, Preising, N. P.1, Hill, C.3 and Riedel, C. U.1 50 1. Institute of Microbiology and Biotechnology, University of Ulm, Germany 2. Trinity College Dublin, Ireland 3. University College Cork, Ireland Autoinducing peptides are signalling molecules used in population-dependent gene regulation of Gram-positive microorganisms. The best studied example of bacterial peptide sensing is the agr system of staphylococci. An operon with high structural and sequence similarity to the staphylococcal arg system was recently identified in the genome of L. monocytogenes EGDe. The listerial agr system is composed of a four gene operon with agrB involved in the proteolytic processing/export of the gene product of agrD, the posttranscriptionally modified signalling peptide, and agrA/agrC encoding a typical two-component system with histidine kinase (AgrC) and response regulator (AgrA). Here, we present our recent advances in identifying and characterizing the native agr peptide of L. monocytogenens from spent cell culture supernatant and the effects of agr peptide sensing on biofilm formation, virulence and global gene expression. Furthermore, the mechanisms of agr-dependent gene regulation in L. monocytogenes were investigated. A ∆argD mutant showed a significant defect in biofilm formation. Invasion of EGDe ∆argD into Caco-2 and Hep-2 cells was significantly reduced compared to EGDe wt. Moreover, the ∆argD mutant showed an unusual subcellular distribution after primary invasion. In line with these observations promoter activities of important virulence genes were reduced in the ∆argD mutant. Using bioluminescence in vivo imaging, a significant attenuation of EGDe ∆argD in a murine model of listeriosis could be shown. More- over, microarray analysis revealed that expression of a large number of genes belonging to all functional categories was affected in EGDe ∆argD both in exponential and stationary growth phase indicating a global impact of agr-dependent peptide sensing on all aspects of physiology in L. monocytogenes.

* Participation Supported by IUFoST.

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REFERENCE agrD-deletion affects InlA- and InlB-regulation via B / P a temperature-dependent and -independent mechanism 51 Waidmann, M. S.1*, Monk, I. R.2 and Riedel, C. U.1 1. Institute of Microbiology and Biotechnology, University of Ulm, Germany 2. University College Cork, Ireland Quorum sensing via the secretion of autoinducing peptides is a commonly used mechanism for gene regulation among Gram-positive bacteria. A well known example is the accessory gene regulator (agr) in Staphylococcus aureus, which is known to affect virulence. Following the sequencing of Listeria monocytogenes EGDe genome and the identification of a homologous gene locus, suggestions of a regulatory role in virulence arose. The first step of listerial pathogenic lifecycle is the 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 epithelial and endothelial cells. To testify the role of agr in regulation of these virulence factors, we evaluated the invasive capability of an agrD deletion mutant, which lacks the putative autoinducing peptide, into Caco-2 and HEp-2 cells. These assays showed that the Internalin A-dependent invasion of the mutant was significantly decreased in comparison to the wild type. The temperature chosen for growing pre-cultures did not influence this defect. By contrast, the effect of the agrD deletion for Internalin B-mediated invasion into HEp-2 cells was only impaired in pre-cultures grown at 37°C. Furthermore, the total numbers of invaded bacteria were much higher as compared to 30°C. These results indicate a temperature-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 affecting either the expression itself or the activation of the identified regulators B PrfA and σ . According to these results agr seems to be an additional player for listerial virulence.

* Participation Supported by IUFoST.

REFERENCE B / P Bovine cranial nerve Schwann cells express E-cadherin, a candidate key-player in the brainstem invasion of Listeria monocytogenes in cattle 52 Madarame, H.1, Seuberlich, T.2, Vandevelde, M.2, Zurbriggen, A.2, and Oevermann, A.2 1. Veterinary Teaching Hospital, Azabu University, Japan 2. 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 into cells and for the crossing of the intestinal and placental barrier during infection. In ruminants, listeriosis occurs most commonly as rhombencephalitis, specifically targeting the brainstem, and it is believed that LM enters the brain via cranial nerves. 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 cattle brain, trigeminal nerve (TN) and its ganglion (TG), and thus its putative role in brain 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, strong membranous E-cadherin expression was observed in choroid plexus epithelial cells by IHC, whilst no other brain structures were labeled. TN and TG exhibited strong E-cadherin expression in Schwann cells and satellite cells (which are specialized Schwann cells), respectively. The labeling was particularly evident at the intercellular boundary of satellite cells and at the Schmidt-Lantermann incisures. Confirming the IHC results, E-cadherin specific bands of molecular masses of approximately 37kDa and 130kD were evident in the WB of the TG, but were absent in the WB of the brainstem. These results indicate that E-cadherin expressing Schwann cells of cranial nerves might be the initial port of entry for LM in cattle rhombencephalitis. From there, LM might gain access to the brain by cell-to-cell spread via axons. However, this hypothesis needs to be confirmed by complementary in vitro investigations of the molecular host-pathogen interactions.

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REFERENCE Pathogenic potential of Listeria monocytogenes isolates / from New Zealand seafood premises: implications for control B P Durante Cruz, C. and Fletcher, G. 53 The New Zealand Institute for Plant and Food Research Listeria monocytogenes is likely to persist in food premises, leading to contamination of products processed therein. It is therefore important to assess the virulence potential 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 isolates obtained from New Zealand (NZ) seafood premises and correlated this with their persistence and genetic profile. From 120 isolates obtained from four seafood premises, 30 (28 from environmental and 2 from product samples) belonging to different serotypes and pulsotypes were compared in this study. These were compared with five human isolates obtained from the NZ Reference Culture Collection, two isolates (human and food) from a NZ seafood-related outbreak of listeriosis and one meat isolate shown to be resistant to high pressure conditions. ScottA was used as a reference strain and assigned an invasion index of 100%. In vitro studies were conducted using Caco-2 cells (100:1 Listeria:Caco-2) followed by 40 min incubation at 37°C. Three independent experiments were performed on each isolate in duplicate. In Greenshell™ mussel premises, isolates from Plant I showed higher invasiveness than those from Plant II. All isolates were serotype 1/2a, except for one 1/2b from Plant I. The persistent and predominant pulsotype from Plant II had a low invasion index. Serotype 1/2b and 4b isolates from the two fish premises showed higher invasiveness than the serotype 1/2a isolates from mussel plants. Product (serotypes 1/2a and 1/2b) and one human (serotype 1/2a) isolates showed higher invasion indices than environmental ones. The seafood outbreak strains had an intermediate invasion index. This study improves our understanding of L. monocytogenes present in NZ seafood premises and highlights the need to improve control. Although some environmental factory isolates were potentially pathogenic, persistent strains were generally less invasive in Caco-2 cells.

Copper homeostasis and virulence in Listeria monocytogenes REFERENCE B / P David, C.1, Schuler, S.1, Glenn, S.2, Jen, C.1, Andrew, P.2 and Roberts, I. S.1 54 1. University of Manchester, UK 2. University of Leicester, UK Copper is essential for many bacteria being an important prosthetic group for certain enzymes. However at high levels copper is toxic in part due to its ability to redox cycle and catalyse the formation of oxygen derived free radicals. As such bacteria need to be able to maintain copper homeostasis. In L. monocytogenes the problem of copper homeostasis is particularly acute. It inhabits a range of environments including soil, effluents and food where it will be exposed to fluctuating copper levels. The use of copper as an anti-microbial in animal feeds, as a disinfectant in factory-based farming and as a biocide in fruit production means L. monocytogenes will have to combat potentially toxic levels of copper during food production and processing. Adapting to fluctuations in the level of copper is important during infections. L. monocytogenes replicates in a number of host tissues that will offer different challenges with regard to copper availability. Early in infection L. monocytogenes grows in the gall bladder, where there are high levels of copper, whereas in the liver and spleen it replicates in an environment in which there may be little free copper. Adapting to these different niches within the host requires effective copper homeostasis. In this paper we describe and analyse the operon that encodes for the only P1 type-ATPase copper exporter in L. monocytogenes and describe the molecular basis by which transcription of this operon is regulated by environmental copper and identify the role played by this operon in infection of L. monocytogenes.

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REFERENCE Pattern of cytokine production during murine listeriosis B / P 55 Dussurget, O. Institut Pasteur, France Listeria monocytogenes causes listeriosis, an infection whose manifestations extend from gastroenteritis to septicemia, meningitis, encephalitis, abortions and perinatal infections. Cytokines are important mediators of host defense against pathogenic microorganisms. They control innate immune responses and contribute to shape adaptive immune responses. Several studies have previously analyzed the production of one or several cytokines in response to Listeria monocytogenes in vitro and also in vivo. In this work, we used a murine model of primary listeriosis to systematically follow in the same animal the protein levels of 20 cytokines 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 major activator of macrophage antimicrobial activity, interferon (IFN) g, in blood, liver and spleen 24h after intravenous inoculation. The early response to infection was also characterized by a significant increase in proinflammatory cytokines tumor necrosis 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 cytokines important for the T cell response, e.g. IL2, and more specifically for T helper 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, also increased upon infection. The level of most cytokines increased in correlation with the number of bacteria in each organ during the first three days of listeriosis. In contrast, a strain of L. monocytogenes which does not produce the major virulence factor listeriolysin O (LLO), and is cleared very early from infected mice, failed to stimulate cytokine production. Together, this first multiplex analysis of cytokine production in vivo represents an important basis to identify virulence determinants involved in the modulation of host immune response.

REFERENCE / Analysis of the post-translocation chaperone PrsA2 and its unique role B P in facilitating Listeria monocytogenes pathogenesis 56 Alonzo, F. and Freitag, N. University Of Illinois at Chicago, USA Listeria monocytogenes is a Gram-positive bacterial pathogen whose ability to cause disease is dependent upon the coordinated expression and activity of secreted products that promote virulence. We have shown that the post-translocation chaperone PrsA2 is critical for L. monocytogenes pathogenesis and that prsA2 mutants exhibit a substantial cell-to-cell spread defect in tissue culture. We therefore hypothesized that a loss of PrsA2 results in perturbation and/or altered activity of factors responsible for intracellular survival. PrsA2 was found to promote the activity and stability of two secreted virulence factors: listeriolysin O (LLO) and the broad range phospholipase PC-PLC. Culture supernatants from strains containing deletions of prsA2 exhibited reduced hemolytic activity as measured by the lysis of red blood cells in vitro, and this reduced activity appeared to be the result of altered stability and increased proteolytic cleavage of LLO. prsA2 mutants 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 vitro as well as within infected host cells. PrsA2’s role in bacterial virulence was found to be unique from the related L. monocytogenes chaperone PrsA1, a protein that shares a high degree of sequence similarity with PrsA2 but makes no discernable contribution to pathogenesis. Proteomic analysis of L. monocytogenes secreted proteins suggests that PrsA2 is required for the correct localization of additional bacterial gene products. Taken together, these data indicate that PrsA2 has been functionally adapted to promote the secretion and activity of multiple L. monocytogenes factors that play important roles in bacterial virulence.

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REFERENCE CtaP is a multifunctional cysteine-transport associated protein / required for Listeria monocytogenes pathogenesis B P Xayarath, B. 57 University of Illinois at Chicago, USA The bacterial pathogen Listeria monocytogenes survives under a myriad of conditions in the outside environment and also within the human host where infections can result in severe disease. Bacterial life within the host requires the expression of genes with roles in nutrient acquisition as well as the biosynthesis of bacterial products required to support intracellular growth. A gene product identified as the substrate-binding component of a novel oligopeptide transport system (encoded by lmo0135) was recently shown to be required for L. monocytogenes virulence. We report here that the gene product encoded by lmo0135 makes multiple contributions to L. monocytogenes growth and survival in environments both inside and outside of host cells. The lmo0135 gene product was required for bacterial growth in the presence of low concentrations of cysteine in vitro, suggesting lmo0135 and its associated transport system function in high affinity cysteine transport. We have therefore designated the lmo0135 gene products as CtaP for cysteine-transport associated protein. Interestingly, CtaP was not required for bacterial replication within the host cytosol, but was required for bacterial adhesion implicating a role for this protein in bacterial attachment to host cells. CtaP appears to function directly as an adhesin as latex beads coupled to CtaP were found to specifically bind epithelial cell monolayers in tissue culture assays. In addition, loss of CtaP increased membrane permeability and acid sensitivity, resulted in altered bacterial surface hydrophobicity, and severely attenuated virulence following both intragastric and intravenous inoculation of mice. Taken together, the data presented indicates that CtaP is part of a unique ABC transport system 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 pH REFERENCE B / P Forster, B. M.*, Pavinski Bitar, A., Slepkov, E. R. and Marquis, H. 58 Cornell University, USA The broad-range phospholipase C of Listeria monocytogenes, PC-PLC, contributes to escape from vacuoles. During infection, PC-PLC accumulates at the membrane cell wall interface until a decrease in vacuolar pH triggers its proteolytic maturation and release. PC-PLC maturation is mediated by the metalloprotease of Listeria (Mpl), which is produced as a zymogen and matures via intramolecular autocatalysis. We tested the hypothesis that Mpl activity is regulated by pH. Radiolabeled infected cells were perfused to manipulate host cell cytosolic pH, and secreted Mpl was immunoprecipitated. Mature Mpl was detected in samples treated at pH6.5, but not at pH7.3. To distinguish whether pH regulates autocatalysis or cell wall translocation of bacterium-associated Mpl, we inserted a Flag tag at the N-terminus of the pro or catalytic domain of Mpl, and used an antibody that recognizes only N-terminal Flag to detect Mpl. Results from fluorescence microscopy indicated that the zymogen is bacterium-associated at physiological pH, but not following acidification of the host cell cytosol. Mature Mpl was not detected bacterium-associated 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 occurs only at acidic pH. Together, these results indicated that Mpl enzymatic activity is regulated by pH, whether it undergoes autocatalysis or mediates the maturation of PC-PLC. Future studies will aim at assessing the possibility that specific amino acid residues act as pH sensors in the regulation of Mpl autocatalysis.

* Participation Supported by IUFoST.

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REFERENCE Identification of propeptide residues regulating the B / P compartmentalization, maturation, and activity of the broad-range 59 phospholipase C of Listeria monocytogenes Slepkov, E. R., Pavinski Bitar, A. and Marquis, H. Cornell University, USA The broad-range phospholipase C of Listeria monocytogenes, PC-PLC, is made as an inactive proenzyme whose activation is regulated by pH and by the metalloprotease of Listeria (Mpl). The propeptide of PC-PLC is comprised of 24 amino acid residues (C28-S51) and regulates PC-PLC compartmentalization and activity. In this study, we generated a series of propeptide mutants to determine the minimal requirement to prevent PC-PLC activity, and to identify amino acid residues regulating compartmentalization and maturation. To determine how many residues were required to prevent PC-PLC activity, nested deletions were generated in the PC-PLC propeptide of an Mpl-minus strain and phospholipase activity was assessed on egg yolk plates. We found that a single amino acid residue at position P1 (S51) of the cleavage site is sufficient to prevent activity, which is consistent with P1’ (W52) being located within the active site pocket. The requirements to retain the proform of PC-PLC bacterium-associated were assessed by immunoprecipitating secreted PC-PLC from radiolabeled infected cells maintained at physiological pH, whereas the influence of the propeptide on the ability of Mpl to mediate PC-PLC maturation was assessed by immunoprecipitating secreted PC- PLC from radiolabeled infected cells perfused with buffer at acidic pH. We observed that the triple mutant E31A Y32A L33A is translocated across the cell wall more effectively than wild-type at physiological pH, and that Y32A and L33A single point mutants are less effectively processed by Mpl at acidic pH. These results indicated that the N-terminus of the propeptide regulates PC-PLC compartmentalization and the ability of Mpl to process PC-PLC. Considering that Y32 and L33 are located 19 and 18 residues upstream of the propeptide cleavage site, we suggest that these two residues initiate the interaction with mature Mpl leading to processing of the propeptide at S51.

REFERENCE A mouse model of fetoplacental Listeria monocytogenes infection B / P and abortion 60 Poulsen, 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 results in abortion, stillbirth,or premature parturition. The latter carries with it a high risk for neonatal sepsis andmeningitis. Alterations in cellular and molecular components of the immune systemoccur during pregnancy to prevent rejection of the maternal allograft (i.e. fetus). How these pregnancy associated changes affect 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 abortion and fetal death. Intragastric infection of pregnant mice resulted insevere infection of the maternal and fetal tissues, in both C57BL/6J and A/J mousestrains. Use of a luciferase tagged L. monocytogenes strain and bioluminescent technology allowed us to visualize infection of maternal and fetal tissues and shedding of L. monocytogenes cells in feces and vaginal secretions of aborting mice. These data support the use of pregnant mice as a model for maternal and fetal L. monocytogenes infection. This model will prove useful in future investigations of the maternal and fetal immune response to listeriosis during pregnancy.

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Listeria infection of the insect model system Galleria mellonella REFERENCE B / P Joyce, S. A. and Gahan, C. G. APC and department of Microbiology UCC, Ireland 61 With the exception of mice, alternative model systems are constrained by an upper temperature 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. This limitation questions the relevance of these models for use with human pathogens where virulence gene expression is temperature dependent. Galleria mellonella, the Greater Wax Moth Larvae, is well documented as an infection model amenable to elevated temperature of 37°C. Here, we examine and establish Galleria mellonella as a relevant model for infection by Listeria species. Mutants attenuated for virulence in mice are similarly attenuated in this model system. We demonstrate that haemolysin production is the dominant factor in the pathogenic process of L. monocytogenes to G. mellonella. Real time visualization of infection reveals that the same sub-sets of virulence genes required for L. monocytogenes infection of both humans and mice are expressed (differentially) and required for insect infections. L. monocytogenes replication occurs in G. mellonella. On examining the host response we find that L. monocytogenes is mainly internalized within one hour of infection. In the presence of hemolysin, hemocyte viability decreased eight- fold and in the absence of PrfA a four-fold increase in viability was evident relative to WT strain. Bacterial load in hemocytes was significantly increased on infection with a mutant in hemolysin (DhlyA) production and a mutant in stress factor production (DsigB) compared to wt infection. We found that the Phenyloxidase (PO) system mounts a response to the presence of Listeria species within 4 hour of insect infection and that infection by L. monocytogenes results in the production of anti microbial peptides (AMPs). Taken together, we propose that G. mellonella is a relevant model for infection by L. monocytogenes at 37°C.

Construction of a murinised Listeria monocytogenes H7858 (4b) strain REFERENCE B / P for improved murine infection Cummins, J. and Gahan, C. 62 Science Foundation Ireland Listeria monocytogenes is Gram-positive food borne pathogen that is capable of causing listerosis culminating in various diseases in humans such as gastroenteritis, meningitis, encephalitis and spontaneous abortions. L. monocytogenes is capable of internalisation into non-phagocytic cells and crossing the intestinal, the placental and the blood-brain barriers. Internalisation into non-professional phagocytic cells is mediated in particular by the surface protein, InlA. InlA promotes listerial uptake into enterocytes by targeting the N-terminal domain of the human E-cadherin. However, this interaction is species specific with its interaction completely impaired in the rat and mouse models due to the absence of a proline at position 16 of the N-terminal E-cadherin repeat. A landmark result was the development of a transgenic mouse line expressing human E-cadherin by intestinal enterocytes. However, an alternative approach to overcome the lack of appropriate animal models has been the creation of a murinised strain of L. monocytogenes EGDe (Wollert et al., 2007 Cell. 129(5):891-902). By the substitution of two amino acids within the InlA protein we have created a murnised strain in the H7858 4b background. Our results have demonstrated that this mutant has an increased ability to infect mice by the oral route and does not increase invasion within human cells. The benefits of this approach will be discussed.

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REFERENCE The role of a phosphoinositide phosphatase in the intracellular survival B / P of Listeria monocytogenes 63 Wang, 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 recruiting actin 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 regulatory roles in orchestrating cell physiology. They control a broad range of processes including organelle identity, signal transduction and cell migration. The basis of this control is their relative state of phosphorylation that is controlled through the action of host kinases and phosphatases. The central role played by phosphoinositides inside cells make them a prime target for subversion by intracellular pathogens wishing to hijack the cell’s physiology. Recently we discovered that L. monocytogenes expresses a phosphoinositide phosphatase, capable of removing phosphate groups from a number of important phosphoinositides. Importantly we demonstrated that this enzyme was essential for the growth of L. monocytogenes inside infected cells and probably is important in escaping the phagosome. This is the first identification of a phosphoinositide phosphatase enzyme playing a key role in the intracellular survival of L. monocytogenes.

REFERENCE / Virulence gene expression in Listeria monocytogenes strains isolated B P from different sources 64 Alessandria, V. University of Turin, Italy Listeria monocytogenes is an important food-borne pathogen that has the capacity to cause severe infections. Ubiquitous microorganism, it is commonly isolated from foods of animal origin, mainly meat and milk products. However, due to its capacity to develop at refrigeration temperatures, human listeriosis outbreaks are most often associated with ready-to-eat food products that are consumed without prior cooking. The incidence of the disease depends on different factors including the infective dose and the immunity conditions of the host. The present work focuses on the expression analysis 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, SCOTT A), 7 isolated from food matrices (4 of which isolated from meat products and three from diary products) and 1 isolated from humans. In the first step, the expression analysis 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 different strains. The combined use of reverse transcription and quantitative PCR (qRT-PCR) was used to evaluate gene expression. As a second step, we wanted to evaluate the 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 differences emerged for the different genes, without showing any significant association between the expression of the genes and the origin of the different strains.

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REFERENCE Targeted Signature-tagged mutagenesis for phenotype screening / of Listeria monocytogenes mutants B P Henriques, A., Carvalho, F. and Cabanes, D. 65 IBMC, Portugal Listeria monocytogenes is a foodborne facultative intracellular human pathogen possessing a wide range of virulence factors tailored for pathogenesis. Common strategies used to identify new virulence factors are based on mutagenesis and in vivo phenotype analysis, which requires testing of generated mutants versus wild type strain in separate mice cohorts. To streamline this analysis, a targeted signature-tagged mutagenesis (T-STM) method was developed. Based on the concept of STM, where random mutants identifiable by specific tagging are negatively selected, we developed a method that enables specific gene targeting in L. monocytogenes genome for the creation of insertional mutants and their simultaneous phenotypic analysis in mice. Tagged “wild type” and mutants for prfA and two unknown LPXTG genes were constructed by inserting differentially tagged suicide vectors. T-STM uses defined tags that allow the discrimination between tagged- strains in a pool by PCR. The four tagged-strains were pooled in equivalent amounts into one single inoculum (Input) used for oral and intravenous infection of mice. Three days after infection, intestine, spleen, and liver of mice were collected, homogenised and plated on rich medium. Resultant bacterial growth (Out- put) was used as template for PCR screening and evaluation of the relative proportion of each tagged-strain. Mutants were considered less virulent when undetectable (or less detectable) by PCR in the output. This technique allowed us to clearly distinguish the severely attenuated prfA mutant, validating the method. PCR products for the two LPXTG protein-encoding mutants appeared in equal proportion to the wild type strain in the output, suggesting that these proteins are not involved in Listeria virulence in this infectious model. This study shows the feasibility and advantage of the developed T-STM method for simultaneous virulence analysis of pools of Listeria tagged mutants. This approach should be now used for larger-scale phenotypic analysis of specific L. monocytogenes mutants.

Listeria monocytogenes cellular infection triggers tyrosine- REFERENCE B / P phosphorylation of Myosin IIA, a new protein involved in invasion Almeida, M. T., Cabanes, D. and Sousa, S. 66 IBMC, Portugal Listeria monocytogenes is a human food borne pathogen that may lead, in particular in immunocompromised individuals, to a severe disease characterized by sep- ticemias, meningitis, meningo-encephalitis and abortions. The study of the cell biology of the Listeria infectious process provided insights in the way bacteria manipulate the host and revealed unsuspected functions of cellular proteins. To cause infection pathogens interfere with crucial host intracellular pathways, and different pathogens often hijack the same signaling pathways. In particular, host phosphorylation cascades are preferential targets of infecting bacteria. In this study, using L. monocytogenes as a pathogen model, we showed that eukaryotic cells present a variable protein phosphorylation pattern upon infection. We addressed in particular the tyrosine-phosphorylated protein profile triggered by Listeria infection and identified the motor protein, Myosin IIA (MyoIIA), as differentially tyrosine-phosphorylated in response to Listeria uptake. We demonstrated that My- oIIA is not only tyrosine-phosphorylated over the time of infection, but is also recruited with actin at the bacteria entry site. In addition, we were able to show that the inhibition of MyoIIA activity affected Listeria entry into non-phagocytic cells. The reduction of MyoIIA expression using RNAi techniques resulted in an increased Listeria uptake. Together these data point to the role of a novel myosin class in the internalization of Listeria, correlating for the first time, myosin post- translational modifications and Listeria infection.

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REFERENCE Invasion profile of Listeria monocytogenes strains involved B / P in invasive and gastroenteritis listeriosis outbreaks 67 Laksanalamai, P., Sahu, S. and Datta, A. Food and Drug Administration, USA Listeria monocytogenes (Lm), the causative agent of foodborne human listeriosis, is a serious public health concern. The disease is characterized by meningitis, septicemia, abortion and death in immuno-compromised population. Growing numbers of evidence have revealed that Lm can also cause self-limited febrile gastroenteritis in healthy individuals. To understand the mechanisms underlying two different disease outcomes several efforts have been made to differentiate gastroenteritis and invasive strains of Lm. Since the difference between these two types of outbreaks is Listerial ability to invade, in this study we have examined the expression of two internalin genes, inlA and inlB which encode virulence proteins required for the internalization process. The inlA and inlB gene expression profiles were determined by quantitative RT-PCR from several strains representing two groups of Lm that cause either invasive listeriosis or febrile gastroenteritis. Analysis of the inlA and inlB gene expression from food and patient isolates showed similar levels of expression from isolates originated from the same outbreaks. Surprisingly, the inlA and inlB gene expression from Lm isolated from invasive listeriosis outbreaks appears to have lower expression that those that caused gastroenteritis listeriosis. In contrast the expression of iap, a gene involved in host cell invasion, was higher in invasive strains. To understand the correlation between the inlA and inlB gene expression and invasion of Lm, we also investigated the efficiency of invasion of these strains by in vitro invasion assay using eukaryotic cell lines, Caco2 and HepG2. The results appeared to correlate with the inlA and inlB expression in that the invasion efficiency is higher in the gastroenteritis strains. Global gene expression profiles of these strains are currently being investigated using a DNA microarray.

REFERENCE Molecular characterization of the Vip-Gp96 interaction B / P 68 Martins, M., Cabanes, D. and Sousa, S. IBMC, Portugal The study of mechanisms exploited by Listeria monocytogenes to cause infection provided new insights in the way bacteria manipulate the host cell machinery. To subvert the host cell signalling pathways, Listeria uses a complex set of surface proteins called virulence factors that act in concert to allow bacterial infection and 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 of the Gp96 domain involved in the interaction with Vip. Using protein-protein interaction and invasion assays, we demonstrated that the region 22-411 amino acids of Gp96 (N-terminal) seems to be exposed outside of the cell thus available for Vip binding. This interaction is required for Listeria uptake into host cells. Al- though this entire region participates in the interaction, the domain comprising the 22-192 amino acids seemed to be the one crucial for the interaction. The existence of cell signalling events downstream Vip-Gp96 interaction was also investigated. Signal transducer and activator of transcription 3 (Stat3) activation during L. monocytogenes invasion was addressed, suggesting that Stat3 was not activated in response to Listeria entry. In addition, we demonstrated that Vip seems to have no role in Listeria phagocytosis by bone marrow-derived macrophages (BMMØ) and in bacterial survival within them.

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REFERENCE Investigation of the molecular mechanisms by which / Listeria monocytogenes grows in the mammalian gall bladder B P Dowd, G., Joyce, S., Casey, P. G., Hill, C. and Gahan, C. G. 69 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 demonstrated that the pathogen grows in the mammalian gall bladder and that gall bladder growth forms a significant phase of the infectious process. However, relatively little is known about the mechanisms that underpin the growth of the pathogen in this milieu. Here we have performed basic experiments which demonstrate that L. monocytogenes grows well in bile from the porcine gall bladder and can utilize bile as a source of energy and nutrients. We used bioluminescence-labeled L. monocytogenes to allow visualization of bacterial growth kinetics in porcine gall bladders infected ex vivo. We then performed random mutagenesis of L. monocytogenes and selected mutants that grow well under laboratory conditions but fail to grow in bile. Se- quencing of the genes mutated in this experiment demonstrated that the pathogen requires genetic loci that are involved in central metabolism for growth in bile, including genes encoding proteins required for amino acid biosynthesis and biotin metabolism. The majority of mutants that were unable to grow in bile were also attenuated for virulence in the mouse model of infection. Overall the work is the first to demonstrate the requirement for specific gene sets for the growth of L. monocytogenes in the specialized environment of the mammalian gall bladder.

REFERENCE Role of cadmium efflux system in Listeria monocytogenes virulence B / P Camejo, A. and Cabanes, D. 70 IBMC, Portugal Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. We found that the shift of the Listeria genome expression during infection is characterized by the activation of genes involved in virulence, stress, subversion of the host immune system, and by the adaptation of the bacterial metabolism to host conditions. Mutagenesis of genes highly induced in vivo allowed the identification of novel L. monocytogenes virulence factors, including cadC. Cadmium resistance in Listeria and other gram positive bacteria is an energy-dependent cadmium efflux system, involving two proteins, CadA and CadC. CadA has been shown to be a cadmium efflux P-type ATPase; cadC encodes a transcriptional regulator that is a member of the ArsR metalloregulatory proteins. The strong in vivo activation of cadC and the significant impaired virulence of the cadC mutant suggest that this heavy metal resistance system constitutes an advantage for in vivo Liste- ria survival. In addition, this system has never been previously implicated in the virulence of other pathogens. The detailed regulation of the CadAC system role as well as its role in cadmium resistance and in the Listeria infectious process will be presented and discussed.

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REFERENCE Investigation of chitinase as a potential virulence factor B / P in Listeria monocytogenes 71 Chaudhuri, S. University of Illinois in Chicago, USA Listeria monocytogenes is an environmental pathogen that causes disease in humans primarily following the consumption of contaminated food products. While considerable attention has been given to the identification of bacterial virulence factors specifically expressed within host cells, relatively little is known regarding gene products that may contribute to bacterial life in the outside environment while also potentially serving a role for virulence within the host. L. monocytogenes has been shown to produce and secrete two chitinases, ChiA and ChiB, as well 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 outside environment where chitin is plentiful. Recently however, chitinases and chitin binding proteins have been reported to contribute to the pathogenicity of two unrelated environmental bacterial pathogens, Legionella pneumophila and Vibrio cholerae. The chitinases produced by these bacteria appear to enhance bacterial colonization of mouse lung (L. pneumophila) and intestine (V. cholerae). To determine if chitinases or chitin binding proteins contribute to L. monocytogenes pathogenecity, in-frame deletion mutants were constructed in chiA, chiB, and lmo2467, and a triple gene deletion mutant was also constructed. All mutants were found to grow similarly to wild type L. monocytogenes in BHI broth culture and within infected tissue culture cells. However, following intravenous infection of mice, two of the 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 spleens of infected animals. As mammals do not produce chitin, these results suggest that L. monocytogenes has adapted its chitinases to exploit recognition of host substrates, potentially carbohydrate-linked molecules, to enhance bacterial survival within infected animals.

REFERENCE Sub-lethal concentrations of common disinfectants do not influence B / P survival and growth of Listeria monocytogenes in whole blood 72 Holch, A., Gaedt Kastbjerg, V. and Gram, L. Technical University of Denmark Listeria monocytogenes is a serious food borne bacterial pathogen that can colonize food processing environment. Virulence gene expression is influenced by several environmental factors e.g. temperature and oxygen-concentration. It has recently been shown that sub-lethal concentrations of disinfectants used in the food industry affect the expression of virulence genes in L. monocytogenes (Kastbjerg et al. 2009). The expression is either enhanced or reduced depending on the active compound in the disinfectant.The purpose of the study was to determine if these changes in virulence gene expression influence the virulence potential of L. monocytogenes in a more complex biological assay. We chose to study the ability of L. monocytogenes to grow and survive in whole blood as several of the virulence genes are important for survival and growth in blood. Two strains of L. monocytogenes (EGD and a maternofetal strain) where grown to exponential phase and exposed to two different disinfectants in a non-inhibitory and a sub-lethal concentration for one hour. The disinfectants contained either peroxide or quaternary ammonium compound (QAC) as their active compound. These cause enhanced or reduced virulence gene expression, respectively. The exposed bacteria were mixed with whole blood and the survival and growth was followed for 50 hours by plate counting. L. monocytogenes was able to grow in whole blood after exposure to the two different disinfectants in two different concentrations. However, pre-exposure to peroxide or QAC did not cause any difference in growth for both strains of L. monocytogenes as compared to exposure to water (control). In all experiments, the maternofetal strain grew slightly better than the EGD strain. Hence, the effect of sub-lethal concentrations of disinfectants on virulence gene expression did not affect the survival and growth in a complex biological model.

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REFERENCE Internalin LRR domain variability in Listeria monocytogenes isolated / from different hosts B P Zaytseva, E. A.1,2, Ermolaeva, S. A.2 and Somov, G. P.1 73 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 suggested to depend on several factors of L. monocytogenes pathogenicity. A number of surface and secreted proteins from internalin family, of which A and B internalin 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 family is presence of so called LLR domains involved in the direct interaction with eukaryotic receptors. The purpose of this research is to analyze distribution of various internalin gene alleles among L. monocytogenes isolated from different sources. Eighty-six L. monocytogenes cultures were used belonging to serovariants 1/2a, 1/2 b, 4b, isolated from various sources in the Far East and European part of Russia in the period from 1952 to 2005. Primers, gene restricting fragment and coding LRR domain were selected with the help of Oligo38 application. Gene presence in L. monocytogenes was determined with the help of PCR. DNA fragment sequencing was performed in Genom Center (Moscow). Molecular genetics features of 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 of LRR domains of genes inlA, inlB, inlC and inlE was determined for all the isolates. L. monocytogenes isolates demonstrated specific distribution of the examined gene alleles depending from isolation source. Among clinic isolates (group 1) low variability was recorded for inlA and inlC (p < 0.01). L. monocytogenes isolates obtained from wild rodent organs showed low variability with for inlB (p < 0.01). Specific alleles of inlA, inlC and inlE were found in Listeria cultures isolated from marine hydrobionts. Alleles of inlE were uniform in all the compared groups. In control group all gene alleles were distributed uniformly too. The obtained results confirm internalin role in L. monocytogenes interaction with a certain host. A microbe with specific gene allele may be more virulent and invasive for a certain type of host even at low infection doses.

REFERENCE Reduced virulence of an adenylosuccinate lyase transposon mutant / of a serotype 4b strain of Listeria monocytogenes B P Faith, N. G.1,2, Kim, J.-W.3, Kathariou, S.3, Sahaghian, R.1 and Luchansky, J. B.4 74 1. School of Veterinary Medicine, Univ. Wisconsin-Madison Madison, WI 2. Food Research Institute, Univ. Wisconsin-Madison Madison, WI 3. Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State Univ., Raleigh, NC 4. Eastern Regional Research Laboratory, USDA-ARS, Wyndmoor, PA A severe outbreak of listeriosis occurred in 1998–99 as a result of contamination of hot dogs with serotype 4b Listeria monocytogenes. We compared several characteristics of strain H7550, implicated in the 1998–99 outbreak of listeriosis, and a plasmid-free derivative (H7550cds) of that strain lacking the cadmium resistance plasmid 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 transposon mutant of strain H7550 that lacks adenylosuccinate lyase activity (strain J22F), 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 derivative H7550cds were equally virulent for mice. We observed no significant difference in the 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 on plastic surfaces within 24 hr in vitro. Strain J22F was better able to form biofilms in vitro than its parent strain H7550, whereas the LLO-negative mutant strain J29H was not significantly different from its parent strain H7550cds in biofilm formation. These results provide the first evidence that adenylosuccinate lyase activity is required for virulence of L. monocytogenes in mice. They also demonstrate that LLO is not required for biofilm formation in vitro.

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REFERENCE Manifestations and outcome of listeriosis in adult patients B / P 75 Fernández 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, Spain 2. Hospital Severo Ochoa, Madrid, Spain Infections caused by Listeria monocytogenes in adult patients are rarely reported despite the increasing prevalence of these infections in western countries. Hence, the manifestations, optimal treatment, prognosis and risk factors for mortality are not entirely known. We herein report a review of the most relevant clinical aspects, treatment and outcome of a large series of patients with listeriosis studied in two hospitals in Madrid, over a period of 15 years. Patient with isolation of L. monocytogenes 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) without differences in distribution by genders. Seventy percent of cases had comor- bidities: cirrhosis of the liver, hematologic neoplasms, immunosupressive disorders and corticosteroid therapy were the most common. Primary bacteremia (48%) and meningoencephalitis (37%) were the most common presentations. Ten patients (16%) presented with focal infections such as bacterial peritonitis (4), endocarditis (3) and brain abscesses (3). Ampicillin alone or in combination with gentamicin and cotrimoxazol were the antimicrobial treatments most frequently used. We did not find differences in the mortality rate among patients treated with monotherapy in comparison with those that received combined therapy. Cotri- moxazol was successfully used in 10 out of 13 patients (77%) treated. Twenty-three patients (37%) died within 30-days of diagnosis. Mortality increased according to the severity of the underlying disease and was significantly higher in immunocompromised patients (66%) than in those with chronic debilitating conditions (20%; p < 0.001). Human listeriosis is a severe disease producing high mortality particularly in immunocompromised patients with hematologic neoplasms and corticosteroid therapy. Despite synergistic activity of combined ampicillin/gentamicin therapy, the survival benefits of this combination remain theoretical. Cot- rimoxazol may be a useful alternative treatment for human listeriosis.

REFERENCE B / P Model for human Listeriosis: in vivo monitoring of orally infected mice using bioluminescent Listeria monocytogenes 76 Bergmann, S., Lengeling, A., Pasche, B. and Schughart, K. Helmholtz-Centre for Infectious Research, Germany The ubiquitous Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis. After ingestion of contaminated food, this pathogen is able to cross the intestinal, blood-brain and placental barrier and leads to gastroenteritis, meningitis and maternofetal infections which may result in abortion and spontaneous stillbirth. We have generated a bioluminescent Listeria monocytogenes strain which carries two amino acid substitutions in the bacterial invasion protein internalin A (InlAS192NY369S) and enables the bacterium to bind to the murine E- cadherin receptor with increased affinity as compared to wildtype Listeria. The genetic modification allows the bacteria to invade intestinal epithelial cells and to cross the murine intestinal barrier with high efficiency. The new bioluminescent Listeria strain was used to monitor bacterial dissemination in orally infected mice. For visualization of bioluminescent bacteria during infection we have used the Xenogen IVIS system, a highly sensitive, low light system optimized for in vivo imaging. 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 infection and show a time variation regarding bacterial spreading, the crisis of infection and bacterial clearance. We found CD1 and C3HeB/FeJ mice to be resistant to orally transmitted listeriosis, they showed reduced bacterial dissemination in the early phase of infection (during first 72 hrs), and a fast clearance of the pathogen from day 5 p.i. on. In contrast BALB/cByJ and C57BL/6J mice we found to be more susceptible after oral infection. This is reflected by slower bacterial clearance and a reduced survival. We further give an outlook how we will use this new approach to obtain a detailed insight in the process of crossing the feto-placental barrier in pregnant mice as well as the blood-brain barrier after oral infection with our modified Listeria monocytogenes strain.

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REFERENCE Galleria mellonella as model system to study Listeria species-specific / and Listeria monocytogenes serotype-specific pathogenesis B P Mraheil, M. A., Krishnendu, M., Hain, T. and Chakraborty, T. 77 Germany Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. This has generated interest in using insects as model organisms to study host-microbe interactions. We used the greater wax moth Galleria mellonella, which can be reared at 37°C, as a model host for examining virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and non-pathogenic Listeria and even between virulent and attenuated Listeria monocytogenes strains and seotypes. Virulence required listerial genes hitherto implicated in the mouse infection model, and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. We conclude that severity of septic infection of L. monocytogenes is primarily modulated by innate immune responses and suggest the use of Galleria as a relatively simple, non-mammalian model system that can be used to assess the virulence of strains of Listeria spp. isolated from a wide variety of settings from both the clinic and the environment.

Listeria monocytogenes ActA is a key player REFERENCE B / P in evading autophagic recognition Pillich, H., Loose, M., Hain, T. and Chakraborty, T. 78 Germany Autophagy is a pivotal bulk degradation system that eliminates undesirable molecules, damaged organelles, and misfolded protein aggregates in response to diverse stimuli, including infection. Autophagy acts to limit intracellular microbial growth but intracellular pathogens have evolved strategies to subvert host autophagic responses for their survival. We found that Listeria monocytogenes ActA, a surface protein required for actin polymerization and actin-based bacterial motility, plays a pivotal role in evading autophagy, but in a manner independent of bacterial motility. We show that L. monocytogenes exploits the biomimetic prop- erty of ActA to camouflage itself with host proteins comprised of Ena/VASP and the Arp2/3 complex, thereby escaping recognition by autophagy.

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REFERENCE Non-haemolytic and hypovirulent Listeria monocytogenes became B / P haemolytic and virulent after passage through mice 79 Secic, I., Lindbäck, T. and Rørvik, L. M. Norwegian School of Veterinary Science Two different isolates (from a fish processing plant and a broiler abattoir) showing typical appearance for L. monocytogenes on OCLA and ALOA, did not express haemolysis on blood agar with blood from different sources (sheep, horse, bovine, human) and appeared as L. innocua on RLM. However, biochemical reactions and 16S RNA analysis identified them as L. monocytogenes. High levels (109) of each isolate were injected i.p. into five mice. Three of the mice died after 1-3 days for both strains. Cultivation from livers and spleens revealed a mixture of haemolytic and non-haemolytic colonies. Non-haemolytic isolates showed typical appearance for L. monocytogenes on OCLA and ALOA, but were identified as L. innocua on RLM. Haemolytic and non-haemolytic isolates from each mouse, as well as the respective mother strains were identical pulsotypes. In contrast to passage through mice, 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 isolate had a seven base insertion in the prfA gene which introduced a stop codon, resulting in a truncated PrfA protein. The seven base pair insertion was deleted during infection in mice, resulting in a normal PrfA protein in the haemolytic version. This study shows that during passage through mice hypovirulent L. monocytogenes can change to a virulent phenotype. It is not known if this phenomenon may occur in humans. These strains would not have been recognized as L. monocytogenes un- less OCLA or ALOA had been used as isolation agar.

REFERENCE Mutants of Listeria monocytogenes (Lm) resistant to the polycationic B / P peptide protamine appear to be attenuated for virulence 80 Schlech, W. Dalhousie University, Canada Previously, we reported the isolation of Lm mutants resistant to protamine, a relatively 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-resistant mutants are attenuated. We also report that p60 mutants, isolated independently from the protamine-resistance phenotype and previously reported to show virulence defects, are also resistant to protamine. Protamine-resistant (PtmR) mutants from the Canadian Maritimes outbreak strain 15U and from the Massachu- setts outbreak strain Scott A, were picked as isolated colonies from TSA plates containing 1 mg/ml of protamine. Electron microscopy and SDS-PAGE were extensively applied to the initial characterization of the mutants. Swiss-Webster mice were infected by direct gastric inoculation with different Lm doses and spread to the spleen and liver monitored at 3 days after inoculation. Survival of Lm in diffusion chambers surgically implanted in the peritoneal cavity of rats for 3 days, was also evaluated. By electron microscopy and SDS-PAGE, the PtmR mutants displayed significant differences in their surface proteins and structure. However, both showed a reduced ability to infect mice (particularly the liver) and a reduced survival in the intraperitoneal diffusion chambers. p60 mutants, which are unable to efficiently infect cells in culture, turned out to also be PtmR. It seems that PtmR mutants, in spite of their different origin and surface characteristics, are attenuated for virulence. This possibly represents an example of an inverse fitness-virulence relationship that could be exploited to select against highly virulent Lm strains in food products treated with inhibitory concentrations of polycationic peptides.

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REFERENCE The role of plasmacytoid dendritic cells in the course of Listeria mono- / cytogenes infection B P Solodova, E., Lienenklaus, S., Jablonska, J. and Weiss, S. 81 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 of the immune system. There are more that 13 IFN-α and a single IFN-β, all using a common 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 clearance of the pathogen from the host. But the impact of these cytokines during bacterial infection is less defined. Plasmacytoid dendritic cells (pDCs) are known to be major natural type I IFN producing cells during viral infections, but not much is known about their impact in the course of bacterial infections. Listeria monocytogenes is one of the bacteria known to induce type I IFN synthesis. In contrast to viral infections these cytokines were shown to have detrimental effects for the host during infection with this bacterium. In our study we used tissue specific conditional reporter and knock-out mice showing that LysMcre-expressing cells but not pDCs are responsible for type I IFN production during Listeria monocytogenes infection. We have also shown that depletion of pDCs revealed a phenotype showing the ability of pDCs to protect mice infected with Listeria monocytogenes. The aim of my present work is to investigate this phenomenon und to understand how pDCs are able to provide protection to mice during Listeria monocytogenes infection.

Oxygen restriction increases the infection potential REFERENCE B / P of Listeria monocytogenes – a transcriptional analysis Andersen, J. B., Bergstrøm, A., Knudsen, G., Bak Christensen, B., Ebersbach, T., 182 Boye, M. and Rask Licht, T. Technical University of Denmark, National Food Institute, DTU, Division of Microbiology and Risk Assessment, Denmark Listeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease during the last two decades. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis. This is highly relevant for safety assessment of this organism in food. We have previously shown (Andersen et al., BMC Microbiology; 2007, 7:55) that the environmental conditions to which L. monocytogenes is exposed prior to ingestion are decisive for its in vivo infective potential in the gastrointestinal tract after passage of the gastric barrier. Infection of Caco-2 cells revealed that Listeria cultivated under oxygen-restricted conditions were approximately 100 fold more invasive than similar cultures grown without oxygen restriction. This means that not only the number of Listeria present in a given food item, but that also the physiological condition of these bacteria is important for food safety. The in vitro and in vivo data suggest that an oxygen-restricted L. monocytogenes cell represents a significantly higher risk than a cell grown without oxygen restriction. In order to identify transcriptional differences contributing to different invasiveness, microarray gene chip technology was applied to cDNA created from RNA isolated from oxygen restricted and non-restricted cultures. The analysis confirmed several relevant genes to be differentially transcribed in the two environmental conditions e.g. genes related to virulence potential of L. monocytogenes.

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REFERENCE Semi-automated repetitive sequenced-based PCR compared to pulsed C / P field gel electrophoresis for Listeria monocytogenes sub-typing 82 Roussel, 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, neonates and immuno-compromised adults. The commercially available, semi-automated rep-PCR assay system, DiversiLab, has been successfully used for subtyping several species of bacteria. We compared here the DiversiLab System with macrorestriction analysis by pulsed field gel electrophoresis (PFGE) which is currently the gold standard for molecular subtyping of Listeria monocytogenes. We used a panel of 116 human and food L. monocytogenes strains for the comparative evaluation. Among these strains, there were 4 pairs of duplicates; 13 strains were epidemiologically related and the remaining food isolates were epidemiologically unrelated. The strains of different serotypes revealed distinct ApaI-PFGE types, Rep-PCR types (RT) and AscI- PFGE types except for one RT and one AscI- PFGE type. The four doublets displayed a unique RT, AscI and ApaI PFGE pattern showing the good reproducibility of the three methods. The epidemiologically related strains were clustered in the same RT and PFGE types. The Simpson’s index of diversity was 0.954; 0.988; 0.994; 0.998 for DiversiLab, AscI-PFGE, ApaI-PFGE and AscI/ApaI-PFGE respectively. Thus, PFGE was more discriminating than Diver- siLab. However, for 1/2a serotype strains, six AscI-PFGE and three ApaI-PFGE types were divided into different RT. DiversiLab allowed a good discrimination between serotype 1/2a strains. This investigation also demonstrated the ability of the DiversiLab to differentiate serotypes 4b and 1/2b strains. DiversiLab is less labour-intensive than PFGE and provides results in less than 24 hours compared with 30 hours to 3 days for PFGE from the time a pure culture of the bacteria is obtained. Based on these results, DiversiLab may be useful for tracking the source of contamination in food processing facilities and their environments.

REFERENCE C / P Listeriosis: a frequent cause of fatal encephalitis in France with high case fatality 83 Mailles, A.1, Vaillant, V., Lecuit, M.2 and Stahl, J.-P.3 1. Institut de Veille Sanitaire, France 2. Institut Pasteur, France 3. University Hospital of Grenoble, France In 2007, we carried out a national prospective study in 106 hospital units to assess the aetiology, clinical patterns and outcome of infectious encephalitis in France. Among 253 case-patients, encephalitis due to L. monocytogenes was identified 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 of the CSF, and fever and decreased consciousness or seizures or altered mental status or focal neurological signs. All case-patients had a complete biological investigation to assess the etiologic diagnosis. Clinical data were collected using standardised questionnaires. Listeriosis case-patients were compared to other case-patients enrolled in the study. Thirteen case-patients were identified with encephalitis due to L. monocytogenes among 253 case-patients (5%) and 131 case- patients with an etiological diagnosis (10%). Listeriosis was the fourth most frequent 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 from 1 case. XX cases had a positive PCR on CSF. The last patient was diagnosed on a combination of clinical and epidemiological criteria. Eleven listeriosis case-patients were confirmed cases, 1 was a probable case and 1 was a possible case. Their mean age was 71 years and 9 were men. Listeriosis case-patients were more likely to have a current treated cancer (23% vs 5%, p=0.005) and to present with cranial 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). These results showed that L. monocytogenes is a frequent cause of encephalitis in France and confirmed its severity. Listeriosis should be considered early in the course of encephalitis, as specific and efficient treatment can be proposed.

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

Listeria monocytogenes: identification and subtyping REFERENCE C / P Favretti, M. Istituto Zooprofilattico Sperimentale delle Venezie, Italy 84 Listeria monocytogenes is a common environmental organism and an important food-borne pathogen too. Pregnant women, neonates, elderly and immunocompromised patients are at greatest risk of acquiring listeriosis. Since the recognition of Listeria monocytogenes as a food-borne pathogen there have been rapid advances in the development of suitable methods for isolation and identification. Although all Listeria monocytogenes strains are considered pathogen, only some serotypes are predominant and frequently involved in outbreaks. So it is very important to obtain viable isolates for typing, elucidating outbreaks and tracing the source of infection. The differentiation of Listeria monocytogenes isolates with phenotypic or molecular subtyping methods has provided useful information related 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 underline any relationship between them. Although there are several techniques for identification and typing available at the moment, it is not possible to identify the “gold standard” method yet. Therefore, using in parallel different methods ensures a correct and complete identification of strains. We present an overview of the techniques used to isolate, to identify and to subtype Listeria monocytogenes; the various subtyping systems provide different degrees of discrimination among isolates. 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 analysis REFERENCE C / P Amar, C. F.1, Tamburro, M.2, Dear, P.3 and Grant, K.1 85 1. Health Protection Agency, Centre for Infections, UK 2. University of Molise, Campobasso, Italy 3. 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 methods do not provide information on strain pathogenicity. Six key L. monocytogenes virulence genes, clustered together on pathogenicity island, LIPI-1 (9Kb), are known to be essential for intracellular survival, whilst two other virulence genes, internalin A and B, are responsible for host cell invasion. Many food isolates produce truncated internalin proteins due to point mutations and thus have reduced pathogenicity. Detection of these mutations and those in other virulence genes are likely to provide valuable information on strain pathogenicity. High Resolution Melt (HRM) analysis is a powerful technique capable of identifying sequence variations in PCR amplicons by accurately determining their melting temperature (Tm). This technique was used to generate virulence fingerprints, using 81 markers spanning LIP1 and Internalin A and B genes. Strains of L. monocytogenes from human cases, food and the environment were compared with results generated using L. monocytogenes EGDe. Considerable variations in Tm were found across markers for LIP1 and internalin A and B genes which were confirmed by sequencing. 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 spanning the hlyA gene being the most conserved. In the housekeeping gene, prs, marker variations were only detected in serotype 4b strains. Tm variations were detected in markers for actA, intA and intB genes in which point mutations are associated with the expression of truncated proteins and a concomitant reduction in virulence. HRM is an accurate and rapid technique for detecting sequence diversity within bacterial genes and not only presents a novel method for strain characterisation but also offers a unique potential to inform on strain pathogenicity.

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REFERENCE Risk factors for nonperinatal listeriosis mortality C / P in Los Angeles County, California, 1992–2004 86 Guevara, 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 health implications as it has a case-fatality rate of 20% and it causes 28% of all foodborne disease related deaths. Yet, data on the risk factors for listeriosis mortality have been limited. A 13-year retrospective cohort study was performed to describe nonperinatal listeriosis mortality in Los Angeles County, California, during 1992–2004. A nonperinatal listeriosis case was defined as a non-pregnant person >42 days old with a positive culture of Listeria monocytogenes and residence in Los Angeles County. Causal modeling based on prior knowledge and bivariable analyses was performed to determine a comprehensive unconditional multivariable logistic regression model with 29 main effect variables. With 281 nonperinatal 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 kidney disease (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 included blood transfusion, asthma, black race-ethnicity, Asian race-ethnicity, antibiotics, hypertension, chemotherapy, and Hispanic race-ethnicity. Cases admitted with sepsis only had the highest mortality (23.7%), while cases with meningitis only had the lowest mortality (3.13%). The findings of this study should highlight the importance of certain underlying risk factors and their association with nonperinatal listeriosis-related deaths. (This study was published in Clinical Infectious Diseases 2009; 48:1507-15 ©2009 by the Infectious Diseases Society of America. http://www.journals.uchicago.edu/loi/cid).

* Participation Supported by IUFoST.

REFERENCE Molecular typing of Listeria monocytogenes isolated from ovine sausage C / P 87 Nives, 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, Italy 2. Istituto Zooprofilattico Sperimentale of Apulia and Basilicata Italy Sardinia, an island located in the middle of Mediterranean sea, has about four million of milking sarda sheep. In recent years in addition to milk and cheese production other products from sheep meat have been commercialized (ham and sausage). In this survey, between 2007 – 2008, five sampling were performed inside a factory located in the northern side of Sardinia. We monitored the different stages of sausage production for detecting the presence of Listeria monocytogenes in food matrices, the equipment and the environment. Isolation and identification of L. monocytogenes were carried out according to ISO 11290-1/2:1996/98 amendment 1-2004. The isolates were characterized using serotyping and three different molecular methods: PFGE, MLST, VNTR according to previous published protocols. Moreover five virulence genes (actA, hlyA, plcB, prfA, iap) were detected by 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 desiccation time, sausage during different seasoning periods) and from the floor drain. On the whole 21 out of 126 samples resulted contaminated. The L. monocytogenes count 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 one batch samples whereas the type B (n=16), belonging to the serotype 1/2b, was isolated from three different food batches and from the floor drain. Our results demonstrate an high occurrence of L. monocytogenes in the studied facility. So the sheep sausage production must be object of a careful and constant vigilance activity and the isolates should be characterized using molecular methods for timely monitoring the contaminations and to play out the due prevention actions.

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REFERENCE A novel phage-PCR assay for the rapid detection of viable Listeria mono- / cytogenes in food within 24 h C P Elemam, M. M. 88 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 of particular concern in the food industry due to its widespread distribution in nature, its ability to survive in a wide range of environmental conditions, and its ability to grow at refrigeration temperatures. The standard microbiological method for L. monocytogenes detection in foods (ISO 11290-1/ A1 - 2004 and NHS-2009) is costly and time consuming; require at least 6 days obtaining a results. The development of a rapid and reliable test capable of detection very low numbers of the organism 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 detection of L. monocytogenes. We have been using the broad host range phage A511 to develop such a method for detection of Listeria monocytogenes in both cheese and stomached food samples. Successful development of the assay requires a viru- cide to achieve differential inactivation of the phage without affecting the viability of the target cell to be detected. Several different chemicals were evaluated as potential virucides, but tea extracts were found to be the most effective virucidal agent. The efficacy of the new assay was tested using cheese samples and was to shown to be able to detect low numbers of cells, in only 24 h in compared to the 6 days to conventional culture methods. Because of the broad host range of the phage the identity of the cell detected must be confirmed using PCR amplification of signature sequences from the phage plaque and several different PCR strategies have been evaluated. The combined phage-PCR method would allow rapid screening for food products prior to release from the factory.

REFERENCE Perinatal listeriosis in Los Angeles County, California, 1992–2004 C / P Guevara, R. E.* and Mascola, L. 89 County of Los Angeles Department of Public Health, USA In Los Angeles County (LAC), California, physicians and laboratories are required to report all listeriosis to the LAC Department of Public Health. From this passive surveillance, a population-based epidemiologic study was performed to further describe perinatal listeriosis. Perinatal listeriosis was defined when Listeria monocytogenes was identified from a normally sterile site between a mother-fetus or mother-infant (infant age < 43 days old) pair. During 1992–2004, of 413 listeriosis cases reported, 122 (30%) were perinatal listeriosis cases. Perinatal listeriosis had a higher average annual incidence rate (5.9 cases/100,000 live births/year) and mortality (32.2%) than nonperinatal listeriosis (2.5 cases/million people/year, and 18.6%, respectively). Disease onset during pregnancy defined intrapartum cases (n=93, 76%), during 0-6 days after birth defined early onset cases (n=21, 17%), and during 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 at admission of intrapartum cases was 28 weeks (range 10-40 weeks) with maternal symptoms commonly including fever (84%), abdominal pain (35%), chills (29%), back pain (24%), and headache (20%). Intrapartum listeriosis cases had maternal infections 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-negative or not cultured. Among intrapartum cases with hospital admission for listeriosis before 38 weeks gestation, 33 (35%) had fetuses continue into pregnancy for more than 7 days (6 fetal demises/stillbirths with median of 13.5 days after onset, 4 sick newborns with median 30.5 days after onset, 17 healthy newborns with median 25 days after onset, and 6 unknown birth outcomes). Although annual incidence of perinatal listeriosis has been decreasing, opportunities such as culturing symptomatic mothers for L. monocytogenes exist to further prevent perinatal listeriosis morbidity and mortality.

* Participation Supported by IUFoST.

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REFERENCE Antimicrobial resistance of Listeria monocytogenes human strains C / P isolated since 1926 in France 90 Morvan, 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 foodborne infection caused by Listeria monocytogenes (Lm). Lm is considered universally susceptible to antibioticsactive against gram-positive bacteria, but resistant strains have been reported. Lm is considered universally susceptible to antibiotics active against Gram-positive bacteria except 3rd generation cephalosporins, but resistant strains have been reported. We have studied retrospectively the antimicrobial resistance of Lm human strains isolated between 1926, the year of its discovery, and 2007. 4 816 human Lm strains were picked randomly in the strain collection of the WHO collaborating for Listeria. Their susceptibility to 23 clinically relevant antibiotics was determined by disc diffusion. MICs of fluoroquinolones and peni- cillins were determined by E-test. All resistant strains were typed by PFGE and those displaying a unique profile were screened for the presence of antibiotic resistance genes by PCR. The mechanisms leading to detected resistance was studied for each resistant strain. Sixty-one human strains (1.27%) were resistant to one or 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, respectively. Resistance to fluoroquinolones was attributed to active efflux. Resist- ance to the tetracyclines was encoded by tet(M), presumably carried by a conjugative transposon in the 14 strains which also harboured the int gene. We also detected the first human isolate with high-level resistance to trimethroprim, due to the dfrD gene. Although no penicillin-resistant strain was identified, MICs have significantly increased since 1926 with values up to 2 µg/mL. In conclusion, L. monocytogenes has not acquired significant clinically-relevant antibiotic resistance. Importantly, no resistance to amoxicillin and gentamicin, the recommended antibiotic regimen for listeriosis, was found. Yet, significant increase of penicillin MICs and the description of a strain resistant to trimethoprim reinforce the need for surveillance and are in favour of systematic antibiotic susceptibility testing including penicillin MICs determination.

REFERENCE C / P Today and tomorrow: The molecular epidemiology of listeriosis in the UK 91 Grant, 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 monocytogenes: in the UK it is the leading cause of death due to a foodborne pathogen. Since 2000 there has been an increase in the number of cases associated with patients aged > 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. In the UK, the number of pregnancy related cases of listeriosis has remained relatively 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 origin. Particularly concerning is that since the beginning of 2009, there has been an increase in the total number of pregnancy related cases. Because the incubation period for listeriosis is long and those at risk of infection are often ill with serious underlying conditions the investigation of individual cases and outbreaks is often difficult with an increased emphasis on microbiological evidence. Approximately 170 human and 900 food Listeria monocytogenes isolates from across England and Wales are received annually by the Laboratory of Gastrointestinal Pathogens. All isolates are identified and characterised using molecular methods including PCR– based serotyping and amplified fragment length polymorphism. This data is used in conjunction with standardised epidemiological data to confirm and link sporadic cases; to identify and investigate clusters and outbreaks; to provide information on routine food testing as well as for national surveillance purposes such as identifying risk exposures for human infection. This paper will demonstrate, with recent examples from the UK, the contribution of molecular typing to epidemiological investigations and will outline a new direction for typing Listeria monocytogenes which will not only be discriminatory but provide additional information on the relatedness of strains and their pathogenic potential.

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REFERENCE Use of Fluorescent Amplified Fragment Length Polymorphism / for improved typing of Listeria monocytogenes C P Matos, J., Amar, C., Desai, M., Cross, L. and Grant, K. 92 Health Protection Agency, UK Listeria monocytogenes is a ubiquitous, psychrotrophic, Gram positive foodborne pathogen which has the ability to persist in food processing environments and grow in a range of ready-to-eat foods. It causes the disease listeriosis which mainly affects those with a weakened immune system including pregnant women, neonates, the elderly and immunocompromised individuals. Although a rare disease it has a high mortality rate (20 –30%) and is currently responsible for more deaths than any other foodborne pathogen in the UK. Typing of L. monocytogenes strains is critical to investigating both sporadic cases and outbreaks enabling the identification of the food vehicle and tracing the origin of contamination. The success 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 epidemiological investigations involving L. monocytogenes. However, it is not without its drawbacks including being lengthy and labour intensive to perform, making it un- suitable for real time analysis. PFGE also suffers from a lack of interlaboratory reproducibility 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 clinical cases, foods and food processing environments using two restriction enzymes (HindIII and HhaI). All isolates were previously subtyped by multiplex PCR-based serotyping 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, reproducible and readily amenable to automation. Cluster analysis of isolates showed that fAFLP efficiently separated lineage I isolates from those of lineage II and that there was a high level of interlineage discrimination.

REFERENCE Towards an application for field samples of a multipathogen platform C / P for molecular detection of raw milk pathogens 93 Omiccioli, E1, Amagliani, G.2, Brandi, G.2, Tonucci, F.3, Foglini, M.3 and Magnani, M.2 1. Diatheva s.r.l., Italy 2. Department of Biomolecular Science, University of Urbino, Italy 3. 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, which commercialisation for human direct use has been encompassed by the 853/2004/EC. Since this product can constitute a risk for human health, the absence 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 identification of food contaminations caused by more than one microbial species. As part of a European Research project (DIAGNOSIS), we developed an enrichment multiplatform Real-Time PCR, including an internal amplification control ensuring the reliability of results, for the detection of all three pathogens. The assay combines an enrichment step in a medium properly formulated for the simultaneous growth of target pathogens (Multipathogen enrichment medium), a DNA isolation method, and a multiplex Real-Time PCR detection system based either on dual-labelled probes (MultipathogenFLUO), or on melting curve analysis (Multi- pathogenHRM). The entire system enables the detection of as few as 1 CFU of each pathogen in 125 ml milk in only two working days and it is now available from Diatheva srl, Fano, Italy. An application study for field samples is currently in progress. A local dairy farm, licensed to the direct selling of raw milk through automatic distributors, was selected for the collection of raw milk samples and swabs from filters located between milking rooms and storage tanks. Samples are analysed by the official reference ISO methods and also culture-enriched in the Multipathogen enrichment medium. Aliquots from all enriched cultures are subjected to DNA column-based extraction and PCR amplified by both the Multi- pathogenHRM and MultipathogenFLUO kits. The comparison of results will allow the estimation of method equivalence and the compliance of the molecular kits with law provisions, contributing to promote the adoption of PCR-based methods alongside the traditional diagnostic procedures.

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REFERENCE Detection and recovery of Listeria species from stainless steel C / P 94 Boone, R., Iugovaz, I, Trottier, Y.-L. and Pagotto, F. Health Canada The 2008 Canadian listeriosis outbreak in ready to eat meat raised many issues over the safety and reliability of the national food supply, including the reliability of detection methodologies aimed at food-contact surfaces made of stainless steel. Five methods pertaining to the recovery of Listeria species including L. monocytogenes from stainless steel were assessed. A cocktail of L. monocytogenes (serovars 1/2a, 1/2b, 4b), L. innocua, plus a Gram-positive organism (Staphylococcus aureus) was inoculated at different levels onto stainless steel. Inocula were recovered using MFLP-41 and assessed using different detection methods, including Petrifilm (3M), BAX Genus Listeria (Qualicon), BAX Listeria monocytogenes, and four VIDAS kits (BioMérieux) (LDUO, LIS, LMO2 and LSX). Direct plating onto selective and chromogenic agars were done simultaneously from each broth and each kit was compared to the “gold standard”, MFHPB-30. Experiments using skim milk 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 following 52 hours of incubation in LX Broth while the BAX Genus Listeria had 72 positive samples out of the 82 tested. The VIDAS strips DLMO and LMO2 yielded 52 and 51 positive samples out of 79 tested, respectively; whereas the BAX L. monocytogenes detected 50 of 81 samples tested. Petrifilm modifications to increase incubation time from 30 h to 48 h are being suggested due to 27 out of 84 samples were negative at 30 h but positive at 48. Differences amongst methods were noted at the single cell inoculum level. None of the methods had false positive results. Through the study of different rapid detection methods, options can be brought forward to improve and ensure optimal conditions for Listeria detection (and recovery) from stainless steel environments.

REFERENCE C / P Natural carriage of Listeria in fresh-water fish in Russia 95 Egorova, I.1, Voronin, M.2, Selyaninov, Y.1 and Kolbasov, D.1 1. National Institute of Veterinary Virology and Microbiology Pokrov Russia 2. National Park “Zavidovo”, Russia To study the extent of Listeria natural carrier stage in fresh-water fish, the Ivankovo water storage reservoir was observed. In the period of 2006 to 2008, 642 fish specimens belonging to 18 fresh-water fish species were examined. In total, 34 listerial 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. monocytogens were isolated predominantly from plant-feeder fish like bream, crucian carp and silver bream. L. innocua - in bream, pike, crucian carp, redeye and perch of all age groups. Listeria carriers were found most often in areas where animal farm buildings and treatment facilities were located. The primary Listeria locations in fish were muscle tissues, gill rakers and parenchymatous organs. In two cases 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 periods the detection rates were 28%. In spring the index dropped to 4%. All the cultures are quite typical specimens of their species. L. monocytogenes cultures produced hemolysins and phospholipases, were agglutinated with specific sera and lysed with phage L2A, and induced purulent conjunctivitis in guinea pigs. L. innocua cultures were hemo- and phospholipase-negative and were agglutinated with a serum of serogroup II, were lysed with phages L4A and did not induce mice death due to intraperitoneal inoculation at a dose of 109 m.b. The experimentally determined diversity of the of Sma I pulse electrotypes suggests there are both associated and independent Listeria reservoirs and there is a circulation of several clone variants of the pathogen in the fresh-water fish population.

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Prevalence of Listeria in wild fauna in Russia REFERENCE C / P Egorova, I.1, Fertikov, V.2 and Kolbasov, D.1 1. National Institute of Veterinary Virology and Microbiology Pokrov Russia 96 2. National Park “Zavidovo”, Russia Monitoring of Listeria in environmental objects and also among ungulate wildlife inhabiting forest hunting ranges of the Central region of Russia has been carried out. 662 samples of soil from feeding grounds, decaying plant residues, forages and wildlife faeces, and also brain, muscle tissue and/or parenchymal organs of the axis deer, the maral, the elk and the wild boar, were tested and 68 listerial cultures were found. The organism carriage indices for the axis deer, the maral and the wild boar in the areas of Tver, Moscow and Kaluga regions were at a similar level making up 2.0 to 2.7%. In Vladimir region the indices turned out to be a little higher gaining 12% for the wild boar and 5.45% for the axis deer, which is most likely due to some differences in the forms of economic activities and inadequate observance of sanitary-and-hygienic requirements. Examinations of elk faeces revealed Listeria in none of the cases which is probably explained by specificity of the elk nutrition. Examinations of environmental objects showed that all of them were to a variable extent contaminated by Listeria of both pathogenic and non- pathogenic species. Listeria were mainly isolated in soil samples taken from feeding grounds used for seasonal feeding of the wildlife. In samples from wild animals Listeria were found in two cases in axis deer brain and lymph node, and also in boar liver and lung. The isolate taken from the axis deer is of a certain research interest, as according to all the phenotypic indications it was classified to L. innocua, although investigation of its genetic structure showed presence of pathogenicity genes typical for pathogenic Listeria species in its genome. The remaining isolates of Listeria found in the course of the monitoring investigations belonged basically to two species, namely L. monocytogenes and L. innocua.

Quantitative assessment of the exposure to Listeria monocytogenes REFERENCE C / P from soft-ripened cheese consumption in North America: a joint FDA/Health Canada project 97 Gendel, 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, USA 2. Health Canada The United States and Canada continue to experience sporadic illnesses and outbreaks of listeriosis associated with the consumption of cheese, particularly soft and soft-ripened cheese. Therefore, the US Food and Drug Administration and Health Canada jointly conducted a risk assessment to assess the public health impact of Listeria monocytogenes in soft-ripened cheese in the US and Canada, and to evaluate the impact of current and potential mitigation and control strategies. To support this risk assessment, a probabilistic model was developed to assess changes in L. monocytogenes prevalence and levels from “farm to fork” in multiple scenarios. This model evaluated the impact of contamination of the milk used for cheese making, in-plant environmental contamination, partitioning, mixing, bacterial growth and bacterial inactivation though the complete product pathway. Model calculations and parameters for a base model were derived from data in the published literature and from specific databases from both countries. The general framework of the model was a second-order simulation that allowed separate evaluation of variability and data uncertainty. An original back-calculation was used to evaluate the frequency and level of environmental in-plant contamination based on published data on L. monocytogenes prevalence and levels in soft-ripened cheeses at retail in the US. This back-calculation model was integrated using the Monte-Carlo method. The results of the back calculation suggest that in-plant contamination is infrequent and generally leads to a low levels of L monocytogenes per cheese (<25 cfu in each whole 250g cheese). Because data on production levels and practices for the cheese making industry are limited, the model was used to compare exposure estimates obtained using the base model to estimates obtained using alternate scenarios. For example, scenario analysis suggested that, for contaminated cheese, the level of exposure is highly sensitive to storage conditions.

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REFERENCE Human listeriosis in England, 2001-2007: association with neighbour- C / P hood deprivation 98 Gillespie, 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 immunocompromised, and, to a lesser extent, pregnant women, unborn or newborn infants. Despite a high mortality rate, the socioeconomics of listeriosis has not been studied in detail, meaning that health inequalities which might exist in relation to this disease are not apparent. Laboratory surveillance data on cases of Lis- teria monocytogenes infection reported in England between 2001 and 2007 were linked to indices of deprivation and denominator data using patients’ postcode. Incidence relative to increasing quintiles of deprivation was calculated by fitting generalized linear models whilst controlling for population. Patient exposure data were scrutinised and compared with commercial food purchasing denominator data to further quantify the risk. For all patient groups, listeriosis incidence was highest 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 from local services (bakers, butchers, fishmongers and greengrocers) than the general population. Patients’ risk profile also changed with increasing neighbourhood deprivation. With increased life expectancy and rising food prices, food poverty could become an increasingly important driver for foodborne disease in the future. Whilst Government policy should continue to focus on small food businesses to ensure sufficient levels of food hygiene expertise, tailored and targeted food safety advice on the avoidance of listeriosis is required for all vulnerable groups. Failure to do so may enhance health inequality across socioeconomic groups.

REFERENCE Characterisation of antibodies for use in an antibody-based sensor for C / P on-site detection of L. monocytogenes 99 Gilmartin, 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 major causative agent of listeriosis, an infection which can kill vulnerable people such as 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 and very low recovery rates of the microorganism (due to the formation of biofilms). Antibody-based sensors permit the rapid and sensitive analysis of a range of pathogens and associated toxins. They offer many advantages such as improved sensitivity, real-time analysis and quantification. An anti-L. monocytogenes monoclonal 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 related Listeria spp. and non-Listeria spp. The mAb was further evaluated in both enzyme-linked-immunosorbent assay (ELISA) and fluorescence-linked- immunosorbent assay (FLISA)-based formats and in a surface plasmon resonance based 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 to monitor the contamination levels of L. monocytogenes in industrial food-producing plants. Preliminary work on the use of this antibody in the detection of L. monocytogenes in biofilms will also be presented.

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Food Investigation of sporadic cases of neuroinvasive listeriosis REFERENCE C / P Goulet, 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.3 100 1. Institut de Veille Sanitaire, France 2. National Reference Centre and WHO collaborating centre for Listeria, France 3. Direction générale de l’Alimentation, France Listeriosis in France is mandatory notified. For each case, a detailed food history is collected. For cases with central nervous system involvement who consent, samples are taken from foods and the environment in the kitchen of their residence or of hospital for patients hospitalized before disease onset. We present results of investigations 2003-2008. Samples are taken by officers of inspection services of the ministry of agriculture, within 14 days after diagnosis of the case, and sent to their authorised laboratories. Strains of Listeria monocytognes (Lm) isolated from these samples are sent to the National Reference Centre who performs PCR serotype, and PFGE (Apa1 and Sma1) typing of the food and patient isolates. Lm contaminated food is traced back with further investigations at the production sites. 140 cases have been investigated. Lm was isolated in 63 (45%) investigations, from cheeses, meat products, and the environment. For 23 investigations, the food or environmental strains had the undistinguishable PFGE profiles of the patient strain. At 2 occasions, the patient strains were isolated from the kitchen environment in their hospital. In 3 other investigations, the patient strain was also isolated at the production site of the contaminated food. At one occasion, the early investigation of a case later shown to be part of a cluster, allowed the prompt investigation in a cheese producing factory and the confirmation of the source of the outbreak. Food investigation of listeriosis cases are of interest because they can lead to control measures in contaminated production sites and limit the size of outbreaks of listeriosis by rapidly identifying their sources. However the yield of these investigations is limited because numerous unpacked food products in the patient’s refrigerators can not be easily identified. An important finding is that Lm strains can colonise the environment of hospital kitchens.

REFERENCE Evaluation of the antimicrobial activity of Vaccinium myrtillus, Prunus C / P domestica and Myrtus communis against Listeria monocytogenes 101 Serio, 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 food preservatives. They are considered consumer-friendly and can be used without any specific authorization or labelling change. In this study, three aqueous plant extracts were evaluated for their antimicrobial activity against 45 Listeria monocytogenes strains. Among blueberry, plum and myrtle extracts, the latter was the most effective against the strains isolated from smoked salmon and meat products, with MIC (Minimal Inhibitory Concentration) values generally ranging from 0.5 to 2.0%. Interesting results were also obtained with the plum extract, although with MIC values from 1 to 4%. Blueberry was effective only at high concentration levels, even above 10%. Isolates from the smoked salmon industry were generally less sensitive, suggesting that resistance to plant extracts may somehow be related to strain origin. A 3-factor-5-level Central Composite Design was used to evaluate the extracts activity in combination with compounds commonly present in some food products, where they can constitute a hurdle for bacterial growth. Myrtle extract activity was boosted by lactic acid and salt, while ATCC7644 strain, used as control, was particularly susceptible to all combinations. These results are in line with our previous research on volatile compounds, where L. monocytogenes was inhibited at low extract concentrations, without significantly affecting food fla- vor. Further studies will be carried out to evaluate the potential of myrtle extract in food biopreservation.

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REFERENCE High throughput quantitative detection of Listeria monocytogenes C / P in food by RealTime PCR 102 Cammà, 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 and can cause a severe disease in human. The Commission Regulation (EC) N° 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs allows the presence of 100 CFU/g in some ready-to-eat products. Conventional bacteriological 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 and automated 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 consists of two automated steps: DNA extraction, and set up of a fast RealTime PCR mix. The whole process could be completed within few hours minimizing the risk of carry-over contamination. To evaluate the inhibitor effect of food matrix on the PCR an internal amplification control was included in each reaction and coampli- fied using the same primer pair used for the target amplification. The specificity of the assay was confirmed on 3 L. monocytogenes strains and 26 other bacterial strains. The method was able to detect 2 genomic equivalents (GE) in 33/42 replicates (LOD= 4.8 GE/reaction with a probability of 95%) and the efficiency resulted between 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. The quantification range was linear over 6 log units with a square regression coefficient > 0.99. Application of the assay was assessed with 12 artificially contaminated samples of canned meat and with 8 real samples (smoked salmon, fresh fish and salami). The microbial load was tested in comparison with a pour plate counting method. The RealTime PCR was able to define an accurate quantification up to 103 CFU/g both in naturally and in artificially contaminated samples.

REFERENCE Bibliographic study concerning procedures for preparing environmental C / P samples for analyses, regarding to L. monocytogenes 103 Barre, L., Carpentier, B. and Gnanou Besse, N. AFSSA, France Listeria monocytogenes (L. monocytogenes) is a bacterium that can contaminate foods 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 isolated from humans, domestic animals, raw agricultural commodities, and food processing environments. Sanitation controls include effective environmental monitoring programs designed to identify and eliminate L. monocytogenes in and on surfaces 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 hygiene. This document is a bibliographic study concerning procedures for preparing environmental samples for analyses, regarding to L. monocytogenes. Different procedures for collecting environmental samples are described: several procedures for sampling methods are compared (cotton swab, sterile sponge, composite-ply tissues, sonication, direct contact agar…). We have also compared different detection and enumeration methods. We concluded that swabbing is known not to detach all micro. The removal efficiency depends on the swab material. Swabbing in moistened condition, as processed here, is more efficient than dry swabbing. Use of ultrasound is still considered as a good method for detaching microorganisms. However, ultrasounds can be lethal to bacteria, particularly when used at low frequency and are more detrimental to attached bacteria than to suspended ones. We concluded that it is important to adapt the type of sampling tools and techniques to the type of surfaces and sampling locations.

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REFERENCE Persistent L. monocytogenes isolates from Austrian and Irish dairies / show the same phenotypic and genetic background C P Stessl, B. 104 Veterinary University Vienna, Austria The food borne pathogen Listeria monocytogenes continues to be of major epidemiological interest to food industry and food sciences. The Zoonosis Report 2004 of EFSA (European Food Safety Authority) listed 1267 cases of listeriosis in Europe 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. This study is focused on the PFGE (Pulsed Field Gel Electrophoresis) and FTIR (Fourier Transform Infrared Spectroscopy) typing of L. monocytogenes strains isolated at distant sampling points (1996–2007) in the Austrian and Irish dairy chain. The aim of this research was to analyse if genetically indistinguishable (“persistent“) L. monocytogenes clones can be differentiated with respect to their phenotype (adaptation towards sanitizers and hygienic measures). A set of L. monocytogenes isolates from seven different European dairies was analysed with two epidemiological techniques: PFGE and FTIR. The PFGE method was performed according the CDC PulseNet Standardized Protocol for Molecular Subtyping of L. monocytogenes. Further data analysis was completed by Fingerprinting II Cluster Analysis using Dice coefficient and UPGMA logarithm (Biorad, Austria). The FTIR sample preparation and spectra analysis were performed as described by Ober- reuter et al. For data processing the program OPUS FT-IR (Bruker, Germany) was used. Cluster analysis was performed using Ward’s algorithm. The PFGE as well as the FTIR cluster analysis suggests the ability of short and long-time persistence of L. monocytogenes isolates in soft, semi-hard and hard cheese manufacturing. An explanation could be that the tested persistent L. monocytogenes isolates were not exposed to selection due to sanitation (colonization of ecological niches characterized by a low hygienic pressure). Additional studies will concentrate on the testing of a L. monocytogenes strain subset against their disinfectant susceptibility.

Epidemiological data on listeriosis in Portugal: 2003 – 2008 REFERENCE C / P Almeida, G, Magalhães, R., Hogg, T. and Teixeira, P. CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal 105 The invasive form of listeriosis although rare is a very serious disease with a high mortality rate. Young, Old, Pregnant, Immuno-compromised, sometimes referred as YOPI, are identified as at risk groups. However the infection may also occur in people with no known predisposing factors. In Portugal listeriosis is a not a notifiable disease and is likely to be under-reported. In 2003 an incidence of 1.4 cases per million inhabitants was reported. As in other European countries the number of cases has increased in the last years. The aim of this study is to present epidemiological on human cases of listeriosis occurred in Portugal between 2003 and 2008. It was possible to gather 107 cases: 16% were maternal/neonatal infections and 84% were non-maternal/neonatal infections. Concerning non-maternal/neonatal cases the sex ratio (M/F) was 1.73, the mean age of the patients was 62 years old with 46 cases (51%) in individuals aged 65 or more. The microorganism was mainly isolated from blood (59%), from CSF (32%) and from other specimen (9%). The incidence 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 active surveillance system of listeriosis was clearly demonstrated.

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REFERENCE Diversity among Listeria monocytogenes isolated from humans C / P Kalekar, S.1*, Rodrigues, J.1, D’Costa, D.1, Malik, S. V. S.2, Kalorey, D. R.3, Chakraborty, T.4 106 and Barbuddhe, S. B.1 1. ICAR Research Complex for Goa, India 2. Indian Veterinary Research Institute, Índia 3. Nagpur Veterinary College, India 4. Institute of Medical Microbiology, Justus Liebig University, Germany Listeria monocytogenes is a food borne pathogen associated with severe diseases in humans and animals. It is associated with neural, visceral and reproductive clinical entities, leading to septicaemia, abortions, stillbirth, meningitis and meningoencephalitis, especially in individuals at risk. We present the genotypic analysis of 17 L. monocytogenes isolates recovered from humans in India during 2006–2009 by multiplex serotyping PCR allowing serovar predictions, conventional serology and by PFGE. Multiplex-PCR serotyping assay revealed 88.24% (15/17) of the strains 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 by serology. Ten ApaI pulsotypes were recognized among the 17 human isolates. PFGE analysis allowed discrimination among isolates of the same serotype and among isolates from the same sampling areas or those isolated from different areas. Thus, PFGE together with multiplex-PCR serotyping allows rapid discrimination of L. monocytogenes strains. In addition, the predominance of L. monocytogenes serotype 4b is of concern, as this serotype has been most frequently associated with human listeriosis outbreaks.

* Participation Supported by IUFoST.

REFERENCE Characterization of Listeria isolated from seafood C / P 107 Rodrigues, 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 transmitted to humans through contaminated food. While several reports indicate that fish and fishery products can be frequently contaminated with L. monocytogenes, no major outbreaks associated with these products have been reported. Since fish and fishery products may be a vehicle for L. monocytogenes, it is important to have information on the incidence of this pathogen. The presence of L. monocytogenes has been documented in the tropical environment but the incidence in fish and fish products is low. We examined the prevalence, molecular characteristics and virulence potential of L. monocytogenes isolates from fishery products to gain further insights on the public health risk caused by this important food borne pathogen. A total of 221 raw seafood samples were examined for the presence of Listeria 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 species detected was L. innocua (33). The hlyA gene was detected in all the L. monocytogenes isolates. All the L. monocytogenes exhibited phosphatidyl inositol specific phospholipase C activity on ALOA agar. Multiplex PCR based serotyping revealed three L. monocytogenes isolates to be 1/2a, 1/2c, 3a, 3c group. One isolate belonged to 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 contaminate and persist in the processing environment.

* Participation Supported by IUFoST.

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Characterization of Listeria species isolated from milk REFERENCE C / P D’Costa, D.1*, Bhosle, S. N.2, Dhuri, R. B.3, Kalekar, S.1, Rodrigues, J.1, Doijad, S. P.1 and Barbuddhe, S. B.1 108 1. ICAR Research Complex for Goa, India 2. Department of Microbiology, Goa University, India 3. Goa State Cooperative Milk Producers’ Union Limited, Curti, Ponda, Goa, India Listeria monocytogenes is a foodborne pathogen commonly found in food and environment. This study aimed to determine the occurrence of L. monocytogenes in raw and market milk. Seven hundred sixty seven raw and market milk samples were collected at different levels of collection and processing. The samples taken included cow milk collected at udder level, from the milk cans, milk receiving centres, processing unit and market. Samples were enriched in Listeria enrichment broth 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 of these 37 (4.82%) were confirmed as L. monocytogenes. Other Listeria species isolated were L. innocua (5.47 %), L. ivanovii (0.13 %) and L. grayi (0.13%). Maximum isolates were recovered from samples collected from market followed by samples at milk processing unit. L. monocytogenes were serotyped using multiplex PCR method. A larger proportion of isolates (26) belonged to group corresponding to serovars 1/2a, 1/2c, 3a, and 3c. Serogroup corresponding to serovars 4b, 4d and 4e was detected in two strains while serogroup 1/2b, 3b, 4b, 4d, and 4e was detected in nine strains. . This study demonstrates the prevalence of L. monocytogenes in the raw and market milk and the need for good hygiene practices to prevent its entry into the food chain.

* Participation Supported by IUFoST.

REFERENCE Prevalence of Listeria monocytogenes in chicken production chain C / P in Thailand 109 Kanarat, S., Nijthavorn, N. and Sukhapesna, J. Veterinary Public Helath Laboratory, Bureau of Quality Control of Livestock Products, Department of 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 primary production stage to further processing plants. Samples were taken from 43 breeder farms, 32 hatcheries, 1331 broiler farms, 22 slaughterhouses and 22 further processing plants. Various types of samples were collected: fecal drops on the litter, drinking water, and chicken feed from breeder and broiler farms; swabs and mecomium from hatcheries; cloacal swabs and fresh chicken meat from slaughterhouses; and ready-to-eat chicken products from further processing plants. The study showed that there was no L. monocytogenes contamination in the chicken production chain except in slaughterhouses and further processing plants. This implies that the chickens did not carry the organism into slaughterhouses, and consequently primary production practices were not responsible for the the contamination of end products. This suggested that the observed L. monocytogenes contamination 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 of good hygienic and/or good manufacturing practices.

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REFERENCE Listeria monocytogenes in Ireland – Epidemiology and Molecular Typing C / P Cormican, M. G.1, DeLappe, N.2, McKeown, P.3 and Garvey, P.3 110 1. NUIGalway, Ireland 2. National Salmonella Reference Laboratory Ireland, Ireland 3. Health Protection Surveillance Centre, Ireland Since January 2004 listeriosis is a notifiable disease in Ireland. Clinicians and laboratory 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 years 2004 to 2007 11, 12, 7 and 21 (total =51) cases of listeriosis were reported to the HPSC. There were 37 cases categorized as adult or juvenile, 10 pregnancy-related and 4 neonatal. Cases were distributed through out Ireland with a slight seasonal peak in Summer in most years. For the years 2004 to 2008 the NSRL has received 4,4,1,12and14(totalof35) L. monocytogenes isolates from humans and 6, 34, 16, 18 and 12 (total of 86) from foods. Isolates from human cases are predominantly serotype 4b (n = 24). Isolates from food are predominantly serotype 1/2 (n= 74). Pulsed field gel electrophoresis (PFGE) performed by the Pulse Net method with ApaI and AscI enzymes and analysed with Bionumerics software ahs allowed the definition of 10 clusters of closely related isolates (2 to 4 isolates per cluster). The clusters link isolates from apparently sporadic human cases, including cases occurring in different years and link isolates from food items with human cases. As the clusters are small and it is not possible to perform typing in real-time epidemiological links between cases have not been identified. The number of cases of listeriosis in Ireland has increased in recent years. There is laboratory evidence linking individual cases and linking cases with food. Real time molecular typing of isolates is essential to identify linked cases in time frame that would allow timely epidemiological investigation and intervention.

REFERENCE Characterization of Listeria monocytogenes isolated from human cases C / P of listeriosis occurred in Portugal in 2008 111 Magalhães, 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 serious infections in humans. High-risk groups include the elderly, immunocompromised individuals, pregnant women and their neonates Clinical manifestations of invasive human listeriosis include meningitis, encephalitis, late-term spontaneous abortion, and septicaemia. The fatality rate is high: 20 to 30%. Foodborne transmission of L. monocytogenes can also cause a self-limiting acute febrile gastroenteritis in immunocompetent persons. Listeriosis is an infection of considerable public health concern. In 2008, 27 isolates of Listeria monocytogenes were recovered from Portuguese human cases of listeriosis and have been characterized by biotyping (cadmium and arsenic sensitivity), genoserotyping and typing by pulsed field gel electrophoresis (PFGE) using the enzymes AscI and ApaI. Strains were aggregated in three multiplex PCR serogroups: IVb (66.7%), IIb (25.9%) and IIa (7.4%). Concerning biotyping four groups were found: sensitive to arsenic and cadmium (51.9%), arsenic sensitive and cadmium resistant (14.8%), resistant to arsenic and sensitive to cadmium (29.6%) and resistant to both heavy metals (3.7%). Twenty two pulsotypes were indentified. The more prevalent pulsotype aggregates three isolates, of these, two were time and geographical related. Two other pulsotypes aggregate two time and geographical related isolates. It was shown that seven pulsotypes (32%) were also found in previous years.

* Participation Supported by IUFoST.

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REFERENCE Post-processing environmental contamination of surface-ripened / soft cheese during affinage C P D’Amico, D. and Donnelly, C. 112 University of Vermont, USA Environmental sampling conducted in a cheese aging room identified extensive contamination with Listeria monocytogenes. Further investigation revealed cross contamination of washed rind cheeses aging on various racks. To determine the extent of post-processing contamination of cheeses in this facility, two commercially available PCR assays were investigated as tools for detection of L. monocytogenes in naturally contaminated surface-ripened soft cheeses. Methods were culture confirmed on CHROMagar Listeria. For analysis, cheeses were composited and 10g sub-samples were assigned to an enrichment broth or Butterfield’s Phosphate buffer for enumeration. Cheeses identified as positive for L. monocytogenes by PCR and/or culture were subsequently enumerated. To achieve a detection limit of ≥5 cfu/g, composited samples were diluted 1:5 followed by direct plating of 1ml on CHROMagar Listeria. Contamination levels ranged from <5 to 1,600,000cfu/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 lower levels (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 cultural results, 10g samples of cheese contaminated at levels as low as <5cfu/g were identified following enrichments of 24 or 26 hours or after secondary enrichment for a total of 44-48 hours. Single enrichment procedures of 24 and 26 hours in Buffered Listeria Enrichment Broth and Oxoid 24LEB, respectively, followed by plating 100µl on CHROMagar Listeria identified approximately 90% of the true positive samples. Secondary enrichment in MOPS-BLEB increased the sensitivity to nearly 97%. PCR detection following single primary enrichment was less sensitive when compared to cultural techniques whereas dual enrichment PCR methods were comparable to their cultural counterparts.

REFERENCE Genetic diversity of Listeria monocytogenes isolated C / P from Portuguese cheeses 113 Almeida, 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 mortality rate. Cheese has been associated with several outbreaks of human listeriosis involving large numbers of consumers, with a wide economic and public health impact. To reduce the incidence of listeriosis it would be necessary to reduce the contamination level at production and at retail, or to inhibit the growth of Listeria monocytogenes in high-risk foods. Typing of bacterial isolates reveals the relationship among isolates. They can be used to determine the primary sources of bacterial contamination and to link people who are ill following the consumption of contaminated foods to the sources of bacterial contamination. Eighty isolates of L. monocytogenes recovered from cheeses (40 from cheeses purchased at retail, 23 from cheeses ready to go to the market, 16 from cheeses analysed at Autoridade de Segurança Alimenter e Económica laboratory and one isolate from cheese in quality control routine activities) were typed by multiplex PCR serogrouping and by PFGE 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 obtained with both restriction enzymes (AscI and ApaI) yielded a total of 26 pulsotypes. Eight of these pulsotypes were previously isolated from clinical cases of listeriosis in Portugal. This study highlights the possibility of cheeses being responsible for human cases of listeriosis in the country.

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REFERENCE Prevalence of Listeria spp. in retail raw ground beef in Izmir, Turkey: C / P A comparison of standard cultural method and Fluorescent 114 in situ Hybridization (FISH) technique for detection Handan Baysal, A.* Izmir Institute of Technology, Department of Food Engineering, Izmir, Turkey Fluorescent in situ hybridization (FISH) is based on the principle that hybridization of a nucleic acid sequence target of a microorganism with a specific DNA probe labeled with a fluorochrome and imaging by a fluorescence microscope. FISH method using a fluorescent labelled oligonucleotide probe designed from specific DNA and RNA sequences has proven to be a useful tool for the detection of a specific microorganism in environmental and clinical samples however the application of the method in food microbiology is investigated by the research studies. It usually involves four steps: sample fixation and permeabilization; hybridization of the target sequence and the fluorescent probe; stringency washes; and detection of the hybridized cells. In this study the possibility to use FISH method for detecting a food-borne pathogen Listeria spp. that used as food safety index in ground meat in market place of Izmir was evaluated. The work consists of two steps. In the first step specified pathogen bacterium was inoculated in ground beef and the applicability of FISH method was evaluated and in the second step the pathogen was detected in fresh ground beef by standard (conventional) cultural methods and FISH method. High correlation (r=0.96) was found between FISH and the conventional plate count method. The FISH protocol revealed to be specific for Listeria spp. detection, since all bacteria showed a positive hybridization signal. Results obtained from the direct application of the FISH technique to cultures show that it allows the detection of Listeria spp. in hours.

* Participation Supported by IUFoST.

REFERENCE C / P Using ListexP100™ for Listeria monocytogenes detection in foods 115 Flores 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, Portugal 2. IBB / Institute for Biotechnology and Bioengineering, Centre of Biological, Engineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal Food types susceptible of being contaminated with Listeria monocytogenes, such as cheese, smoked fish, salads, raw vegetables, sausages and, in general, ready-to-eat foods, pose a serious threat to consumers as this pathogen can cause severe diseases. L. monocytogenes can multiply even at refrigeration temperatures, with the minimum infective dose still remaining unknown. Development of efficient and rapid methods for the a priori detection of contamination by this microorganism in foods is of great significance and is required in order to ensure public health concerning consumption of foods that are considered to be of higher contamination risk. The vast majority of Listeria-specific detection methods, developed as alternatives to the standard selective plating methods, possess drawbacks such as a low specificity or efficiency (which is the case of antibody-based assays), or also detect dead cells (in the case of assays that detect genetic material, e.g. PCR, restriction-enzyme pattern-based assays). The main goal of the research work described herein was to investigate the possibility of using Listex™P100 (a commercial bacteriophage-based preparation for the control of Listeria contamination in foods, gently supplied by EBI Food Safety B.V.) for the design of a fast-response detection method for L. monocytogenes in foods. The procedure developed during our research effort encompassed a bacteriophage amplification assay based on the specificity and lytic activity of the broad-host range P100 listeriaphage with plaque formation as the assay end point. Aliquots of phage P100 were added to the test samples allowing all Listeria cells to be infected. After 15 minutes of incubation at 30ºC, to allow phage adsorption and eclipse phase with the nucleic acid delivered into the host cell, a virucidal agent was added in order to destroy all those phages that have not effectively infected a bacterial cell. Among several effective virucidal agents we selected 4.8 mM FeSO4 and 13 ppm of tannic acid and left standing for 5 min at room temperature. The virucidal activity was neutralized by the addition of 2% Tween-80 and the samples were then mixed with helper cells and spread over the surface of agar plates using a soft agar overlay. After 16h of incubation at 30ºC, the levels of Listeria contamination were determined by the presence of phage plaques. It was demonstrated that the method was able to detect the presence of bacteria, however only in samples containing high numbers of L. monocytogenes.

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REFERENCE Mapping of molecular profiles associated to Listeria monocytogenes / food isolates circulating in Italy C P De Cesare, A.1, Parisi, A.2, Latorre, L.2 and Manfreda, G.1 116 1. Alma Mater Studiorum – University of Bologna, Italy 2. Istituto Zooprofilattico Sperimentale della Puglia e Basilicata, Italy Listeria monocytogenes is a bacterial pathogen transmitted to humans from a variety of foods. Although most human listeriosis infections in Italy are being generally considered to represent sporadic cases, recent results suggest that a considerable number of listeriosis might occur in clusters, many of which could represent single-source outbreaks. To support tracing of food sources of human listeriosis infections, a long term mapping of serotypes, ribotypes and AFLP types associated to Listeria monocytogens isolates collected from different meat and cheese products as well as food production plant environments has been performed. The results achieved show that AFLP profiles of Listeria monocytogens isolates from the same food source cluster together with a similarity ≥ 84 %, whereas AFLP clusters grouping isolates from different sources show a similarity ranging between 60 and 78%. Isolates belonging to the same AFLP cluster show common ribotypes and serotypes. In particular, isolates from meat based products, such as fresh and fermented sausages, are mainly classified within EcoRI ribotypes 14 S2 and 202 S2 and serotypes 1/2c and 1/2b. Furthermore, cheese products are classified within the EcoRI ribotype 204 S5 and serotype 1/2a.

Zoonotic aspects of Listeria monocytogenes isolates REFERENCE C / P from zebu dairy animals Parihar, V. S. 1,3, Barbuddhe, S. B.1, Kalorey, D. R.2, Kotwal, S.4,Danielsson Tham, M.-L.3 117 and Tham, W.3 1. ICAR Research Complex for Goa, Ela, Old Goa, India 2.Department of Microbiology, Maharashtra Animal and Fishery Sciences University, Nagpur, India 3.Örebro University, Sweden 4.Sher-e- Kashmir University of Agricultural Sciences & Technology of Jammu, J&K Listeria monocytogenes is a non acid-fast, Gram-positive pathogen, which is considered food- and feed-borne. Knowledge of the direct and indirect transmission of L. monocytogenes between animals and humans, is however, limited. The aim of the current study was to characterize isolates of L. monocytogenes obtained from zebu dairy animals with mastitis. Fifteen isolates of L. monocytogenes from the same number of clinical and subclinical cases of mastitis were obtained from five farms harbouring altogether 90 zebu dairy animals. The isolates were characterized 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 isolates characterized belonged to serovar 4b and exhibited four virulence associated genes of L. monocytogenes. PFGE revealed that all isolates belonged to the the same pulsovar. One of the reasons that all isolates belonged to the same pulsovar despite being from different farms may be that certain types of L. monocytogenes are adapted to specific niches. The presence of an identical pulsovar could also indicate a common source of infection for the different farms. All the farms delivered milk to the same dairy. The same pulsovar of L. monocytogenes was isolated from zebu dairy animals with mastitis from five different farms.

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REFERENCE Performance of ALOA and Palcam agars for detection of Listeria C / P monocytogenes in naturally contaminated raw meat products 118 Gravato Rowlands, R. E.1, Asturiano Ristori, C., Geraldes Martins, C.1, Jakabi, M.1 and Gombossy de Melo Franco, B. D.2 1. IAL, Brazil 2.USP, Brazil The correct isolation and identification of Listeria monocytogenes in foods heavily contaminated with competing microorganisms may be difficult, especially when L. 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/green colonies, with pathogenic species producing a characteristic opaque halo after 24 h at 37°C. Non-Listeria spp. produce white colonies. The aim of this study was to evaluate the performance of ALOA for investigation of L. monocytogenes in 552 samples of naturally contaminated raw meat products, comparing the results to those obtained using the conventional Palcam agar. Meat samples were collected in supermarkets 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. Using the ISO 11290-1:1996/Amd.1:2004 detection method, L. monocytogenes was present in 234 (42.4%) samples. ALOA was more effective than Palcam agar for isolation of L. 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 two media simultaneously. When the ISO 11290-2:1998 enumeration method was used 95 samples (17.2%) were positive for L. monocytogenes, and among them, 31 samples (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, counts of 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 enumeration of L. monocytogenes increases the effectiveness of the two ISO methods when raw meat products are tested.

REFERENCE Comparison of MOPS-BLEB and Fraser as secondary enrichment C / P broths for Listeria monocytogenes 119 Upham, 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 from foods employ bipartite selective enrichment procedures to increase pathogen concentration prior to detection. The methods use the same bacteriological media for primary enrichment but different secondary enrichment media. Secondary media MOPS-Buffered Listeria Enrichment Broth (MOPS-BLEB) and Fraser broth were compared for their ability to facilitate growth of L. monocytogenes. Foods contaminated with Listeria were subjected to a common primary enrichment followed by parallel secondary enrichment in MOPS-BLEB and Fraser broth. Listeria was isolated and enumerated by plating the broths on selective agar. Of 164 food samples, presumptive L.monocytogenes colonies were isolated from 164 using MOPS-BLEB and 162 using Fraser broth, demonstrating broth equivalence (p = 0.50, Chi- square). Quantitative analysis revealed significantly higher levels of L.monocytogenes in MOPS-BLEB than Fraser broth (log10 CFU, 8.88 and 7.99, p < 0.0001 by paired t test). Interestingly, for foods inoculated with a mixture of L.monocytogenes and a non-pathogenic species of Listeria, L.innocua, concentrations of the later 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.monocytogenes to L.innocua in MOPS-BLEB. Enrichment broths used by CFIA were found to be equivalent for enabling cultural detection of L. monocytogenes, however, MOPS- BLEB produced significantly higher pathogen concentrations and pathogen to competitor ratios and thus has potential to enable earlier detection and facilitate detection when competitors are present.

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REFERENCE Comparison of UVM, Palcam and Oxoid Novel Enrichment (ONE) broth / as primary enrichment broths for Listeria monocytogenes C P Upham, J., Mosher, M., Borza, A., Huszczynski, G., Dorey, M., Eloranta, K. and Douey, D. 120 Canadian Food Inspection Agency The purpose of this study was to compare three primary enrichment broths for ability 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 and incubated for up to 48 hr, and growth curves were constructed by diluting and plating the enrichment cultures at regular time intervals to determine cell concentration (cfu/ml). Growth curves were also prepared from media that had been used to enrich various food products (deli meats, dairy products, fresh produce and seafood) that were artificially contaminated with L. monocytogenes alone, or L. monocytogenes in the presence of the background organisms Escherichia coli and Staphylococcus epidermidis, or in the presence of a non-pathogenic Listeria species, L. innocua. In the absence of food matrices or competitive microorganisms, the lag phase 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 growth rate was significantly greater in Palcam broth (0.371) and ONE broth (0.396) compared to UVM broth (0.277). Similar differences in growth rates were observed when broths were used to enrich L. monocytogenes-contaminated food products. When food were contaminated with a equal mixture of L. monocytogenes and L. innocua, the exponential growth rate of L. innocua was notably faster than L. monocytogenes 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. monocytogenes growth rate in Palcam broth and to a lesser extent in UVM broth, but not in ONE broth. These studies suggest that ONE broth may be preferable to UVM and Palcam broths to facilitate detection of L. monocytogenes when L. innocua is present.

Isolation and characterization of Listeria monocytogenes from asazuke REFERENCE C / P (Japanese light pickles) Maklon, K., Kusumoto, A., Makino, S.-I. and Kawamoto, K. 121 Obihiro Univ. Agri. Vet. Med., Japan Asazuke is a ready-to-eat Japanese light pickle, mainly made of vegetables which are 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 contamination has not been evaluated yet. In this study, we investigated the prevalence of L. monocytogenes, Salmonella spp., Escherichia coli O157 and coliforms in 108 asazuke samples 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 by 1/2a (2 isolates), 1/2b, 3b and 4c (1 isolate each) while Salmonella spp., E. coli O157 and coliforms were not detected. All L. monocytogenes isolates demonstrated hemolytic 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 revealed their potential pathogenicity. Moreover, 7 out of 12 isolates were from asazuke produced by one factory and pulsed-field gel electrophoresis (PFGE) profiles suggested that 6 of them were indistinguishable. Additional investigation of L. monocytogenes contamination was performed in the asazuke factory environment and 23 out of 60 swabs (38.33%) were isolated. Comparison of PFGE profiles showed relatedness between food and environmental isolates. Taken together, our results indicated that asazuke was contaminated with L. monocytogenes, which was probably from vegetables, human sources or from production lines. Further analysis to clarify the source of pathogen contamination is needed to establish a prevention method and consumers should be aware of asazuke consumption that might be contaminated with L. monocytogenes.

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REFERENCE Grouping of human Listeria monocytogenes isolates C / P 122 Lopez-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 restriction enzyme used was AscI. The serovar 4b isolates were divided into 27 PFGE types, the serovar 1/2a and 1/2c isolates into 108, and the serovar 1/2b and 3b isolates into 24. PFGE types with identical profiles of small bands (< 145 kbp) were grouped together with PFGE designations of A-Z. This grouping was based on the assumption 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 108 serovar 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. monocytogenes isolates was analyzed with multiple-locus variable-number tandem-repeat analysis and the clusters were generally in good agreement with both the clusters generated by PFGE and serotyping data. The advantage of dividing L. monocytogenes PFGE types into groups is that the profile of every new isolate characterised with PFGE can easily and quickly be identified by studying the group affiliation of the isolate and then identifying the matching type within the group.

REFERENCE C / P Characterization of Listeria monocytogenes strains isolated from food and environmental samples 123 Nucera, 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, Italy 2.Istituto Zooprofilattico del Piemonte Liguria e Valle d’Aosta, Italy This study aimed to investigate type diversity and distribution over time and across sources by subtyping via Pulsed Field Gel Electrophoresis (PFGE) a set of 300 L. monocytogenes strains collected over a five year period. Five clinical human strains isolated in the same geographic area and period of the study were also included in the analysis. The isolated strains belonged to serotype 1/2a (45% of the samples), 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.= 2 2 13.9; χ p < 0.05) and 1/2c with meat (O.R.= 33.3; χ p < 0.05). PFGE results highlighted 152 pulsotypes shared among two or more samples: 9 pulsotypes were recurrent and 6 were shared between strains isolated from different food and environmental sources. The other generated pulsotypes were source specific and/or retrieved 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 shared between strains collected from food/environmental isolates and human clinical strains. The results of this research underline and confirm the importance of im- plementing a broad typing database which could facilitate epidemiological investigations and enhance novel food attribution modelling approaches for the identification of listeriosis outbreaks and the related sources.These data, even if focused on strains collected in a limited geographic area, may pose the ground to evidence how large subtype databases may aid in the detection of common- and source specific- types. However, more comprehensive databases, both at national and at Eu- ropean level, are needed to reveal L. monocytogenes strains diversity and to provide useful data for epidemiological investigations.

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REFERENCE Development of a multiplex SNP-typing method to identify the four / epidemic clones of Listeria monocytogenes C P Lomonaco, S.1, Civera, T.1, Dalmasso, A.1, Knabel, S. J.2 and Bottero, M. T.1 124 1. Università degli Studi di Torino, Italy 2.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). The current classification of ECs is based on results from different subtyping techniques such as RFLP analysis, hybridization assays, PFGE and ribotyping. Recently, a multiplex PCR able to identify ECI, ECII and ECIII was developed. In addition, sequence-based methods, such as multivirulence-locus sequence typing, were able to further subtype EC strains and also to highlight potentially informative single nucleotide polymorphisms (SNPs). The aim of the present study was to develop an alternative method for the identification of all four ECs by developing a SNP-typing method based on minisequencing. This technique simultaneously analyses several SNPs based on a Primer-Extension Reaction (PER). Extension primers are designed immediately adjacent to the SNPs and extended by a single [F]ddNTP. Results are then visualized with a genetic analyzer as specific patterns. Six SNPs, yielding a specific pattern for each EC, were selected. These SNPs were located on 4 virulence genes (inlA, inlJ, inlB and srtA). Gene fragments were preliminary amplified with a multiplex PCR and then used as template for the PER reaction. Analyzed samples were 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 previously classified as ECs showed the same profile as ECI, ECII and ECIV. These findings were confirmed with other biomolecular tests (e.g. ECs-specific PCR) and by considering available epidemiological data for these strains. The proposed SNP-typing assay is potentially high-throughput and amenable to automation, allowing a rapid identification of all four ECs and can therefore be used as a screening method in the analysis of L. monocytogenes strains.

Listeria monocytogenes incidents reported to the UK Food Standards REFERENCE C / P Agency from 2000 to 2008 Aish, J. 125 Food Standards Agency, UK The UK Food Standards Agency contributes to the management of a wide range of food related incidents involving microbiological, chemical, radiological and physical hazards. One important foodborne microbiological hazard for which the Agency oversees incident investigations is Listeria monocytogenes. When incidents are reported to the Agency the relevant data is recorded on the Agency’s Incidents Database. 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 incidents involving Listeria spp. that were reported to the Agency between 2000 and 2008 and to identify that main food categories that were involved. During the nine year period from 2000 to 2008 approximately 140 incidents caused by the contamination of food with Listeria spp. were reported to the Agency, the majority of which involved L. monocytogenes. Most of the incidents involved ready-to-eat meat/meat products, cheese, fish/shellfish and sandwiches/sandwich fillings. L. monocytogenes is an important foodborne pathogen. Although healthy people are not usually at risk of illness, listeriosis can be an issue for vulnerable groups such as pregnant women and the immunocompromised. In addition to overseeing incident investigations the Agency’s 4C’s strategy (cleaning, cooking, chilling and avoiding cross-contamination) is important in reducing the risk from L. monocytogenes. The Agency also provides food safety advice to vulnerable groups and commissions research to further our understanding of this organism.

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REFERENCE Genetic diversity of Listeria monocytogenes in broiler flocks C / P 183 Courtillon, C. Toquin, M.-T., Le Nôtre, Y., Fravalo, P. and Mansour Chemaly, M. AFSSA, France Listeria monocytogenes is a foodborne pathogen, persistent in industrial environment and is responsible of listeriosis, an infectious and rare disease. The aim of this study was to update and create data set from broiler flocks regarding their contamination by Listeria monocytogenes. 145 flocks have been sampled, 85 conventional and 60 free range flocks. Molecular characterization by PFGE with two restriction enzymes ApaI and AscI was performed in order to establish the clonal relationships of the isolates and to trace the contamination between the flocks. A total of 126 isolates were genotyped. The prevalence in broilers has been estimated at 31.7% (46 positive samples on 145). The serotyping revealed the presence of serotype 1/2a in majority: 50% vs. 31.7% for serogroup 4 (4e and 4b) and 18.3% for 1/2b. Serotype 1/2a is the most present in conventional farms (37.3%) contrary to free range farms (12.7%) where serogroup 4 is dominant (23% vs. 8.7 %). PFGE typing showed a high discriminatory index: 0.971 for both combined enzymes. 47 genetic patterns were identified. Dendrograms, obtained with BioNumerics software, confirmed the high genetic variability between isolates (55.7% of similarity) especially in serotype 1/2a (65.5%). This index (0.954) was similar to the one obtained when studying laying hen flocks. The serogroup 4 and the serotype 1/2b seemed more clonal, 0.892 and 0.798, respectively. Regarding the production system, results between conventional and free range farms are comparable, 0.959 and 0.948, respectively. The PFGE showed that two identical profiles can be found in different flocks and different geographical locations while in a same flock, patterns can be very different. The breeding type is not linked to bacteria genetic identity as are serotypes. In conclusion, this investigation brought new and updated data regarding Listeria monocytogenes in broiler flocks which were not available before.

REFERENCE Tracing Listeria monocytogenes contaminations throughout C / P the processing chain of a typical Italian pork meat product 184 using Pulsed Field Gel Electrophoresis (PFGE) characterization Annunziata 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 performed on 124 Listeria monocytogenes strains isolated from a prevalence study throughout the Parma dry-cured ham production, in order to evaluate contamination patterns. 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 assured for all carcasses, so every strain was tested to evaluate the presence of contaminations in previous or next phases of the production process. Genetic relationships of 56 different pulsotypes obtained from the combination of AscI and ApaI macrorestriction patterns were graphically represented and analyzed. Six different clusters were identified. The analysis of PFGE patterns obtained from the same sample examined at the three different points of the production process (carcass – fresh ham – cured ham) did not produce identical or highly similar pulsotypes. The only pulsotype occurring in a strain from faeces was not recovered from other materials. Strains from the same production plants were usually genetically homogeneous or highly similar. In the plants where there was not genetic homogeneity, one pulsotype or cluster was always sharply prevalent over the others. The presence of single or prevalent pulsotypes in the same processing plants could be associated to persistent strains of Listeria monocytogenes. This is corroborated by the highest prevalence of 1/2c serotype strains, which adhere significantly more to working surfaces, isolated from all stages of the process. Genetically homogeneous or highly similar strains were also recovered in different establishments, at different phases of the production process. This situation could be bounded to previous sporadic contaminations between different production stages, which could have allowed the localization of strains into favorable niches inside the processing environment.

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

REFERENCE Production and validation of capture-ELISA kit based on monoclonal / antibodies specific for Listeria monocytogenes in foodstuffs C P Portanti, O., Di Febo, T., Luciani, M., Pompilii, C., Armillotta, G., Principe, V., 185 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 of foods. Validation was carried out according to the criteria for qualitative analysis set out in ISO 16140:2003. The ELISA kit has satisfied the norm requirements in terms of relative accuracy, relative sensitivity, relative specificity, relative detection level, inclusivity and exclusivity. The kit is a capture-ELISA test applied to the sample enrichment broth, based both on a monoclonal antibody (9B8F7) to L. monocytogenes and a peroxidase-conjugated anti-Listeria monoclonal antibody (6F12C8). The method has been validated in meat, fish and dairy products by means of incurred and artificially contaminated samples with target and non target strains. Results for relative accuracy, relative sensitivity, relative specificity and relative detection levels are in agreement with ISO 11290-1:1996 requirements. Capture-ELISA test is able to detect Listeria with 100% inclusivity and exclusivity in 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 analytical methods. It also shows the same features as the internationally recognised ISO 11290 techniques, as required by UNI EN ISO 17025:2005.

Production of a reference material for microbiological tests REFERENCE C / P containing Listeria innocua Pomilio, F., Ricci, L, Di Giannatale, E., Semprini, P., Candeloro, L. and Migliorati, G. 186 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy The study describes the production of a reference material constituted by glass balls, covered by skimmed milk powder micronized and artificially contaminated with Listeria innocua (ATCC 33090 strain). The lot of prepared material, constituted by 1g unities of balls, has been made in test-tubes, submitted to a stability and homogeneity evaluation through the determination of the mesophilic bacterial count and following Listeria innocua identification. The material stability, kept at –20°C, has been evaluated at 54, 180 and 355 days. Data pointed out the decreasing tendency of the bacterial count, statistically significant, at 54 days. At 180 and 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 period the decrease has been equal to 8.75 UFC/g. Samples examined in the same time lapses resulted homogeneous in the interval between 180 and 355 days. Only a sample out of the 105 examined resulted negative. After 355 days of preservation 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-parametric tests, for independent samples resulted stable at -20°C and +4°C. Furthermore, for the reference value definition, a proficiency test has been organized, which allowed to assign the value of 3.2 UFC/g to the material. In the same study, 35 samples out of 244 (15.4%) resulted negative. Used methodology could be applied to reference materials production also on other micro-organisms. The adopted technology has the benefit of operating in “closed” systems, making bio-safety of operators and environment easily manageable.

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

REFERENCE Colonization of a newly constructed commercial chicken further D / P processing plant with Listeria monocytogenes 126 Berrang, M. E.1, Meinersmann, R.1, Frank, J.2 and Ladely, S.1 1. USDA-Agricultural Research Service, USA 2. University of Geórgia, USA This study was undertaken to determine potential sources of Listeria monocytogenes in a newly constructed chicken further processing plant and document the eventual colonization of the facility by this pathogen. To ascertain the colonization status of the plant, floor drains were sampled after a production shift and again after a clean up shift on a roughly monthly basis for 21 months. Potential sources of L. monocytogenes to the plant included: incoming raw meat, incoming fresh air and 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 processing operations. Within four months, several subtypes of L. monocytogenes were detected in floor drains both before and after cleaning and sanitizing operations. No L. monocytogenes was detected on filters for incoming air, samples associated with plant employees, or a nearby discount shopping center. One subtype of L. monocytogenes was detected in a natural stream near the plant however, this subtype was never detected inside the plant. Eight subtypes of L. monocytogenes were detected in raw meat staged for further processing; one of the raw meat subtypes was indistinguishable from a persistent drain subtype recovered after cleaning on eight occasions in four different drains. Poultry further processing plants are likely to become colonized with L. monocytogenes; raw product is an important source of the organism to the plant.

REFERENCE D / P Episcopic differential interference contrast/epifluorescence microscopy to characterise in situ Listeria monocytogenes biofilms 127 on stainless steel surfaces Gião, M. S. and Keevil, C. W. University of Southampton, UK Listeria monocytogenes is a Gram-positive pathogen associated with the contamination of several food products, including meat, dairy products and vegetables. Al- though this pathogen can be found in natural environments the contamination of processed food can occur after contact with contaminated surfaces. Previous studies routinely focused on biofilm recovery and culture quantification. In situ studies 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 ideally suited to study biofilms in a native state. The aim of this work was to utilise the advances in EDIC/EF microscopy to study the formation of L. monocytogenes biofilms under different nutrient and temperature conditions. L. monocytogenes NCTC 13372 was suspended in Brain Heart Infusion (BHI), Tryptone Soya Broth (TSB), TSB supplemented with 0.6% of Yeast Extract (TSBYE) and filter-sterilised tap water. Five ml of each suspension were transferred for 6 well plates containing 1 cm2 stainless steel coupons and incubated at 4°C, 22°C, 30°C and 37°C. After 4 and 24 hours 2 coupons were removed for each tested medium. One coupon was stained in situ with SYTO 9 and observed under EDIC/EF microscopy and the other coupon was scraped and total and cultivable cells quantified by staining with SYTO 9 and plating onto BHI agar, respectively. Results showed that even for the lowest temperature, after 4 hours of incubation L. monocytogenes biofilms were already developing on the surfaces. Except in the case of biofilms formed in tap water where cells were in general less cultivable, the different media do not influence significantly the formation of biofilm on stainless steel surfaces. Conversely, the in situ observation of the coupons demonstrated that the morphology of the biofilm is temperature dependent with implications for food processing.

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REFERENCE Temperature dependent defect in biofilm formation / by Listeria monocytogenes D P Abdalla, S.1, Glenn, S.1, Shama, G.2 and Andrew, P.1 128 1. Department of Infection, Immunity and Inflammation, University of Leicester LE1 9HN, United Kingdom 2. Department of Chemical Engineering Loughborough University, Loughborough, LE11 3TU, United Kingdom Listeria monocytogenes can cause major problems in food processing industries because of its ability to survive and grow over a wide range of environmental conditions. Furthermore L. monocytogenes can attach to and produce biofilm on wide variety of different surfaces in the food-processing environment, allowing the bacterium to resist to antimicrobial and sanitizing agents. The aims of this study were to investigate the ability of Listeria monocytogenes wide type strain 10403s and Tn917-LTV3 transposon mutants to attach to abiotic surface at a variety of temperatures. A published attachment assay was modified to compare the ability of the WT strain and a number of transposon mutants to attach to polystyrene surfaces over 2 hours incubation. Attached cells were detected using crystal violet staining. Certain transposon mutants were shown to have a significant reduction in the level of attachment, but this defect was temperature dependent, being evident at 30°C and 18°C but not at 37°C. The identity of the transposon insertion site in these mutants is being actively pursued. It was concluded that there is a temperature dependent involvement of some gene production in surface attachment.

Mode of action of Lactococcus lactis Sa31 antimicrobial peptide REFERENCE D / P on Listeria monocytogenes ½ c Barile, M.1, Mormile, A.1, Ceres, C.1, Pepe, O.2, Cortesi, M. L.1 and Murru, N.1 129 1. DISCIZIA, Faculty of Veterinary Medicine, Napoli, Italy 2. Faculty of Agraria Università degli Studi di Napoli, Italy Lactic acid bacteria produce antimicrobial peptides referred to as bacteriocins that have potential as natural food preservatives. Lactococcus lactis Sa31, isolated from gilthead breams fillets packaged in modified atmospheres, normally produces bacteriocin in GM17 broth with maximum activity (2.560 AU/ml) at 11h in the late logarithmic to early stationary phase of growth. In the present work was investigated the Sa 31 ability of bacteriocin production in different liquid media, the partial characterization and mode of action of peptide against Listeria monocytogenes ½ c strain. Overnight extracts of producer strain cultures in Triptone Soya Broth, M17 + 0.5% glucose (GM17) and 0,5% lactose (LM17), MRS, and BHI broth was tested against Listeria monocytogenes ½ c. To evaluate the mode of action, the crude (5.120 AU/ml-1) and partially purificated bacteriocin (10.240 AU/ml-1) by precipitation with ammonium sulfate were inoculated in suspension of 50 mM potassium phosphato buffer pH 7.0 containing Listeria monocytogenes ½ c (1.4 x108 CFU/ml). Finally, for absorption study of partial purificated bacteriocin, 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 residual bacteriocin activity was tested as described above. The strain Sa 31 was able to produce bacteriocin in BHI broth with maximum titre 5.120 AU/ml-1 after short incubation time. The bacteriocin, sensitive to heat and more powerful at acid pH, exhibited a bactericidal mode of action and acted rapidly. The death of Listeria monocytogenes ½ c cells was observed within 10 min. after treatment with 10.240 AU/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. monocytogenes ½ c at pH 7.0. These results demonstrate the effectiveness of this bacteriocin and its producer as an inhibitor of Listeria monocytogenes in vitro, and the possibility of their applying in a food system where post-manufacture contamination by this organism could be problematic as in fishery products as well as in other foods.

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

REFERENCE Tracing the source, epidemiology and persistence of D / P Listeria monocytogenes in Irish Farmhouse cheese processing facilities 130 Jordan, K., Fox, E., O’Brien, M. and Hunt, K. Teagasc, Ireland Many outbreaks of listeriosis have been related to dairy products, although the source of contamination has rarely been determined. Sixteen farmhouse cheesemaking facilities were sampled at monthly intervals over a two-year period. Sam- ples included raw materials, food contact surfaces, non-food contact surfaces, farm samples and product. In order to determine the source, persistence and epidemiology of the strains obtained from 13 cheesemaking facilities, isolates of L. monocytogenes were differentiated using PFGE and serotyping. At 9 of the 13 contaminated processing facilities farm samples were the source of the environmental contamination. We could not determine the source of the contamination at the remaining 4 facilities. Persistent isolates (similar isolates obtained at the same facility for > one year) were found at two cheesemaking facilities. Reduction of the occurrence of L. monocytogenes in milk could reduce the contamination of cheese processing facilities and therefore reduce the risk of cheese contamination. Cheese producers 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 the farm and processing facility will help reduce the risk of contamination.

REFERENCE Phenotypic and genotypic characteristics of Listeria monocytogenes D / P strains isolated from a convenience food-processing plant 131 Blatter, 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 economic impacts. Amongst other foods, ready-to-eat products have been implicated as routes for human infection. The aim of this study was to investigate the occurrence and genetic diversity of L. monocytogenes isolated from the environment and food products in a Swiss sandwich-producing plant. Over a 12-month period, environmental swabs (n=2.028) collected during the working activity and after cleaning and disinfection, as well as ingredient and sandwich samples (n=217) were collected. Swabs originated from the processing room and the equipment of five sandwich production lines. Isolation of L. monocytogenes was accomplished by culture after enrichment (Half-Fraser/Fraser broth, Palcam and Ottaviani-Agosti agar). Isolated strains were characterized by serotyping, determination of genetic lineages, Rep PCR typing, and PFGE analysis after macrorestriction. L. monocytogenes were detected in 70 (3.5%) environmental swabs and 16 (7.4%) samples from ingredients and sandwiches. Of the positive environmental samples, 40% originated from three slicers, which tested repeatedly positive on several occasions. Of the 86 isolated L. monocytogenes strains, 93% belonged to serotype 1/2a and genetic lineage 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 ingredients and sandwiches belonged to only one genotype. Strains of this genotype were found repeatedly on/in slicers, tables, conveyor belts, tables, a bread-feeding machine, spattles, air guns, salmon and egg sandwiches. Based on the genotyping results, indications of persistent contamination of the sandwich processing area with a predominant strain were evident over the whole sampling period. Moreover, in food-processing plants with low Listeria prevalence in the final product, environmental monitoring is more effective than end product testing.

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REFERENCE Influence of flow direction on the adhesion of Listeria monocytogenes to / brushed stainless steel surfaces D P Skovager, A.1, Whitehead, K.2, Ingmer, H.3, Verran, J.2 and Arneborg, N.1 132 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 and Engineering, 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 process equipment constitutes a major health problem for the food industry. When the bacteria have succeeded in forming a biofilm, they are difficult to remove from the 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 disinfection agents. It is therefore extremely important to target the bacteria before they adhere to the solid surface and develop into a biofilm. One factor which may influence the adhesion of L. monocytogenes is the surface characteristics of processing equipment. In the present work, a method using fluorescence microscopy and a perfusion system will be developed to examine the initial adhesion of single L. monocytogenes cells to steel surfaces. Results will be presented showing the influence of flow direction on the adhesion of L. monocytogenes to brushed stainless steel surfaces.

REFERENCE Occurrence of Listeria monocytogenes in raw milk and dairy products D / P in Kazerun, Iran 133 Mehdi 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 milk and dairy product samples including white cheese, yoghurt and Iranian yoghurt drink (Doogh) that were collected during five months from two traditional dairy manufacturer in Southern Iran. The prevalence of Listeria monocytogenes in raw milk 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 of both manufacturer probably because of their low pH values.

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

REFERENCE Antilisterial mode of action of bacteriocin ST182Gu produced D / P by Enterococcus casseliflavus isolated from guava 134 Todorov, 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 , Brazil 2. ESTG, Viana do Castelo , Portugal This research is on the anti-Listeria bacteriocin ST182Gu and studies its mode of action. Bacteriocins of lactic acid bacteria (LAB) are ribosomally synthesized antimicrobial peptides. Their bactericidal mechanisms may include pore formation, degradation of cellular DNA, disruption through specific cleavage of 16S rDNA, and inhibition of peptidoglycan synthesis. In our knowledge this is the first report of the isolation of bacteriocinogenic strain of Enterococcus casseliflavus from guava. Enterococcus casseliflavus ST182Gu, produces a pediocin-like bacteriocin (based on highly homology of PCR product generated with the primers targeting pediocin PA-1 structural gene) with activity against several LAB, Listeria spp, and some other human and foodborne pathogens. No significant differences in growth and production of bacteriocin ST182Gu were observed when the strain E. casseliflavus ST182Gu was cultured for 24h in MRS broth at 20°C, 30°C or 37°C. At this 3 different cultivation temperatures activity was 1.0x109AU/ml against L. ivanovii subsp. ivanovii ATCC19119 and 2.6x104AU/ml against Enterococcus faecium ATCC19443. Low levels of bacteriocin ST182Gu activity (approximately 1.3x104AU/ml against L. ivanovii subsp. ivanovii ATCC19119 were recorded after 3h of growth in MRS broth at 37°C and the maximal bacteriocin ST182Gu production (1.0x1010AU/ml) was recorded in MRS after 27–33h of growth. A decrease in activity was observed after 33h of growth and it was reduced to 1.0x108AU/ml at 48h of incubation. Addition of bacteriocin ST182Gu to exponential or stationary phase cultures of L. ivanovii subsp. ivanovii ATCC19119 inhibited growth for 24h. The effect of bacteriocin ST182Gu on cells of L. ivanovii subsp. ivanovii ATCC19119 was visualised by AFM and indirectly recorded based on enzyme, protein and nucleotide material leakage. Bacteriocin ST182Gu is highly active against L. ivanovii subsp. ivanovii ATCC19119 and exhibits a synergetic effect in the inhibition of this surrogate microorganism when applied together with subletal doses of ciprofloxacin.

* Participation Supported by IUFoST.

REFERENCE Control of Listeria monocytogenes in fresh goat cheese by bacteriocinogenic D / P strain Lactococcus lactis subsp. lactis DF4Mi or commercial nisin 135 Nader 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 bacteriocinogenic strains, favouring the in situ production of the bacteriocin. The aim of this study was to evaluate the capability of the bacteriocinogenic strain Lactococcus lactis subsp. lactis DF4Mi, isolated from goat milk, to control the growth Listeria monocytogenes in fresh goat cheese over 10 days of storage under refrigeration, and to compare 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 the cheese manufacturer practice. Three sets of cheeses were prepared: one with pasteurized milk with starter culture strain DF4Mi (106 UFC/ml), second with bacteriocin (-) strain of L. lactis subsp. lactis and other with addition of 12.5mg/l of pure nisin (Fluka). L. monocytogenes 701 was added to cheeses during the manufacturing, in order to obtain 103 UFC/g. Appropriative controls such as cheese without any starter 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 to counts of lactic acid bacteria and L. monocytogenes every other day for 10 days period. Results indicate that L. monocytogenes decreased 3-log in the cheeses containing either bacteriocinogenic L. lactis subsp. lactis DF4Mi or bacteriocin (-) L. lactis subsp. lactis, showing that inhibition of L. monocytogenes might have occurred due to another factor than the production of bacteriocin. Analysis of the cheeses containing nisin demonstrated that incorporation of this bacteriocin at 12.5mg/l could inhibit the growth of L. monocytogenes allowing a decrease of 2-log counts during storage. The present study showed that the use of purified bacteriocin in fresh goat cheese is more effective in the control of L. monocytogenes than the direct application of the bacteriocinogenic L. lactis subsp. lactis DF4Mi.

* Participation Supported by IUFoST.

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

REFERENCE High pressure processing ensures elimination of Listeria monocytogenes / in sliced dry cured ham D P Stollewerk, K., Jofré, A., Comaposada, J., Aymerich, T., Ferrini, G., Arnau, J. and Garriga, M. 136 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 without altering their sensorial properties. Listeria monocytogenes can contaminate different types of meat products, such as dry cured ham, during slicing. The aim of the present study was to evaluate the behavior of L. monocytogenes in three types of 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 was brine salted, fermented and smoked. Afterwards, slices of ham were spiked with a mixture of 3 strains of L. monocytogenes (ca. 100 CFU/g). Drying was performed by the Quick Dry Slice process®. Pairs of slices were vacuum packed and half of them were submitted to a HP treatment of 600 MPa for 5 min. Packages were stored under refrigeration for 112 days and the evolution of the pathogen was followed by plating in cromogenic agar and species-specific real time PCR. During storage of non-pressurized hams the levels of L. monocytogenes progressively decreased in the 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 the plate detection limit (10 CFU/g) in all the hams. In H3 the pathogen was absent during the whole storage. After 112 days L. monocytogenes was not detected in any of the samples (absence in 25g). The composition and processing of dry cured ham affected the survival of L. monocytogenes. Fermentation and smoking speed up the decrease of the pathogen, but only the application of a HP treatment enabled to achieve a product free of L. monocytogenes during the whole storage period.

REFERENCE Inhibition of Listeria monocytogenes by Lactococcus spp. EU2241 D / P in tropical shrimp 137 Abdoulaye 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 as pathogens and spoiling microorganisms. Growth of these undesirable flora is often delayed by the use of classical techniques such as salting, low storage temperature, modification of atmosphere packaging or use of preservatives. However the human pathogen Listeria monocytogenes (Lm) that was evidenced in raw and ready to eat shrimp by some studies can often grow in those conditions and may reach the tolerated level of 100 Lm g-1. Biopreservation, an alternative technology for food preservation which consists in inoculating a product with selected bacteria to eliminate or prevent growth of undesirable microorganisms, may be useful against Lm. In a previous study, a strain of Lactococcus spp. EU2241 had been isolated from raw salmon and showed capacities to inhibit in model medium some pathogens isolated from seafood product. In this study the inhibition capacity of Lactococcus spp. EU2241 was tested in shrimp artificially contaminated with Lm. Four batches of cooked shrimp were prepared. 1: sterility control; 2: inoculation with Lactococcus spp. (106 CFU g-1); 3: inoculation with Lm (103 CFU g-1); 4: co-inoculation of Lactococcus and Lm. Shrimp were stored at 8°C for 31 days under modified atmosphere (CO2/N2 50-50). When inoculated alone, Lm grew very well in shrimp reaching its maximum level, 109 CFU g-1, in 10 days. An immediate inhibition of Lm was observed in co-inoculated batch, reaching more than 4 logs at the end of storage with no sensory changes, confirming the interest of this bacterium for an application in biopreservation of seafood products against Lm. The use of Seafood Spoilage and Safety Predictor software to predict the growth of Lm in 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 observed in this study. A chemically defined liquid medium is currently developed to study the inhibition mechanisms.

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REFERENCE Listeria monocytogenes and Listeria innocua in slaughter line of a swine D / P meatpacking plant in Rio Grande do Sul State, Brazil 138 Schittler, L.1 and Padilha Silva, W.2 1. UDESC, Brazil 2. UFPeL, Brazil Listeria monocytogenes is bacterium widely distributed in nature which presents high capacity to colonize surfaces and form biofilms in food processing plants. It is the causative agent of listeriosis which may cause a range of diseases from gastroenteritis to death, being the meat an important vehicle for transmission to humans. The aim of the present study was to investigate the presence and characterize L. monocytogenes and Listeria innocua in the slaughter line of a swine meatpacking plant, located in Missões Region, state of Rio Grande do Sul, Brazil. For this was evaluated water, cold chambers, carcasses, knives, faeces, hands and drains and the strains obtained were serotyped and submit to molecular typing through the analysis by random amplification of polymorphic DNA (RAPD) with UBC 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 the major 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. monocytogenes strain was isolated, from a drain, however it belonged to serovar 4b, an important serovar in public health, because it is frequently involved in listeriosis in humans. RAPD of L. innocua strains produced 15 genetic combined profiles, whose index of discrimination was D=0.94, whereas the serotyping presented low discriminatory 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 cross contamination in this plant is considered important, since some L. innocua strains persist in the local.

REFERENCE Listeria monocytogenes in raw meat products marketed in the city D / P of Sao Paulo, Brazil: Incidence and counts data for risk assessment 139 Ristori, 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, Brazil 2. Food Surveillance, COVISA, Sao Paulo, SP, Brazil 3. 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 products. Despite abundance of results on the prevalence of L. monocytogenes in these foods in different countries, quantitative data on level of contamination required for appropriate risk assessments are scarce. This study aimed to evaluate the prevalence and numbers of Listeria monocytogenes in a variety of meat products available in the local market in the city of Sao Paulo, SP, Brazil. Between May 2008 and July 2009, 552 samples of refrigerated raw meat products (138 beef sausages, 138 pork sausages, 138 ground beef samples and 138 chicken leg samples) were purchased in 138 local supermarkets, selected after stratification by region and by random draw. Tests for presence and enumeration of L. monocytogenes were based on 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 highest prevalence occurred in ground beef (59.4%) followed by chicken legs (58%), beef sausages (37.7%), and pork sausages (39.8%). In most samples (67.4%), the level of contamination 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 to 8.9x102 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 Sao Paulo, and for establishing science-based intervention strategies aimed at reducing these risks, specially for pregnant women and immunocompromised individuals.

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REFERENCE Inhibiton of Listeria monocytogenes by Carnobacterium / maltaromaticum in combination with extract of Lippia sidoides Cham. D P in cold-smoked surubim fish broth 140 Barbosa 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 severe foodborne infection caused by Listeria monocytogenes (Lm). In this work, to improve the safety ready-to-eat fish, multiple hurdle experiments were carried out with cold-smoked surubim broth (CSSB) stored at 5°C for 5 weeks. To inhibit Lm, it was evaluated the application of a hidroalcoholic extract of the Brazilian folk plant Lippia sidoides (ELs) (10 µl/ml) in combination with Carnobacterium maltaromaticum (Cm) C2 and A9b+ (bacteriocinogenic strains) and A9b- (non- bacteriocinogenic). CSSB was inoculated with 102 CFU of Lm/ml and/or 105 CFU of 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 plus Cm A9b- plus ELs; 14) Lm plus Cm A9b+ plus ELs. Bacterial populations and bacteriocin 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, listerial population 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 C2 alone and Cm C2 plus Lm, which may explain the stronger inhibitory effect of Cm C2. ELs only did not inhibit Lm (ca. 108 CFU of Lm/ml) and its use in combination with 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, suggesting that ELs can adversely affect the protective culture and decrease the effectiveness of the biopreservation system studied.

Inhibition of Listeria monocytogenes in cooked ham REFERENCE D / P by virulent bacteriophages and protective cultures Holck, A., Schirmer, B. C. and Berg, J. 141 Nofima Mat AS, Norway Listeria monocytogenes is found in a number of raw and ready-to-eat (RTE) products, poultry, seafood and dairy products. RTE products like cooked ham have little inherent stability against the growth of L. monocytogenes. Several studies show reduction of L. monocytogenes in food when exposed to phages. Phage particles, however, become immobilised and are inactivated soon after addition to nonliquid foods and can thus not prevent later outgrowth of surviving Listeria. Here we have shown that protective cultures can be used successfully as an additional hurdle together with phages to reduce growth of L. monocytogenes. Sliced cooked ham was inoculated with cold adapted L. monocytogenes and exposed to bacteriophages. The hams were subsequently inoculated with Lactobacillus sakei TH1 protective culture, vacuum packed and stored at 10°C. Addition of phages gave an immediate 1-2 log reduction in L. monocytogenes. After 14 – 28 days of storage, an additional 2 log reduction was observed in samples with phages and protective culture compared to samples with phages alone. The effect of the protective culture was evident both at 10 and 4°C. Higher inoculation levels of protective culture gave a stronger inhibition of L. monocytogenes. The use of phages in combination with protective cultures is a general method that can potentially be applied to different products where there is a risk for growth of L. monocytogenes.

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REFERENCE Surface colonization by Listeria monocytogenes: role of the flagella D / P in the biofilm formation process 142 Desvaux, 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 the biofilm formation process in Listeria monocytogenes have not been definitively as- certained. So far, the flagellum is the only cell surface factor demonstrated as involved in biofilm formation within this species, even so its exact contribution and role remain controversial. This prompted us to reinvestigate the role of the flagella in the course of biofilm formation at growth temperatures relevant to regulation of genetic expression in L. monocytogenes using both static and dynamic cultures conditions. Biofilm formation was assayed at early and late stages of the process using paramagnetic microbeads and crystal violet methods. Cell morphology and biofilm architecture were determined by confocal scanning laser microscopy using bacterial cells expressing fluorescent markers. Cells were grown statically in microtiter plates and in dynamic conditions using flow cells. As revealed by proteolytic treatments, extracellular/cell-surface proteins rather than exopolysaccharides are crucial determinants involved in listerial biofilm formation. While L. monocytogenes was enable to swim at 20°C, no swarming could be observed. 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 growth temperature tested. Besides crystal violet method, such results were confirmed by confocal microscopy in static and dynamic cultures conditions using fluorescently labelled listerial cells. Biofilm formation was for the first time investigated over a range of growth temperatures relevant to regulation of genetic expression in L. monocytogenes species. The use of complementary growth conditions, i.e. static and dynamic cultures, allowed providing a comprehensive picture of biofilm formation in this species. In those tested conditions it clearly appeared that flagella are not essential but rather detrimental to biofilm formation in L. monocytogenes.

REFERENCE D / P The role of sanitizers in controlling Listeria monocytogenes on stainless steel surfaces: Lessons learned from the 2008 Listeriosis outbreak 143 Hébert, K., Farber, J. and Pagotto, F. Health Canada Proper maintenance and sanitization of food-contact and non-food-contact surfaces remains a high priority for industry in ensuring a microbiologically safe final product. Listeria monocytogenes is often present in factory environments and needs to be controlled to avoid contamination of final product. The national outbreak of listeriosis caused by contaminated deli-meat raised questions about the ability of Listeria to survive on stainless steel surfaces. We evaluated the effectiveness of 11 sanitizers previously and currently used by Canadian industries against strains of L. monocytogenes. Eleven sanitizers were evaluated using the American Society for Testing and Materials standardized method (E2197) using stainless steel disk coupons, according to the recommendations on the labels or from the manufacturer. A soil load consisting of a microbial suspension, BSA, mucin and tryptone was used to mimic environmental conditions. Bacteria were 7 inoculated at 10 CFU per coupon, dried and challenged against 50 µl of the suggested working concentration and exposure time of each sanitizer. The log reduction was calculated by direct plating. Ten sanitizers met the performance criteria for L. monocytogenes, achieving a minimal 6-log reduction. There was no significant difference observed amongst the different active ingredient formulations. A single, alchohol-based hand sanitizer used by the industry did not satisfy the criteria when tested on stainless steel coupons. This study demonstrated that sanitizers are effective in controlling Listeria on stainless steel surfaces when proper adherences to protocols were maintained. Misuse of sanitizers can allow the organism to survive and possibly make its way into the final food product.

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Listeriophage ecology and diversity on dairy farms REFERENCE D / P Vongkamjan, K.*, Moreno Switt, A., den Bakker, H. C., Fortes, E. D., and Wiedmann, M. Department of Food Science, Cornell University, Ithaca, NY, USA 144 Listeria monocytogenes is common in dairy farm environments and contaminated silage appears to be the major source of L. monocytogenes infections in farm ruminants. Improved understanding of the ecology and diversity of phages infecting L. monocytogenes (“listeriophages”) in food associated environments is critical to evaluate the impact, effects, and potential of phage-based pathogen control strategies. We collected silage samples over the course of one year on two dairy farms to perform screening for L. monocytogenes and listeriophages, using standard methods and representative host strains. L. monocytogenes isolates were characterized by molecular methods (e.g., sequencing of sigB) and subsequently classified into lineages I and II. Listeriophages were characterized for host range using a 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), six samples were positive for L. monocytogenes; 43/81 samples from farm 1 and 40/53 samples from farm 2 were positive for listeriophages. Overall 34/134 silage samples contained >100 PFU/g silage with samples containing up to 2x104 PFU/g. For the majority of phage positive-samples, phages were detected on serotype 4a host strain. Phage isolates represented considerable host range diversity (12 distinct lysis groups). Most phages did not lyse lineage I strains, but lysed most lineage II strains 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 listeriophage diversity in dairy farm environments, suggesting that phages play an important role in the ecology of L. monocytogenes on dairy farms.

* Participation Supported by IUFoST.

Evaluation of curative and preventive decontamination treatments REFERENCE D / P on Listeria monocytogenes biofilms with a new screening system Quinon, E.1, Chamot, S.1, Groelly, J.2, Chavant, P.2, Bernardi, T.2, Desvaux, M.1 145 and Hebraud, M.1 1. INRA, France 2. BioFilm Control, France Food safety requires not only interventions on the product but also on all surfaces of the workshops from the receipt of raw materials until the processing and packaging of food products. All surfaces can be contaminated by microorganisms able to adhere, to form biofilms and to disseminate all along the chain. The literature shows that bacterial cells in biofilm, compared with their planktonic counterpart, present a greater resistance to hostile environments and particularly to cleaning disinfection procedures. The efficacy of cleaning disinfection products currently used for surface decontamination is generally evaluated on planktonic cells, which does not predict their efficacy on bacteria in biofilm. We have implemented a screening system, initially developed by the Biofilm Control society to evaluate bacterial ability to form a biofilm on an abiotic surface (BioFilm Ring Testâ), to test the efficacy of 4 commercial decontamination solutions to detach biofilm from the surface and to affect the viability of bacterial cells. Three temperatures (10°C, 20°C and 37°C) and three Listeria monocytogenes strains were used for the tests. The effect of preventive decontamination treatment of the surface on cell adhesion and biofilm formation was also assessed. Two out of the 4 products, containing quaternary ammonium compound (QAC) as biocide, dramatically affected the viability of sessile cells whatever the temperature while only one out of the two was efficient to detach adhered cells. The formulation of this last product also contains a polyenzymatic cocktail. The two other solutions remained inefficient on biofilms. The QAC + polyenzymatic cocktail was also the only product efficient to prevent or delay the biofilm formation after a preventive treatment of the surface. The BioFilm Ring Testâ can be used to sreen the efficacy of biocides on bacterial biofilms in order to improve the microbiological status of the food processing plants.

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REFERENCE Pulsed Field Gel Electrophoresis, conventional and molecular D / P serotyping on Listeria monocytogenes: An European Proficiency 146 Testing Inter-laboratory Trial Félix, 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 sur la Qualité des Aliments et sur les Procédés agro-alimentaires (Lerqap), Maisons-Alfort, France In Europe, the incidence of listeriosis caused by Listeria monocytogenes is relatively low, however an increase in cases, which differ depending of the countries, has been observed since 2000. Different methods are available for first-level characterization in epidemiological surveillance, in particular, conventional and molecular serotyping. Regarding fine characterization techniques, macro-restriction analysis by pulsed-field gel electrophoresis (PFGE) has been described as the most discriminatory methods for L. monocytogenes subtyping. This method is widely used by the European National Reference Laboratories (NRLs) according to a 2009 EFSA survey. AFSSA’s Laboratory for Study and Research on Food Quality and Processing was appointed as Community Reference Laboratory (CRL) for Lis- teria monocytogenes in 2006. Recently, the CRL organized a proficiency test in order 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 same panel of six strains was used for all three methods and was sent to the 17 participating NRLs. This panel included serotypes 1/2a, 4b, 1/2c, 1/2b and 3a strains. Each laboratory was free to choose the protocols used. All participants followed the PFGE PulseNet Europe standardized protocol. PFGE migration parameters were set by the CRL, pattern interpretations were performed following the CRL’s interpretation 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 undistinguishable of the expected profiles The inconsistencies observed were caused by slight standardization defaults or in few cases by a fault in the extraction. This PT trial was a valuable opportunity to improve the typing ability of NRLs and will allow PFGE pattern exchanges.

REFERENCE Detection of Listeria spp. in raw and pasteurized liquid egg-products D / P and in the egg-breaking plants environment 147 Rivoal, K., Fablet, A., Chemaly, M., Salvat, G. and Protais, J. AFSSA, France Listeria monocytogenes has been recognized as a human pathogen for decades and is etablished as an important food-borne pathogen. There is a lack of information about eggs and egg-products contamination by Listeria spp. The aim of this study was to detect Listeria spp. in liquid egg-products (whole, yolk and white with or without salt and/or sugar) collected in 5 egg-breaking plants in north western France, during one year. To study the seasonal variation, each egg-breaking plant has been visited during each season. For a raw egg-product sample, a pasteurized sample has been tested and the egg shells used for theses egg-products have been swabed to detect Listeria spp. The environment of plants was also sampled with swabs during the production of the egg-products tested. At the present time, the project is not achieved. Three seasons have been studied and the last one is now going on. The environment of the egg-breaking plants seems to be very contaminated by Listeria spp. when the egg-products and their shells were slightly contaminated by the bacteria. Moreover, Listeria monocytogenes has been very rarely isolated 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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REFERENCE Biofilm formation by Listeria monocytogenes isolates under conditions / that mimic food and digestive tract D P Kretli Winkelströter, L.1, Oliveira, M. A.2 and De Martinis, E. C.1 148 1. Universidade de São Paulo, Brazil 2. Instituto Adolfo Lutz, Brazil L. monocytogenes is ubiquitous; able to tolerate many adverse conditions found in foods and can persist in processing plants due to formation of biofilms. L. monocytogenes can cause severe foodborne infections in immunocompromised people and in pregnant women. In this work, L. monocytogenes isolates were screened for ability to form biofilm under diverse conditions. Overnight cultures of 51 isolates of L. monocytogenes from food, clinical and environmental sources were inoculated (1:20, v/v) in Brain Heart Infusion Broth (BHI) and under the following conditions: 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 bacteriocin-producing cultures of Lactobacillus sakei 1, Leuconostoc mesenteroides 11A or Enterococcus faecium 130. Aliquots of inoculated broths (200µl) were transferred to 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 well was 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 was measured at 595 nm (CV-OD595) and directly correlated with biofilm formation. L. monocytogenes ATCC 19115 was used as parameter of comparison for biofilm formation. 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 approximately 9.8%, 1.9%, 7.8% and 19.6% for L. monocytogenes isolates incubated respectively in BHI only and under presence of sucrose, NaCl and Oxgall, indicating a higher ability to form biofilm. In BHI pH 2 and BHI plus 50% (v/v) of extract from bacteriocin-producing cultures, L. monocytogenes isolates did not show higher differences in biofilm formation in comparison with L. monocytogenes ATCC 19115 (0.70-0.81 CV-OD595). L. monocytogenes isolates showed different abilities to form biofilm under different conditions probably due to modulation of gene expression, which will be further studied.

Biofilm formation of Listeria monocytogenes EGDe depends REFERENCE D / P on temperature and nutrient availability Auchter, M.*, Endres, J., Waidmann, M. S. and Riedel, C. U. 149 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 phenotype associated with biofilm grown cells. Thus, biofilm formation in the gall bladder could be a strategy of this important pathogen to protect, establish and multiply itself at a site inaccessible to the components of the immune system prior to causing systemic infections. To investigate the mechanisms of biofilm formation, we tested L. monocytogenes EGDe and an isogenic agrD deletion mutant for their ability to form biofilms under different conditions. Biofilm formation was tested in a standard microtiter plate assay at different time points during biofilm growth 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). Biofilm formation at 37°C was poor in rich broth and could be significant enhanced when cells were grown under nutrient limitation. Deletion of the agrD gene resulted in defective biofilms in 10-fold diluted, as well as in full-strength BHI. The biofilm formation 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 adjusted and filter sterilized) of EGDe wild type cells grown in full-strength BHI was added to the ∆agrD strain. Additionally, DDAO staining and DNAse treatments revealed a potential role for extracellular DNA in the initial stages of biofilm formation.

* Participation Supported by IUFoST.

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REFERENCE Antimicrobial activity of lactococcal and enterococcal strains D / P isolated from artisanal products from North West of Italy toward 150 Listeria monocytogenes Dal Bello, B., Rantsiou, K., Ambrosoli, R., Zeppa, G. and Cocolin, L. Department of Exploitation and Protection of the Agricultural and Forestry Resources, University of Turin, Italy Listeria monocytogenes is a foodborne pathogenic Gram-positive bacterium that is widely distributed in soil, sewage, fresh water sediments and effluents, and is frequently carried in the intestinal tract of animals and humans. Biocontrol of L. monocytogenes by bacteriocin-producing lactic acid bacteria or by bacteriocin extracts has attracted great attention in recent years and new preservation strategies to control growth of L. monocytogenes have been developed, including the application of the bacteriocin nisin (Food and Drug Administration, 1988). In this work, we have investigated the potential role as bioprotection agents of autochthonous lactococcal and enterococcal strains isolated from fresh and fermented artisanal products (cheese and meat) of Piedmont region (North West of Italy) determining their antimicrobial spectrum of activity towards different foodborne spoilage and pathogenic microorganisms. Bacteriocin-producing strains were identified by molecular methods and genetic determinants encoding the antimicrobial proteins were targeted by PCR. Thirty-nine strains of Lactococcus lactis exhibited inhibition towards L. monocytogenes NCTC 10527 and most of them (26 strains) showed the presence of the genes responsible for the nisins A and Z production. Regarding Enterococcus spp., 31 strains showed inhibition activity towards L. monocytogenes NCTC 10527 and the presence of the genes responsible for the enterocins A and P production was determined. It is interesting to underline that for some strains it was not possible to identify any known bacteriocins. In this study a high incidence of bacteriocin producing strains was observed. In the future, the possible use of these active strains could be a new way to ensure safety of foods.

REFERENCE D / P Plant-based strategies for Listeria monocytogenes control in foods 151 Paparella, 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 alternative to chemical preservatives. Considering the complex chemical composition of EOs, their antimicrobial action is not likely attributable to one single mechanism, although bacterial cytoplasmic membrane seems to be a specific target. The authors evaluated the antimicrobial activity of thyme, oregano and cinnamon EOs against Listeria monocytogenes strains, isolated from the smoked salmon industry and from meat products; these strains showed variable levels of antimicrobial resistance, belonged to different serotypes and had heterogeneous molecular profiles. Thyme and oregano EOs were particularly effective, with Minimal In- hibitory Concentrations (MICs) correlated with strains biodiversity. Automatic turbidometry was applied to evaluate the effects of EOs on cell physiology; the results documented a lag phase extension and a decrease of maximum growth rate and maximum growth value. The antibacterial effect was evident even after a short contact time with the cells. Using flow cytometry combined with fluorescent techniques, the authors demonstrated that membrane disruption is the primary inactivation mechanism of thyme and oregano oils. A different mechanism might be involved in cinnamon EO, acting on cells enzymatic activity. Further studies, carried out by means of EPR (Electronic Paramagnetic Resonance), highlighted the effect of oregano EO on membrane fluidity and order, variable with increasing EO concentrations. These findings prove that food biopreservation with EOs involves various mechanisms of action and may be considered a safe and effective measure to control Listeria monocytogenes growth, although with different effects depending on strains biodiversity.

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REFERENCE Scientific studies for survival of Listeria monocytogenes / in dairy products and its application in practice D P Cabanova, L., Škuntova, O. and Kantikova, M. 152 State Veterinary and Food Institute, Dolny Kubin, Slovakia European reference laboratory for Listeria monocytogenes in Paris has prepared together with some National reference laboratories from the European Member States the scientific study for Listeria to prove the producer that his products are safe till the end of the shelf- life. In our study we have developed studies for the producer of two different dairy products after several positive findings of Listeria in the final products. As a first product the raw sheep cheese has been selected and as a second one the steamed cheese products have been analyzed. In the first stage the physico-chemical characteristics has been determined (aw, pH) and than the subsamples has been prepared and artificially contaminated with a mixture of two reference Listeria strains and third isolated strain from the previous positive 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 analyzed (aw, pH, Listeria detection and enumeration has been determined). At the end of the shelf – life the growth potential has been calculated and the Listeria numbers at the beginning and at the end of the shelf- life has been given.

REFERENCE The effect of chilling temperatures on the virulence D / P of Listeria monocytogenes isolates with different origins 153 Neves, 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 Universitário de Almada, Instituto Piaget, Portugal 3. Instituto Nacional dos Recursos Biológicos, IP, Portugal 4. 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 the food industry, there are few studies on the effect of these temperatures on the virulence of Listeria monocytogenes. The influence of 1, 7 and 30 days of storage at chilling (7°C) and freezing temperatures (-20°C and -76°C) on the virulence of 19 L. monocytogenes isolates was evaluated in this study. The isolates selected according to their genetic diversity and different initial levels of virulence were from frozen (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 reference strains (n = 2). In the case of the chilling assays, the cells were kept in physiological buffer and culture medium was added just before infection. After the respective storage, the isolates were used to infect HT-29 cell monolayers in a plaque forming assay (pfa), immediately or after thawing the bacterial suspen- sions at room temperature. The pathogenic potential of the strains was expressed as 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 the refrigeration temperature (7°C) acted more significantly (P < 0.05) on the decreasing of the virulence potential of all isolates, although time didn´t show a significant 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 the temperature of -76°C tended to be more conservative of the initial virulence. Bet- ter elucidation of which refrigerated food matrices allow growth of the pathogen may help to evaluate the real risk of the presence of Listeria in food.

* Participation Supported by IUFoST.

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REFERENCE How to improve a sampling plan in order to better assess D / P L. monocytogenes contamination on diced bacon at the plant 154 Bergis, H.1, Commeau, N.1, Zuliani, V.2, Cornu, M.1, Beaufort, A.1 and Garry, P.2 1. AFSSA, France 2. IFIP, Institut de la filière porcine, France Food Business Operators (FBO’s) are legally responsible for the safety of their food products (Regulation CE 2073/2005). The regulation does not specify the frequency of sampling/ testing and it is up to the FBO’s to decide the appropriate level of sampling/testing to help validate and verify their food safety management plans. In this scope, sampling plans are useful tools to control and to assess the contamination of a given foodborne pathogen at the plant and also to help the FBO to take decisions. In the present study, a production of diced bacon in a French plant was monitored to assess the contamination of this product by Listeria monocytogenes. The steps of the process are: tumbling, steaming, dicing, and packaging.The aim of this study was to try, through the elaborated sampling plan, (i) to establish a link between the prevalence of L. monocytogenes in the pork breasts and in diced bacon; (ii) to determine whether the levels of contamination of the pork breasts and in the diced bacon are related; (iii) to estimate the incidence of the tumbling step and of the dicing step on the final product contamination.The food process was studied from the raw material (pork breasts) to the final product (diced bacon) and the steps possibly involved in the contamination identified. An experimental stratified random sampling plan was constructed in two steps of the process: after tumbling (a important step), and after packaging. 106 sampled tumbling breast and 86 packages of diced bacon were analysed. Detection and enumeration of L. monocytogenes were performed on 100cmr of the pork breast and on 100g of diced bacon. The lactic acid bacteria (LAB) were also enumerated. From the 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 a good indicator of the presence of L. monocytogenes in diced bacon. It has been shown that over a certain level of contamination of the breast, the diced bacon from the same batch are also contaminated with L. monocytogenes.

REFERENCE Contamination of Listeria monocytogenes in a cold-smoked D / P pork processing plant using brining injections 155 Berzins, A.1, Silins, I.1 and Korkeala, H.2 1. Faculty of Veterinary Medicine, Latvia University of Agriculture; University of Helsinki, Latvia 2. University of Helsinki, Finland Contamination of L. monocytogenes was studied in a cold-smoked pork processing plant using brining injections. Overall prevalence of L. monocytogenes in cold- smoked pork products during a 5-year period was 26%. Environmental sampling combined with PFGE subtyping and serotyping was applied to investigate the genetic diversity of L. monocytogenes in the brining facilities and alongside premises. A total 183 samples were collected for contamination analyses, including samples of the product at different stages during manufacture (n = 136) and environmental samples (n = 47) covering most of the manufacturing surfaces during a 3-month period. Overall, the prevalence of L. monocytogenes in raw pork before processing was 18%. The prevalence of L. monocytogenes in pork increased to 60% after being in brining area and brining machine. Two different L. monocytogenes PFGE types belonging to serotypes 1/2a and 1/2c were recovered from the different sites of the brining machine (feeding teeth and smooth surfaces), thus causing persistent contamination of RTE cold-smoked pork products over period of 5-years. In addition, brining machine harboured two PFGE types belonging to serotypes 1/2a and 4b, which were found on different contamination sites: feeding teeth, smooth surfaces and spaces of the machine. Brining injections increased L. monocytogenes contamination in finished RTE cold-smoked pork products and processing environment. Brining area, and specifically, brining machine should be subjected for disassembling and extensive cleaning and disinfection to eliminate any persistent contamination with L. monocytogenes.

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REFERENCE Effects of GRAS products on growth of Listeria monocytogenes during / cold storage of salmon fillets D P McCarthy, S. and Johnson, D. 156 Food and Drug Administration, USA Microbial growth can affect the quality and safety of prepared, processed, and ready-to-eat (RTE) meat, poultry, and seafood. Pathogenic bacteria, including Lis- teria monocytogenes (Lm), can be resistant to preservatives, sanitizers, and antibiotics. The presence of Lm on RTE products results from cross-contamination from contact surfaces in food processing plants. This study examined the ability of three GRAS products to inhibit the growth of Lm on salmon fillets during cold storage. Twenty-five-g portions of raw salmon fillets were inoculated externally with six log10 CFU Lm 1a1/g. Inoculated fillets were incubated at 25°C for 20 minutes and at 4°C for two hours to allow attachment. Filets were then treated with sterile 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 filets were analyzed immediately after exposure (T0) and after storage at 4°C for up to 12 weeks. Fillets were shaken in wash buffer and cell counts in washates were determined by spread plating on chromogenic agar. Numbers of Lm increased by one log10 during 9 weeks of cold storage of fillets treated with SDW. In contrast, TM MOstatin VS reduced the numbers of Lm by two logs10 during storage for 9 weeks. IONALTMLC and levulinic acid prevented the growth of Lm for up to four weeks. Each of the GRAS products reduced densities of salmon bacterial flora by two to four logs10 at two weeks compared to SDW; however, no difference was observed at 12 weeks. The use of GRAS products could prevent an increase in numbers of Lm that are associated with cross-contamination in processing plants.

Heavy-metal and detergent resistance of Listeria species isolates from REFERENCE D / P milk processing environments Doijad, S.*, Garg, S. and Barbuddhe, S. B. 157 ICAR research complex for Goa, India Listeria monocytogens is an important food-borne pathogen responsible for varied clinical forms in animal and humans. The resistance of Listeria strains to cadmium, arsenic and quaternary ammonium compounds was studied and it was correlated with resistance to quaternary ammonium compounds used as disinfectants in the food-processing industry. Limited information is available on the prevalence of resistance among isolates from the environment of food-processing plants. In this study, a total of 27 Listeria species isolates from the milk processing plants were included. Out of 27, 14 (51%) found to be Listeria monocytogenes,12(44%)were L. innocua and1(3%)isolatewasof L. ivanovii. All the isolates were subjected for determination of the resistance to cadmium, arsenic and quaternary ammonium disinfectant (benzalkonium chloride (BC)). Isolates were not inhibited at 40µg/ml of cadmium chloride, while 12 (44%) of them grew upto 200µg/ml. Isolates grew well in 40µg/ml of sodium arsenite while 3 of them could tolerate 200µg/ml. The isolates could grew at 1µg/ml of BC while only 7 could tolerate 5µg/ml. The amount of BC tolerated by these isolates are well above the amount used in milk processing environment. There was no co-relation found between resistance of isolates to cadmium, arsenate and BC, all isolates were independently resistance to metal and detergent. It was interesting to note that none of the isolates contained plasmid. This suggests that resistance against metals and detergent in Listeria spp. is not mediated by the plasmid and need further investigations. Our findings suggest that the milk processing environment constitute a reservoir for L. monocytogenes and other Listeria species. Resistance to heavy metals and quaternary ammonium disinfectant is of significance to food processing industry.

* Participation Supported by IUFoST.

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REFERENCE Detection of Listeria monocytogenes in lettuce sold at markets D / P and supermarkets in Porto, Portugal 158 Noronha, 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 particularly among the elderly, very young and immunocompromized individuals and has also been associated with late-term miscarriages in pregnant women. The high incidence of L. monocytogenes in foods and the high fatality rate associated with listeriosis, has contributed to L. monocytogenes being considered a public health hazard and a continuing source of loss to food processors due to the large number of voluntary and obligatory recalls. Listeriosis outbreaks have been linked to the consumption of raw vegetables including lettuce. According to the European Food Standards Agency, lettuce supports L. monocytogenes growth and can be related with the transmission of listeriosis. Twenty different lettuce samples collected in markets or supermarkets located in the North of Portugal, were analyzed for the presence of L. monocytogenes by the VIDAS method. Of the tested lettuce samples, 30% were positive for the presence of L. monocytogenes. Three isolates were collected from each of the positive samples. They were serotyped by Multiplex PCR and 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 of washing raw vegetables thoroughly before eating and preparing green salads and vegetable dishes shortly before eating in order to reduce the risk of listeriosis.

REFERENCE Preliminary analysis of structure and chemical composition of extra- D / P cellular polymeric substance produced by Listeria monocytogenes 159 Nwaiwu, 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 forming biofilms, it does not produced extensive amounts of extracellular polymer sub- stances (EPS) that contribute to adhesion and persistence in the environment. We present here evidence that under specific growth conditions L. monocytogenes is capable EPS synthesis and this does promote surface adhesion. After EPS extraction, analysis by NMR, ATR-FTIR and SEM indicated that the material is high molecular 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-positive bacteria). Further amino acid analysis also ruled out this material being a polymer of other amino acids, while elemental energy dispersion x-ray analysis showed very low nitrogen content with carbon and oxygen the predominant elements. The as yet unidentified polymer has unusual physical properties, and scanning electron microscopy (SEM) showed rapid absorption of moisture by the EPS as relative humidity increased. Hence it is likely that this polymer will also contribute to desiccation tolerance of the bacterium.

* Participation Supported by IUFoST.

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REFERENCE Ecology and persistence of Listeria monocytogenes strains in fermented / meat sausage processors from the Northern region of Portugal D P Ferreira, V.1, Barbosa, J.1, Vongkamjan, K.2, Moreno Switt, A.2, Hogg, T.1, Gibbs, P.1, 160 Wiedmann, M.2 and Teixeira, P.1 1. CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal 2. Cornell University, USA In this study, 202 Listeria monocytogenes isolates were recovered from product samples representing 7 processors of Alheira, a traditional fermented meat sausage from the Northern region of Portugal. Samples were collected from each processor in separate dates, either at retail establishments or at processing plants. Isolates were characterized by molecular serotyping and DNA macrorestriction analysis by pulsed field gel electrophoresis (PFGE). Characterization by PFGE suggested persistence of particular strains over time in the 7 different processors, the apparent time of persistence ranged from 14 to 32 months. While a number of studies have evaluated associations of different strain characteristics, including biofilm formation etc., with strain persistence in processing plants, no convincing and consistent evidence for specific strain characteristics that facilitate establishment of persistence have been found so far. We thus investigated whether phage susceptibility and presence of lysogenic prophages in L. monocytogenes may be associated with strain persistence in the environment. A subset of 41 L. monocytogenes isolates representing sporadic and persistent PFGE patterns was screened for lysogeny. Twenty six prophages where induced and their lytic spectrum against the 41 strains of L. monocytogenes was investigated. Lysogens included both sporadic 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 and 3a) or B (1/2b, 3b, and 7) isolates, there was no evidence for differences in phage susceptibility between persistent and sporadic strains. While our findings support that L. monocytogenes serotypes differ in phage resistance, strain persistence does not seem to be associated with enhanced phage resistance.

REFERENCE Biofilm formation and survival of L. monocytogenes and slaughter house D / P bacteria on surfaces at relevant environmental conditions 161 Langsrud, 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 surfaces are of concern in the food processing industry. Surface associated bacteria can lead to cross contamination of food and have serious consequences for human health and food quality. Persistence of Listeria monocytogenes in the food processing environment is a well known food safety concern. Also, other environmental bacteria may cause problems when persisting in the food industry. A better knowledge on the ability of dominant bacteria to produce biofilm and survive on food contact surfaces under relevant conditions is therefore important. We have studied biofilm formation and survival of L. monocytogenes and environmental bacteria isolated from surfaces in a meat slaughter house. Bacteria were isolated after cleaning and disinfection and prior to slaughter on a regular working day and identified by 16S rDNA sequencing. Selected isolates from dominating genera and L. monocytogenes were tested for their ability to form biofilms under various temperature conditions in a microtiter plate assay. We also studied survival on stainless steel under controlled temperature and humidity conditions. The susceptibility of surface adhered bacterial cells to four commercial disinfectants commonly used in the meat industry was investigated. Predominant bacteria in the slaughtering line belonged to the genera Pseudomonas, Serratia, Acinetobacter, Citrobacter, Aerococcus, Kocuria and Staphylcococcus. The data indicated low biofilm forming abilities of L. moncytogenes at relevant food industry temperatures, while other environmental bacteria showed variable biofilm formation at 12 and 20°C. Gram-positive bacteria showed excellent survival on stainless steel during incubation at 12°C, 70% relative humidity. The bactericidal effects of disinfectants on bacteria adhered to surfaces were low (< 3 log kill) for all tested disinfectants. In conclusion, survival of bacteria after cleaning and disinfection in the meat industry can be explained by resistance to disinfectants when dried on surfaces and either ability to survive at dry conditions or biofilm formation in the presence of water.

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REFERENCE Control of L. monocytogenes by lysozyme combined with olive leaf D / P extract in edible pullulan film coated on chicken breast fillets 162 Handan Baysal, A.* Izmir Institute of Technology, Department of Food Engineering, Urla, Izmir, Turkey In recent years, naturally occurring antimicrobial and antioxidant compounds have been preferably employed in meats because of their potential health benefits and safety compared with synthetic preservatives. The objective of this study was to evaluate the antimicrobial effect of olive leaf extract (OLE) and lysozyme added to pullulan film coatings against Listeria monocytogenes in raw chicken breast meat stored aerobically under refrigeration (4°C). The effectiveness of these compounds in a meat model system was evaluated by surface inoculation (approximately 106 CFU/g) of L. monocytogenes onto chicken breast fillets. Fresh chicken breast fillets used in this research was purchased from a local supermarket. The inoculated raw chicken breast fillets were treated before storage by dipping into 5% (w/v) pullulan film-forming solutions with and without the addition of antimicrobial agents: (a) deionized water, (b) 5% (w/v) pullulan + 20% (w/v) water extract 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 moisture, the samples were stored at 4°C. The inhibitory effects of OLE and lysozyme containing 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 research has demonstrated that the use of an edible film coating containing natural extracts or the application of a lysozyme solution is a promising means of controlling the growth and recontamination of L. monocytogenes on raw chicken breast meat stored under refrigeration.

* Participation Supported by IUFoST.

REFERENCE Evaluation of antilisterial activity by lactic acid bacteria D / P 163 Borges, 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 Listeria monocytogenes. It occurs primarily in elderly patients, immunocompromised individuals, pregnant women and their neonates. Some lactic acid bacteria (LAB) are able to produce antimicrobial compounds with activity against several pathogens, such as organic acids (lactic and acetic acids), hydrogen peroxide, antimicrobial enzymes and bacteriocins. The objective of this work was to evaluate the antimicrobial activity of selected LAB against L. monocytogenes. Thirty-five isolates of LAB, available at the culture collection of Escola Superior de Biotec- nologia, demonstrated inhibitory activity against 29 clinical isolates of L. monocytogenes. To characterize this antilisterial activity the antagonistic spectrum of each LAB culture, neutralized cell-free supernant and neutralized cell-free supernant treated with catalase or trypsin were investigated. All isolates of LAB showed antibacterial effect probably due to the production of bacteriocins. The bacteriocin activity (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 this study, the use of bacteriogenic LAB can be important to control L. monocytogenes of clinical origin.

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REFERENCE Molecular methods to assess Listeria monocytogenes route / of contamination in a dairy processing plant D P Cocolin, L., Alessandria, V., Dolci, P. and Rantsiou, K. 164 Univeristy of Turin, Italy In this study we investigated the occurrence of Listeria monocytogenes in a dairy processing plant during two sampling campaigns in 2007 and 2008. Samples represented by semi-finished and finished cheeses, swabs from the equipment and brines from the salting step, were subjected to analysis by using traditional and molecular methods, represented mainly by quantitative PCR. Comparing the results obtained by the application of the two approaches used, it became evident how traditional microbiological analysis underestimated the presence of L. monocytogenes in the dairy plant. Especially samples of the brines and the equipment swabs were positive only with qPCR. For some equipment swabs it was possible to detect a load of 104-105 cfu/cm2, while the ISO method employed gave negative results both before and after the enrichment step. The evidences collected during the first sampling year, highlighting a heavy contamination of the brines and of the equipment, lead to the implementation of specific actions that decreased the contamination in these samples during the 2008 campaign. However, no reduction in the number of L. monocytogenes positive final products was observed, suggesting that a more strict control is necessary to avoid the presence of the pathogen. All the isolates of L. monocytogenes were able to form biofilm, and, interestingly, considering the results obtained from their molecular characterization it became evident how strains present in the brines, were genetically connected with isolates from the equipment and from the final product, suggesting a clear 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 producing companies.

REFERENCE Persistence of L. monocytogenes in artisanal cheese producing plants D / P Almeida, G., Santos, I., Magalhães, R., Barbosa, J., Hogg, T. and Teixeira, P. 165 CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal The consumption of raw milk or raw milk products has caused several listeriosis outbreaks resulting in several hundred cases. This highlights the risk of these products and led public health officials to recommend that raw milk and dairy products prepared from raw milk should not be consumed by susceptible populations, particularly pregnant women. The presence of L. monocytogenes in cheeses has been widely reported in the literature over the years. The prevention of Listeria contamination is difficult to achieve in raw milk cheeses. Also, contaminated raw milk can contaminate the plant environment and some strains can colonize and persist for long periods. The present work aimed to characterize L. monocytogenes isolates recovered between 2004 and 2007 from an artisanal ewe’s raw milk cheese producing plant. PFGE using restriction enzymes AscI and ApaI was performed in forty isolates: 18 from cheese, one from raw milk, 20 from environmental sites, and one from whey. Six combined pulsotypes were obtained, one of them aggregating 20 isolates. This is an evidence of the presence of a resident clone with a persistence time estimated in at least, 14 months. The profile obtained from cheese isolates was also encountered in isolates from ewe’s raw milk and from raw milk reception’s floor suggesting that raw milk could be the source of contamination. Control L. monocytogenes in the environment plays an important role in reducing its presence in foods, which is necessary to reduce the incidence of listeriosis.

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REFERENCE Occurrence of Listeria monocytogenes in food products collected in D / P Portugal from retail establishments and food plants 166 Mena, 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. The ingestion of contaminated food with this microorganism may cause serious health problems for consumers. During two years (2007 and 2008) the presence of L monocytogenes was evaluated in a total of 1476 food samples, collected from retail and food plants. The detection of the microorganism was performed using the automated VIDAS system and the positive results were confirmed following the ISO 11290 standard. L. monocytogenes was detected in 134 (9.1%) of the analyzed samples. Most of positive samples were from foods normally submitted to heat treatment 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), vegetables 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 temperatures and could achieve levels of contamination that can cause disease. Also, foods that will have a heat treatment at consumer’s home could represent a hazard if cross contamination of food items that will be consumed without any further step of destruction occurs.

REFERENCE D / P Listeria monocytogenes biofilms grown at 12°C showed reduced susceptibility to sanitizers 167 Lourenço, A., Machado, H.* and Brito, L. CBAA – Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia, Technical University of Lisbon, Portugal Listeria monocytogenes may form biofilms on food contact surfaces of difficult sanitization which may lead to recurrent contamination of food products. The eradication of biofilms can only be achieved by using adequate hygienization routines which will ultimately ensure food safety. The biofilm forming ability of four L. monocytogenes strains from different origins, cheese, dairy environment and from human cases of listeriosis was evaluated, either in pure culture or in co-culture with Pseudomonas aeruginosa, at 37°C and 12°C using the Calgary Biofilm De- vice® (CBD). The minimum biofilm eradication concentration (MBEC) was determined for four commercial dairy sanitizers (one alkyl amine acetate based, T99; two chlorine based, T66 and DD and one phosphoric acid based, BP). Co-culture biofilms had an average total population of 7 to 8 Log10 CFU/peg. P.aeruginosa was the dominant species, either at 37°C or at 12°C, representing 99% of the total CFU/peg. L. monocytogenes biofilms grown, either at 37°C or 12°C, although with different incubation times (24 hours and 7 days, respectively) reached a similar cell density (6 Log10 CFU/peg). Nevertheless, the biofilms produced at 12°C were generally less susceptible to the sanitizers than when produced at 37°C. One of the strains (3880) retrieved MBEC values of 30720 µg/ml and 16000 µg/ml for T99 and BP, respectively. These values were above the maximum in-use recommended concentrations (T99 – 29700 µg/ml and BP – 11500 µg/ml) for these agents. The growth in co-culture also proved to be relevant regarding disinfectant susceptibility, as the co-cultures were generally less susceptible than L. monocytogenes pure cultures. The MBEC values obtained for the chlorine based agents were never over recommended in-use concentrations which may indicate a more efficient ability to eradicate biofilms, if in-use conditions are met.

* Participation Supported by IUFoST.

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REFERENCE Modelling growth of Listeria monocytogenes in cheese as function / of environmental variables D P Sand Rosshaug, P. and Hallberg Larsen, M. 168 Faculty of Life Science, Department of Veterinary Pathobiology, Denmark This project is part of a larger project with the purpose to develop a predictive model of growth of the pathogen Listeria monocytogenes in a dairy chain producing blue/white mould cheese. This project focuses on the growth kinetics of L. monocytogenes as function of environmental variables. Temperature, water activity, pH, lactic acid, NaCl, and O2 are environmental factors that impact the growth kinetics 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/white mould cheese. The growth kinetics of L. monocytogenes as function of these variables was formulated as an ordinary differential equation using Cardinal Parame- ters to describe the influence of the environmental factors. The growth kinetics was applied in a predictive mathematical, deterministic model of L. monocytogenes. Finally the growth kinetics of L. monocytogenes was investigated using a stochastic predictive model taking into account the stochastic nature of some of the inputs including the lag.

REFERENCE Characterization of anti-Listerial bacteriocin produced by Lactobacillus D / P plantarum ST8SH, a strain isolated from Bulgarian salami 169 Todorov, S. D.1* and Lemos Vaz-Velho, M.2 1. Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Brazil 2. Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Portugal Strain ST8SH, isolated from Bulgarian salami, was identified as Lactobacillus plantarum based on biochemical tests, sugar fermentation reactions (API50CHL), PCR with species-specific primers and 16S rDNA sequencing. Strain ST8SH produces a 3.0kDa class IIa bacteriocin, active against Listeria monocytogenes, Listeria innocua, Streptococcus caprinus, Streptococcus spp., Lactobacillus casei, Lactobacillus curvatus, Lactobacillus salivarius, Lactobacillus pentosus, Enterococcus mundtii, Enterococcus faecalis and Lactococcus lactis subsp. lactis. No change in activity was recorded after 2h at pH values between 2.0 and 12.0, and after treatment at 100°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-leakage and was visualised by atomic force microscopy. The highest level of activity (25600 AU/ml) was recorded when cells were grown at 37°C or 30°C in MRS broth (pH 6.5). Peptide ST8SH adsorbs at low levels (400 AU/ml) to producer cells. High cell numbers of L. plantarum ST8SH and L. innocua LMG13568 were recorded at beginning when co-cultured. However, the cell numbers of L. innocua LMG13568 decreased from 1.6x104 CFU/ml to 2.5x102 CFU/ml in 12h and to undetectable levels after 24h. Plantaricin ST8SH production was stimulated by presence of L. innocua LMG13568 (102 400 AU/ml). Similar results were obtained with addition of 10% autoclaved overnight culture of L. innocua LMG13568 to MRS growth media on production of plantaricin ST8SH. Based on the genetic approach strain ST8SH harbours associated genetic determinants for production of a variation of the well known plantaricin 423. Future purification of the produced bacteriocin need to be performed to determine if Lactobacillus plantarum ST8SH produces this bacteriocin (plantaricin 423 – like) or harbour more then one bacteriocin operons.

* Participation Supported by IUFoST.

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REFERENCE EFSA’s proposal for an EU-wide retail survey on Listeria monocytogenes D / P in selected categories of ready-to-eat food products 170 Boelaert, 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 technical specifications on an EU-wide retail survey on Listeria monocytogenes in selected categories of ready-to-eat (RTE) food products that should take place during the whole year of 2010. The proposed technical specifications focus on sampling those categories of RTE food in which the highest L. monocytogenes contamination have been observed in the European Union (EU): soft and semi-soft cheeses, smoked and gravad fish, and heat-treated meat products that are handled after heat treatment. Sampling of these RTE food categories would be targeted at retail outlets serving the final consumer, with catering and wholesale establishments excluded. Food products are suggested to be tested at the end of the shelf-life and additionally in the case of smoked and gravad fish, immediately after sampling. At the Community-level a total of about 3,000 samples should be taken for each RTE food category, per analyses stage. A specific number of samples is proposed to be proportionally allocated to the Member States according to the size of their human populations. Standardised analytical L. monocytogenes detection and enumeration 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 the L. monocytogenes prevalence at the Community level. A modelling and simulation approach should be applied in the analyses of the results so that the effectiveness of the implementation of Community L. monocytogenes criteria may be assessed. A similar model-based approach will also be used to estimate the growth potential of L. monocytogenes in smoked and gravad fish.

REFERENCE D / P Ripening conditions: an asset to control L. monocytognes in cheeses 171 Callon, C., Picque, D., Corrieu, G. and Montel, M.-C. INRA, France The EC regulations for dairy products require the absence of L. monocytogenes output production with a possible derogation at < 100cfu/g if it is demonstrated that there is no evolution during storage until consumption. To identify the microbial 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 following steps: 1) selection of milk with anti-Listeria properties, 2) identification of microbial communities and simplifications of their composition, 3) cheese-making experiments in different conditions of ripening. This strategy was successfully applied to the inhibition of L. monocytogenes in cheese core. Indeed, a microbial community of raw milk, composed of 7 strains of lactic acid bacteria (Lactobacillus and 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 community. The decrease of relative humidity from 98 to 93% can also act as a barrier against L. monocytogenes, especially at the surface of cheeses with or without microbial consortium. It led also to an increase of dry matter at the surface of cheeses and 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, by favouring the growth of Lactobacillus and Leuconostoc composing the microbial consortium, was more effective than 9°C for inhibiting Listeria. This can be explained by the highest galactose metabolism and acid production. On the contrary in the control with only S. thermophilus, the ripening temperature of 13 °C favoured the growth of L. monocytogenes. The ripening conditions reasoned according to the composition of microbial communities could be a promising strategy in the control of L. monocytogenes.

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REFERENCE Deterministic and stochastic behavior of Listeria monocytogenes / suspended cells or detached from stainless steel surfaces during D P cheese manufacturing 172 Belessi, 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, Greece 2. Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland Growth probability and kinetic models for Listeria monocytogenes in response to multiple hurdles occurring during cheese manufacturing are mainly focused on suspended L. monocytogenes cells. This study aimed to compared: (i) the growth/no growth interface of L. monocytogenes cells attached on stainless steel (SS) surfaces, or in suspension, within adjusted media and (ii) the behavior of planktonic and detached Listeria cells during manufacturing and ripening of two popular Greek cheeses: Feta and Graviera. A multi-strains composite of L. monocytogenes isolates from cheese, factory and farm in Greece and Ireland, were grown in 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), and cells 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 in TSBYE, 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 inoculated at three simulated stages of Feta and Graviera manufacture: in pasteurized milk, after cutting the curd and after the first ripening. The growth of D cells slightly delayed compared to P cells while it was more affected by aw than pH. On cheese, L. monocytogenes survived throughout the ripening at low levels. The differences in probability of growth of single cells for both inocula (P and D) were assessed by stochastic approaches. Furthermore, PFGE analysis resulted that 91% of the cells of any tested condition belonged to the cheese factory isolate. The results may address safety implications relevant to the potential of attached cells to proliferate, whereas data may contribute to filling data gaps on risk assessment of L. monocytogenes isolates from the dairy industry.

REFERENCE Prevalence of Listeria monocytogenes in game meat D / P Atanassova, V. 173 Institute of Food Quality and Food Safety, Germany Listeria spp. are widespread in the environment. Foods of animal origin like raw meat, milk, soft cheese and smoked fish are often contaminated by Listeria. During slaughtering and food processing, these bacteria can distribute and establish as specific contaminants in difficult to clean areas of the plant and the processing environment. Freshly shot game meat can be readily contaminated during the cutting and trimming steps. Listeria endures most non heat processing steps and will be present in packaged retail meats. Chill storage for long periods of time can result in a considerable increase in numbers. In this study 797 (roe deer, red deer and wild boar) samples of wild game from freshly shot whole carcasses and 481 samples from packaged game meat stored under chill conditions (+ 4°C) were analyzed for the presence of Listeria spp. and Listeria monocytogenes. Standard methods according to ISO 11290 were applied. Enrichment of meat samples was performed first in half Fraser, followed by full Fraser broth. Enrichment cultures were streaked to OCLA plates. In total 7.3% (n=58) of the meat from whole carcasses was positive for Listeria spp., with a total of 39 (4.9%) samples being contaminated by Listeria monocytogenes. There was no difference in the prevalence in meat from different game species. Chill stored packaged game meat showed higher prevalence rates of 20.6% (n=99) Listeria monocytogenes positive samples. The results 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 meat cuts. To reduce the risk of infection for the consumer, proper cooking is advised during meal preparation.

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REFERENCE Relationship between pathogenic profile and in vitro biofilm formation D / P capacity of Listeria monocytogenes strains isolated from meat, fish and 174 processing plants Meloni, 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 vitro biofilm formation of 106 Listeria monocytogenes strains having no epidemiological correlation and isolated from different environmental and food sources, were analyzed. The isolates were grouped into different categories based on the source of isolation: 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 salmon processing plants environments (24 and 16% respectively). The quantitative assessment of the in vitro biofilm formation was carried out by using a microtiter plate assay with spectrophotometric reading (OD620). The cut-off value (ODc) was equal to three time the standard deviation of the negative controls plus the average OD 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 producers (ODc

REFERENCE D / P Prevalence and molecular characterization of Listeria monocytogenes in traditional fermented pork sausages produced in Italy 175 Mazzette, 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 monocytogenes in fermented sausages produced in the Sardinia region (Italy). The environments and products from two large and two small processing plants, representative of the regional supply chain, were sampled to investigate prevalence and genetic profile of Listeria monocytogenes. A total of 170 samples were collected: 132 environmental samples, 12 from swine carcasses, 16 from ground meat and 10 from fermented sausages at the end of ripening. Detection and enumeration of Listeria monocytogenes were carried out by using the ISO 11290-1:1996 and 11290- 2:1998 protocols respectively. A subset of 28 strains, randomly selected between those isolated from the positive samples, were further characterised by multiplex PCR-based serogrouping. The contamination routes of Listeria monocytogenes in the plants were traced by use of PFGE restriction analysis. In addition, a quantitative assessment of the in vitro biofilm formation was also carried out. The results underlined the presence of Listeria monocytogenes in all the fermented sausages processing plants (overall prevalence in the environments, 15%). The prevalence of Listeria monocytogenes was37%ingroundmeatand80%inthedry sausages. These products did show detectable levels, always ranging below 100 CFU/g. No specific serotype was recovered during the processing and ripening of the dry sausages. The serotypes 1/2b - 1/2a showed better ability in biofilm formation. A high heterogeneity of pulsotypes (n=14) occurred within the plants, with subtypes appearing to be specific to each processing plant. These results may be due to the great diversity of the Listeria monocytogenes strains collected from raw meat, but also to the quantities used by each plant. The large plants exhibited the highest PFGE profiles heterogeneity, supporting this hypothesis, since these plants also used the greatest amount of raw meat from several different sources.

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REFERENCE Minimum Biofilm Eradication Concentration (MBEC) / of different antimicrobials on Listeria monocytogenes D P and Salmonella enterica biofilms 176 Rodrigues, 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 do Minho, 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 foodborne disease outbreaks, particularly those concerning L. monocytogenes and Salmonella enterica. The ability of these bacteria to adhere and form biofilms on several surfaces makes sanitation more difficult and challenging, reason why susceptibility tests must cover not only planktonic cells but also adhered cells and biofilms. Through MBEC assessment using Calgary Biofilm Device (CBD), this work aimed at comparing the performance of four antimicrobials on L. monocytogenes and Sal- monella enterica biofilms and the disinfection efficiency between strains and species. Three L. monocytogenes strains (994, 1562 and CECT 4031T) and five S. enterica strains (355, CC, NCTC 13349, LT2 and ATCC 140285) were used. Biofilms were grown on CBD for 24 hours, in Mueller-Hinton Broth, at 37°C with shaking at 125 rpm. Disinfection was performed with sodium hypochlorite, benzalkonium chloride, hydrogen peroxide and triclosan, while bacterial death was assessed by standard plate method on Trypticase Soy Agar after sonication. Results showed that sodium hypochlorite had the lowest MBEC values while triclosan had the worst performance, since no S. enterica biofilm eradication was achieved even at the maximum concentration used. It was also found that, except for hydrogen peroxide, MBEC values for L. monocytogenes biofilms were similar or inferior to those found for S. enterica. Significant differences on minimum survival biomass results were also observed between strains of the same species. Summarizing, this work has pointed out chlorine agents as the most effective on disinfecting biofilms of both species used and revealed a higher susceptibility of L. monocytogenes biofilms to disinfection in general when compared with S. enterica biofilms. Moreover, not only interspecies but also intraspecies variability were found to influence disinfection efficacy.

REFERENCE Examination of the ability of adherence, biofilm formation and sensi- D / P tivity to some disinfectants of different Listeria monocytogenes strains 177 Milanov, 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 Serbia 2. Faculty of Veterinary Medicine, Republic of Serbia The objective of this work was to examine the capabilities of 14 Listeria monocytogenes 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. monocytogenes was determined after 3-hours of attachment and after 48-hour incubation in tryptone soy broth at 25°C and 37°C by the use of standard count technique on blood agar from tenfold dilution. Three days old biofilm formed on glass surfaces of the selected L. monocytogenes strains was treated with paracetic acid and phenol disinfectants for 5 and 10 minutes. On the stainless steel surface the biofilms were formed during 7 days of incubation in a tryptone soy broth supplemented with 0.6% yeast extract (TSB-YE) at the temperature of 25°C. The developed structures were examined using scanning electron microscopy. For all the examined L. monocytogenes strains the number of attached bacteria to glass slides for 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 slides did 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 of their ability to form biofilm were found. Seven of 14 investigated strains of Listeria monocytogenes did not form biofilm, and only individual bacterial cells were distributed over the stainless steel surface. The strains classified as biofilm producers formed structures of different appearances, from a uniform, confluent monolayer of bacterial cells to individual large, three-dimensional cell aggregates. L. monocytogenes cells attached to glass surface expressed higher resistance to disinfectants than the cells in suspension.

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REFERENCE Evolution of Listeria monocytogenes contamination D / P in poultry production: from the farms to the processing levels 187 Mansour 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 update data regarding L. monocytogenes in poultry flocks (laying hens, broilers, turkeys) and the impact of their introduction in the slaughtering process. Samples were collected from the flocks (5 bootswabs) which were followed up to the slaughterhouses where sampling consisted of swabs from the environment (before and after cleaning and desinfecting procedures), caeca and products (neck skins and fillets). The isolation of L. monocytogenes was performed according to the NF EN ISO11290-1 method and the identification included serotyping. RFLP-PFGE was carried out in order to establish the clonal relationships and to trace the contamination between the farms and the slaughterhouses. 32% of the flocks were tested positive for Listeria monocytogenes. The serotyping of L. monocytogenes strains showed that the majority belonged to the type 1/2a which presented high genetic diversity (Simpson Index: 0.95). At the slaughterhouses, the contamination varied between 4 and 26% of the total sampling. Positive samples were found in the environment after cleaning and desinfection and the same isolates were found in the environment between the slaughtering of other batches and on the products. Strains found at the farm level were clearly different from those found at the slaughterhouses. Our work highlighted the spreading of L. monocytogenes in poultry flocks, residual and cross contamination at the slaughterhouses. Despite a high level of faecal contamination at the farm level, no relation could be established with the contamination of slaughterhouses. This suggests that the primary production is not the main source of contamination of poultry products. Efforts should be focused on the slaughterhouses where inefficient C&D procedures and cross contamination were the main identified sources of contamination.

REFERENCE D / P 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., 188 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. The consumption of food products contaminated with this bacterium can cause listeriosis, a disease with a high mortality rate that can affect especially vulnerable groups. The ubiquitous nature of Listeria, its resistance to several environmental conditions and the ability to grow at refrigeration temperatures, promote the occurrence of contamination in any stage of the food chain. Taking simple precautions like thoroughly cooking foods, respecting the chill chain, proper washing fruit and vegetables eaten raw and washing hands repeatedly, reduce contamination chances. The main activity of our Laboratory, it is the control of the microbiological quality of ready-to-eat foods served in canteens. This regular survey includes 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 and swabs in surfaces and utensils. This work evaluate the presence of Listeria monocytogenes (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 establishments surveyed by INSA Lisboa, between 2004 and 2009. In a total of 5450 ready-to-eat foods 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 consumed by risk populations, such as those of kindergarten, schools, hospitals and homes for the elderly, and that our positive results correspond to 58.9% of samples collected in these type of establishments, they could stand as a useful tool, demonstrating that if additional precautions are not implemented, Listeria monocytogenes can be a potential risk for these particularly susceptible groups, even at concentrations less than 100 cfu/g.

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Incidence of Listeria monocytogenes in Queijo Fresco REFERENCE D / P Rosa, N., Campos, L., Correia, C., Ferreira, I., Furtado, R., Maia, C., Santos, S., Cunha, C. I. and Santos, M. I. 189 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 cases and outbreaks of listeriosis in humans, usually transmitted by the consumption of 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 a major concern to Public Health. It was the purpose of this study to evaluate the presence of Listeria monocytogenes in “Queijo Fresco” from a number of brands with 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 examined for the presence of Listeria monocytogenes. Secondary enrichments, in Fraser broth, were analysed by the mini-VIDAS LMO® (bioMérieux, Durham, France), enzyme-linked fluorescent immunoassay method for Listeria monocytogenes detection. Positive samples were confirmed by isolation on ALOA® (AES CHEMUNEX) selective agar followed by biochemical characterization with API LISTERIA® (bioMérieux, Durham, France). Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS v13.0) software. Of 125 samples, 13 (10.4%) were positive for Listeria monocytogenes. It was statistically proved that the Commercial Presentation influences the presence of Listeria monocytogenes, being the cheeses sold packed less contaminated than those sold unpacked (p =,049). This study demonstrates that Listeria monocytogenes is present in “Queijo Fresco” from a range of brands commercialized in Lisbon. The contamination observed, represents a potential risk for the Portuguese consumer due to his natural taste for this product.

Risk factors for Listeria monocytogenes contamination REFERENCE D / P in French broiler flocks Aury, K., Le Bouquin, S., Toquin, M.-T., Petetin, I., Le Nôtre, Y., Allain, V., 190 Fravalo, P. and Mansour Chemaly, M. AFSSA, France Despite a decreasing incidence of Listeriosis in France since 1999, Listeria monocytogenes continue to be a public health concern. In order to update data relating to L. monocytogenes in France in poultry production, an epidemiological study was conducted in broiler chicken flocks between October 2005 and September 2006 aiming to identify the potential risk factors associated to the presence of L. monocytogenes. 142 broiler chicken flocks were included in this study. The L. monocytogenes status of these flocks was based on 5 samples of bootswabs per farm. The flocks were considered positive if at least one sample was tested positive for L. monocytogenes. Information on potential risk factors was collected by questionnaire at the same time as sample collection. The association between characteristic management practices and L. monocytogenes status was assessed by logistic regression. The prevalence of L. monocytogenes contamination in these flocks was 31.7%. The risk of L. monocytogenes contamination was increased when farmer did not respect the principle of the two areas (clean and dirty) at the poultry house entrance. The absence of spraying at the first disinfection and/or pest control of the poultry house before the arrival of the next flock was found to increase the risk of being infected. When litter storage was not protected and when farm staff take care of the other broiler chicken houses of the holding, the risk of L. monocytogenes contamination increased significantly. For the watering system, pipettes without recuperator were found to be associated with a higher risk of contamination than pipettes with recuperator or drinkers. This study brings new insights for the risk management of L. monocytogenes infection in broiler flocks.

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REFERENCE Portuguese sushi: is it contaminated with L. monocytogenes? D / P 191 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 the consumption of ready-to-use products of seafood, like sushi, that were previously restricted to oriental countries. Sushi is a traditional Japanese food, mostly composed of rice and raw fish. The sushi trend involves a higher consume of raw fish, particularly among young, urban people including pregnant women. Although fish is considered a healthy food, as with other animal products, consumption of raw muscle incurs potential health risks such as ingestion of pathogenic bacteria. Ad- ditionally, the preparation practices involved in the production of sushi has the potential to allow contamination, namely with Listeria monocytogenes, the agent of listeriosis, an infection that targets mainly pregnant women (and their fetuses), children, the elderly and immunocompromised individuals. In spite of the number of cases per annum is relatively low, these infections can be acute, with mortality up to 30%. Due to the seriousness of clinical manifestations and high rates of mortality in populations at risk, control and prevention of this disease, represents an important challenge to sanitation authorities and deserves the attention of food microbiologists and health professionals. In this study, 90 samples of sushi collected from different establishments in Lisbon, Portugal, were analyzed for their microbiological status and the prevalence of pathogenic bacteria. For L. monocytogenes detection the ISO 11290-2:1998/Amd 1:2004 method was performed. The results obtained showed that all samples were negative for L. monocytogenes and just one of them revealed the presence of Listeria seeligeri. Although the number of samples studied was small, we believe this study provides important information concerning the incidence of L. monocytogenes in Portuguese sushi and also evidences that the consumption of this type of food probably is not a major problem in relation with this pathogen.

REFERENCE D / P Prevalence of Listeria monocytogenes throughout the production process of Parma ham: tracing contaminations from slaughterhouses 192 to the final product Prencipe, 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”, Italy 2. University of Bologna - Veterinary Medicine, Italy 3. Consorzio del Prosciutto di Parma, Italy 4. Centro Ricerche Produzioni Animali S.p.A., Italy In order to evaluate Listeria monocytogenes prevalence and contamination levels throughout the Parma Ham production chain, 774 swine carcasses were traced along the whole process until the final product. Analyses were carried out on samples originated from the same carcass but taken at three different points of the process (as carcass, fresh ham and dry-cured ham). Fecal samples were also taken from 498 carcasses (about 2/3 of the whole sample). The prevalence of Listeria monocytogenes, calculated at 2.9% in the carcasses swabbed in slaughterhouses, increased up to 12.5% in fresh hams sampled when entering the manufacturing plants and eventually fell to 2.0% at the end of the production chain (de-boned and packaged dry-cured hams). Listeria monocytogenes was isolated from only one fecal sample (prevalence 0.2%), corroborating the evidence of the low importance of 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/g in fresh hams, 0.04 to 100 UFC/g in dry-cured hams sampled at the end of the production chain. Only the 3.2% (n =3) of contaminated fresh hams came from contaminated carcasses, no one among the 14 contaminated dry-cured hams came from contaminated fresh hams. Certainly, cutting was the stage with the highest contamination risk throughout the Parma Ham production process. However, beside the sharp reduction of contaminations subsequent to dry-curing, seasoning environment should be addressed as the critical step of the whole process for the contaminations found in the final product. The significant differences between plants corroborates the importance of the processing environment as source of contamination from Listeria monocytogenes. Further biomolecular analyses are needed in order to confirm the marginal role of transferring contaminations between different stages of the production chain and to assess the presence of persistent strains inside the processing plants.

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REFERENCE Effect of the inoculum size on growth of L. monocytogenes / in dices of poultry breast D P Lardeux, A.-L., Gnanou-Besse, N., Doux, C. and de Courseulles, E. 193 AFSSA, France Traditionally the microbiological safety of foods has been established via challenge tests. Since the contamination level attained by Listeria monocytogenes may depend on the initial bacterial concentration, it is important to take into account this effect when performing challenge tests. To date, there are, to our knowledge, few studies which have examined its impact in food. The effect of the inoculum size on growth of L. monocytogenes in a matrix of dices of poultry breast has been studied. This matrix 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 tests have been carried out for three batches, according to technical guidance document of Community Reference Laboratory for Listeria monocytogenes. Thus, the fastest strain of L. monocytogenes has been selected by a study of strains growth with potassium lactate at different concentrations. Then, L. monocytogenes growth studies 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 % carbon dioxide). Follow-ups of natural microflora (mesophilic and lactic) have been performed, as well as the measurement of physical-chemical parameters (pH, aw and lactic acid). L. monocytogenes was able to grow in all conditions. However, growth was higher under vacuum packaging conditions, and with a high inoculation level. Moreover, important differences were observed between batches. In parallel, an important growth of background microflora was observed, which probably had a strong impact on L. monocytogenes growth.

Investigation into the mechanisms of detergent induced changes REFERENCE D / P in disinfectant susceptibility of attached Listeria monocytogenes 194 Walton, J., Hayes, R., Protheroe, R., Hill, D. and Gibson, H. University of Wolverhampton, UK The control of Listeria through effective cleaning and disinfectant strategies is crucial to maintaining the safety of food. The aim of this work is to investigate the effect of detergent treatments on the susceptibility of attached Listeria monocytogenes to subsequent disinfectant treatments. While the detergents were prepared to working concentrations, the disinfectants were lower than the recommended in use concentration to allow quantification of changes in susceptibility. Results so far have shown that L. monocytogenes, attached to stainless steel surfaces, became significantly less susceptible (up to 3.5 log10 difference) to benzalkonium chloride (BAC, 0.005% v/v) following treatment with the anionic detergents sodium alkyl sulphate (SAS, 0.2% v/v), sodium dodecyl sulphate (SDS, 0.2% w/v) and sodium lauryl ether sulphate (SLES 0.2% v/v). L. monocytogenes also became significantly less susceptible (0.4 log10 difference) to sodium dichloroisocyanurate (NaDCC, 0.0008% w/v) following treatment with SAS but significantly more susceptible (1 log10 difference) following treatment with SLES. The non-ionic detergent, fatty alcohol ethoxylate (FAE, 0.1% v/v), had no effect on susceptibility to either BAC or NaDCC. The changes in susceptibility may be due to effects on cell membrane permeability, cell surface hydrophobicity and/or reduced uptake due to efflux. Flow cytometry using the fluoresceine propidium iodide revealed significant increases in cell membrane permeability by SAS and FAE while no change was observed with SDS. Hydrophobic interaction chromatography showed that L. monocytogenes became less hydrophobic following treatment with SAS and SDS but FAE had no effect. Current work using ethidium bromide has shown a reduction in fluorescence following treatment with SAS and SDS suggesting that the detergents may trigger an efflux mechanism resulting in reduced uptake of disinfectant. To conclude, detergents can influence the susceptibility of L. monocytogenes to BAC and NaDCC which does not appear to be related to changes in cell membrane permeability. However, susceptibility does appear to correlate with changes in cell surface hydrophobicity and enhanced efflux.

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REFERENCE Prevalence of Listeria monocytogenes in raw milk sold D / P at vending machines in Abruzzo region 195 Prencipe, 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 of raw 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 detect Listeria monocytogenes. Moreover, the presence of the compulsory information for consumer and the observance of the proper storage temperature were verified. 31 samples (10.5%) positive for Listeria monocytogenes were found, taken from 6 distributors supplied by 3 farms. 29 over the 31 positive samples were collected at 4 vending machines supplied by only one farm. All the Listeria monocytogenes strains were identified as serotype 4 b. The resistance to two antimicrobials 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 samples had been found contaminated by Listeria monocytogenes. The combination of the resistance patterns and restriction profile of the 29 samples isolated from A farm, identified three different subtypes. The persistence of Listeria monocytogenes in raw milk samples, supplied by A farm, confirmed that an inadequate hygiene management exposes the consumer to a real risk of infection by Listeria monocytogenes. The absence of information for the consumer and the lack of cold chain maintenance during the distribution step could increase this risk. Therefore, it is necessary that farmers and distributors’ managers apply continuously the own check and its efficacy should be verified by the Competent Authority implement- ing specific surveillance plans. However it is fundamental to inform the consumer correctly and on continuous basis about the potential risks associated to food products and on the right handling procedures during transport, storage and treatment of food products.

REFERENCE Characterization of Listeria monocytogenes strains isolated from soft D / P and semi soft cheeses sampled at retail level 196 Acciari, 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 strains isolated during a survey carried out on cheeses sampled at retail level. Five semi- soft and soft cheeses (gorgonzola, taleggio, asiago, crescenza and brie) were selected among the higher consumption dairy products in Italy and the most frequently contaminated by Listeria monocytogenes. Each strain was serotyped, tested for susceptibility to antimicrobials and pulsotyped using AscI and ApaI Pulsed Field 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 resistance patterns showed that most Listeria monocytogenes strains were resistant to oxacillin (97.6%), lincomicin (80.9%) and clindamycin (78.7%). The resistance to two antimicrobials with two different resistance patterns (OXCC, OXL) was detected in 17% of the strains tested, to three antimicrobials with one resistance profile (OXCCL) in 70.2%. No strains were sensitive to all antimicrobials tested. Combination of AscI and ApaI macrorestriction patterns yielded 11 different pulsotypes clustering in three groups. Two main pulsotypes were found by grouping 21.3% and 57.4% of the strains isolated. Evaluation of PFGE profiles showed no relationship between pulsotypes and cheese type, manufacturer or retail outlet. Temporal distribution of the prevailing pulsotypes showed a persistent profile throughout most of the study period, except from August to September, when a different pulsotype was found. Therefore the temporal variation in the prevalence of specific strains, as reported in this survey, could be the consequence of factors able to influence the product contamination pattern. Large scale studies will contribute to assess the dynamics of Listeria monocytogenes contamination.

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

REFERENCE Preliminary report on the organisation of a food microbiology / proficiency testing program as a tool to guarantee the equivalence D P of the US and IT official control systems 197 Di 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 2002 the Italian Ministry of Health developed an integrated approach to assure the equivalence between the Italian and the US official control systems. A key part of the ongoing program has been carried out thought an ad hoc laboratory proficiency testing with the aim of monitoring the technical skills of the official laboratories in charge of testing Listeria monocytogenes on meat products to be exported in the USA. This study describes the organization of a proficiency testing and the protocol experimented for preparation and verification of samples consisting in food matrix with a basic microflora contaminated with Listeria monocytogenes at two different level of contamination. The proficiency testing is an indispensable tool to assess the performance level of a testing laboratory and to demonstrate its reliability at national and international framework. In our specific context, the proficiency program has also concurred as one of the effective measures to guarantee the equivalence within the certifications issued by the Italian and US official laboratories.

High nisin susceptibility of Listeria spp. wild-type strains isolated REFERENCE D / P from dairies with traditional cheese preservation in Portugal Pintado, C. M. B. S1,2 and Ferreira M. A. S. S.2 198 1. Escola Superior Agrária, Instituto Politécnico de Castelo Branco, Castelo Branco, Portugal 2. Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Lisboa, Portugal Evaluation of nisin susceptibility of 219 Listeria spp. wild-type strains isolated from milk, cheese and cheese processing environment at given conditions was the aim of this work. Minimum inhibitory concentrations (MIC) were evaluated by the agar incorporation method, on TSYEGA medium. The variability of Listeria spp. susceptibility was very low especially at pH 5.5, with 98% of isolates showing MIC values between 10 and 50 IU ml-1 at 37°C. The increase of pH from 5.5 (average of cheese core pH) to 6.8 (average of cheese rind pH) at 37°C resulted in an increase in the MIC values by 5 to 20 times (from 10 – 50 to 50 – 100 IU) or more (> 100 IU ml1) in 68% of isolates. An increase in temperature from 20°C to 37°C at pH 5.5 resulted in an increase on MIC values only in 8% of all isolates tested. Nisin spontaneous resistance at a frequency of 10-3 to 10-4 was observed more frequently at pH 6.8 and 37°C. Exposure to combined stress conditions different from the ones tested herein was previously reported to increase cell resistance mechanisms and indicates that changes in virulence may occur concomitantly. Nevertheless, the majority of strains tested were able to grow at 10 IU nisin ml-1 but not at 50 IU ml-1 at pH 5.5, 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 demonstrated a good potential for use in bioactive coatings as a combined hurdle effect to control L. monocytogenes strains in dairy products.

AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control POSTERPRESENTATIONS // P / 178–181

REFERENCE What is an appropriate level of protection for Listeria monocytogenes E / P in foodstuffs consumed by vulnerable groups? 178 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 important route of transmission of listeriosis and a wide range of food products have been shown to be associated with both outbreaks and sporadic cases. Food safety criteria for L. monocytogenes in Regulation (EC) No. 2073/2005 are applicable to three categories of ready-to-eat (RTE) foods: absence of L. monocytogenes in 25g is required 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 within shelf-life. However, vulnerable groups (pregnant women, the immunosuppressed, the elderly, and many patients in hospitals) are at particular risk of infection, and consumption of low levels of L. monocytogenes may be of greater risk when eaten by these groups. Source attribution of L. monocytogenes infections in England and Wales to RTE food sources has shown that the most important source for the overall population were multi-component foods (e.g. sandwiches, pre-packed mixed salads) (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 infection. Concurrently, a number of outbreak investigations in England, Wales and Northern Ireland have revealed that infections were linked to consumption of sandwiches served to the vulnerable group population in hospital settings. Sand- wiches tested in these investigations contained the outbreak strain of L. monocytogenes at very low concentrations (present at <10/g) and legally would be deemed safe by the Regulation. However, the legal limit of 100 cfu/g is not based upon formal dose-response formulas and given the recognised rise in listeriosis observed in the EU, it is questionable whether the legal limit affords the level of protection deemed appropriate to protect the vulnerable group population.

REFERENCE ILCD: An Interactive Listeria culture diversity knowledgebase E / P Ashok Kumar, J.1, Barbuddhe, S. B.1, Kalekar, S.1, Rodrigues, J.1, Chopade, N. A.2, Hain, T.3 179 and Chakraborty, T.3 1. ICAR Research Complex for Goa, Ela, Old Goa 2.Department of Pathology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur, India 3.Institute of Medical Microbiology, Justus Liebig University, Giessen, Germany Indian Listeria Culture Database (ILCD) is an online databank of profiles developed for the Listeria strains isolated in India from various sources. ILCD is based on a relational database management system that is hyperlinked to visualize phe- notyping and genotyping results in the form of tables and DNA fingerprint images for individual strains. The system has been developed using open source LAMP (Linux, Apache, MySql and PHP) tools. The database contains geographical source of the strain, its lineage, serotype, source of isolation (animal/human), year of isolation, phenotypic and genotypic characteristics, antibiotic sensitivity patterns, and DNA fingerprint (PFGE image). This is an interactive web based database so that the data can be exchanged between laboratories electronically. A flexible search system based on systematic comparisons of the database entries allows inter-laboratory comparison of profiles. There are search options, state wise, serotype wise and so on. The parameters can be selected to search the information required. Whole description of a particular strain can be visualized by clicking on identity number of the strain. The data can be stored and accessed via internet browser. The web interface of ILCD allows users with no special programming background or bioinformatics experience to examine the results. The user is able to view the description of any particular strain e.g. isolated from human or animal or food source; orretrieve all information about a particular strain. Being the first of its kind, ILCD is expected to be a very helpful tool in strengthening the concept of ‘geographic genomics’ and will be very helpful to molecular epidemiol- ogists and those interested in research on Listeria.

AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control POSTERPRESENTATIONS // P / 178–181

REFERENCE Listeria spp. and the domestic environment: consumer knowledge, / attitudes, risk perceptions and food-handling behaviours E P Redmond, E. C. 180 Cardiff School of Health Sciences, UK In Europe, since 2000 incidence of listeriosis has increased by >59%. This increase has occurred almost exclusively in adults aged > 60 years with listerial bacteremia. The importance of the home as location for acquiring foodborne disease has prompted numerous studies to understanding of consumer food safety practices in the domestic environment. Cognitive and behavioural data is required to develop effective communication initiatives to improve food-handling practices and decrease incidence. This study aims to review microbiological studies and determine consumer risk perceptions, knowledge and domestic food-handling behaviours of that may contribute to the risk of listeriosis. Electronic searches of the Internet and library databases, personal-communication with food safety professionals and attendance at international conferences has facilitated the collection of 143 cognitive and behavioural consumer food safety studies and 16 microbiological surveys of the domestic environment carried out over the past 35years. Listeria has been isolated from up to 62% domestic kitchens and frequently contaminated items include refrigerators (2-10%isolations) and kitchen dish- cloths/sinks (10-84%isolations). Cumulatively, 8% of all consumer food safety studies 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% temperatures exceeding recommendations. Observed risk-related handling malprac- tices implemented by adults aged 60-75years are linked to pathogenic contamination of kitchen surfaces and ready-to-eat (RTE) foods. Considerable inadequacies in food safety practices and behavioural influences have been identified that may increase risk of listeriosis. Data comparisons between consumer groups will be presented, including likelihood of consuming RTE foods past use- by-dates, frequency of checking refrigerator temperatures and cleaning of refrigerators. Findings will be discussed in the context of strategy development and future consumer food safety communication iniatitives designed to decrease incidence of listeriosis.

REFERENCE Do you know the temperature in your refrigerator? E / P Røssvoll, E.1, Jacobsen, E.2, Ueland, Ø.1, Einar Granum, P.3 and Langsrud, S.1 181 1. Norwegian Institute of Food, Fisheries and Aquaculture Research 2.SIFO, National Institute for Consumer Research, Norway 3.Norwegian School of Veterinary Science The use of legislation to reduce risk is at present used at all steps of the food production chain from the primary producer to the stores. It is, however, both difficult and undesirable to manage consumer food handling through legislation. As soon as the products are bought by the consumer, the knowledge about how the product is treated further terminates. The aim of this study was to improve food safety in the domestic environment by risk analysis of consumer food handling and evaluation of risk-reducing measures. We wanted to get a better understanding of the interaction between consumer food handling practices and the potential growth of pathogens under domestic conditions. To investigate this we conducted a consumer survey, a case study, and a microbial experiment and showed that 1) consumers were ignorant to or unaware of the temperature in their refrigerator, 2) ready-to-eat (RTE) foods were exposed to great temperature variations by consumers, and 3) the fluctuations in temperature resulted in enhanced growth of psychrophilic pathogens as Listeria monocytogenes. In conclusion, consumers ex- pose themselves to increased risk of contracting food borne diseases by being ignorant to or unaware of the temperature in their refrigerators. The temperature fluctuations found in the case study enhanced pathogenic growth, and can in worst cases lead to outbreaks of food borne diseases.

AREA // A // Biology of Listeria monocytogenes POSTERPRESENTATION// A/ 47

REFERENCE Listeria monocytogenes agr system: Quorum sensing, or maybe not? A / P 47 Garmyn, D., Révelin, C. and Piveteau, P. Université de Bourgogne – INRA, France Communication, often referred as Quorum Sensing, is involved in the adaptation of most bacteria to their environment. So far, the agr system is the sole communication system described in the genus Listeria. The agr communication system affects the biology of Listeria monocytogenes during saprophytic life (biofilm formation) and during infection. We have recently demonstrated that agr expression was heterogeneous during growth of L. monocytogenes EGDe as biofilm. Our observations did not fit the criteria of the Quorum Sensing paradigm. Indeed, Quo- rum sensing means that the whole population presents the same phenotype once the 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 combining gfp reporters, flow cytometry and fluorescent microscopy. This experimental set up was used to revisit auto-induction, Quorum Sensing and more recent theories of bacterial communication during the saprophytic life of Listeria monocytogenes.

AREA // D // Strategies for prevention and control of Listeria monocytogenes POSTERPRESENTATIONS // P / 199–200

REFERENCE Utilization of Lactococcus lactis M104, a wild nisin-producing raw milk / isolate, as an antilisterial adjunct in traditional Greek Graviera cheese D P processing 199 Samelis, 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 contamination is assured during processing and storage of traditional Greek Graviera cheese 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 enhancing L. monocytogenes inactivation by using Lactococcus lactis M104, a nisin-producing (Nis+) raw milk isolate, as an antilisterial adjunct at cheese manufacture. Fresh Graviera curds with added a commercial starter culture (SC), or the SC plus the Nis+ strain (SC+M104), were inoculated (3 log cfu/g) with a 3-strain cocktail of avirulent L. monocytogenes/innocua before molding in a commercial plant. After brining, cheeses were ripened at 17-18ºC and 90% RH for 20 days and stored at 4ºC in 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. Populations of 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. Although Nis+ colonies and the Nis+ gene were isolated from the SC+M104 cheeses only, no major differences in Listeria survival occurred between cheeses with or without the Nis+ strain. All cheeses contained enterocin (A, B and P) plus plantaricin A genes associated with Enterococcus faecium and Lactobacillus plantarum of the NSLAB flora, and showed similar chemical changes. Thus, conditions prevailing during 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 criteria REFERENCE complementing the existing concepts of microbiological criteria. To achieve these D / P objectives it is critically important a harmonisation of food safety control proce- 200 dures. The EU project “Selection and improving of fit for purpose sampling procedures for specific foods and risks”-BASELINE, funded under the FP7 programme, intends to obtain the following objectives: 1) to review the sampling schemes currently available for food authorities and food producers to collect data for quantitative risk assessment at EU level; 2) to assess the relevance and suitable limit 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 the identified biological risks; 4) to develop predictive mathematical models for the target biological risks; 5) to validate and harmonise the sampling schemes and the alternative detection methods developed in the project; 6) to share and disseminate the scientific knowledge coming out from the project to stakeholders. The project results will be translated in clear recommendation to the EC as well as end users and they will have a significant impact on protection of human health. A poster detailing the project objectives, strategies and expected impact will be presented at the conference.

AUTHORS INDEX

Abdalla, S. D/P/128 Bowman, J. P. A/O/03, A/P/17 Abee, T. A/O/05 Boye, M. B/P/182 Acciari, V. C/P/184, D/P/196 Brandi, G C/P/93 Achtman, M. A/P/27 Braun, E C/O/29 Agyekum, K. B/O/14 Bremont, S. C/P/90 Aharonowitz, Y A/O/11 Brennan, O. A/O/09 Aish, J. C/P/125 Briandet, R. D/P/142 Albano, H. D/P/163 Briers, Y. A/O/07 Alessandria, V A/P/32, A/P/38, B/P/64, D/P/164 Brisabois, A. A/P/01, B/P/47, C/P/82, D/P/146 Allain, V. D/P/190 Brisse, S. A/O/06 Allen, V. A/P/29 Brito, L. A/O/02, D/P/153, D/P/167 Almeida, G. A/P/30, A/P/44, C/P/105, C/P/111, Bruno, J. C. B/O/22 C/P/113, D/P/166, D/P/165 Bugarski, D. D/P/177 Almeida, M. T. B/P/66 Burall, L. A/P/26 Alonzo, F. B/P/56 Burgess, S. C. B/P/48 Alvarez-Dominguez, C. A/P/00, B/O/19, C/PL/09, Busia, G. D/P/175 Amagliani, G. C/P/93 Butler, F. D/O/42 Amar, C. C/P/91, C/P/92 Byrne, B. C/P/119 Amar, C. F. C/P/85 Cabanes, D. B/O/23, B/P/65, B/P/66, B/P/68, B/P/70 Ambrosoli, R. D/P/150 Cabanova, L. D/P/152 Ancora, M. C/P/102 Cabo, M.L. A/P/45 Andersen, J. B. B/P/182 Cabrita, P. A/O/02 Andrew, P. B/P/54 Caccia, N. D/P/142 Andrew, P. D/P/128 Calderone, D. D/P/194 Andrew, P.W. A/P/10 Calendar, R. A/O/10 Anita, B. A/P/12, A/P/19 Calvo, E. A/P/43 Arapakh, S. D/P/172 Camejo, A. B/O/23, B/P/70 Armillotta, G. C/P/185 Cammá, C. C/P/102 Arnau, J. D/P/136 Campos, L. D/P/189 Arneborg, N. D/P/132 Campos, M. L. D/P/188 Asakura, H. A/P/22 Candeloro, L. C/P/186 Ašanin, R. D/P/177 Cantinelli, T. A/O/06 Ashok Kumar, J. C/O/25, E/P/179 Carneiro, L. A/P/44, D/P/166 Asséré, A. D/P/146 Caro, V. A/O/06 Atanassova, V. D/P/173 Carpentier, B. C/P/103 Aubry, C. B/O/21 Carranza-Cereceda, C. A/P/00, B/O/19 Auchter, M. B/P/50, D/P/148 Carrasco-Marin, E. A/P/00, B/O/19 Aury, K. D/P/190 Carrera, S. A/P/45 Aymerich, T. D/O/43, D/P/136 Cartinhour, S. A/O/13 Azeredo, J. C/P/115, D/P/176 Carvalho, F. B/O/23 Azevedo, I. A/P/30 Carvalho, F. B/P/65 Bai, F. A/P/36 Casey, P.G. B/P/69 Barbosa, J. A/P/30, C/P/111, C/P/113, D/P/160, Castle, M. E/O/49 D/P/163, D/P/165 Cécile, C. D/P/171 Barbuddhe, S- B. A/P/06, A/P/07, B/P/49, C/O/25, Ceres, C. D/P/129 C/P/106, C/P/107, C/P/108, C/P/117, D/P/157, E/P/179. Ceri, H. D/P/176 Barile, M. D/P/129 Chafsey, I. A/P/08, D/P/142 Barre, L. C/P/103 Chakraborty, T. A/PL/01, B/P/77, B/P/78, Batista, S. A/O/02 C/O/25, C/P/106, E/P/179 Baysal, A.H. C/P/114 Chambom, C. A/P/08 Beaufort, A. D/O/40, D/P/154 Chamot, S. D/P/145 Belessi, C. A. D/P/172 Chaturongakul, S. A/P/31 Belletti, N. D/O/43 Chaudhuri, S. B/P/71 Benjamin, F. D/P/146 Chavant, P. D/P/145 Berg, J. D/P/141 Chaves-Lopez, C. D/P/151 Bergis, H. D/O/40, D/P/154 Chemaly, M. D/P/147 Bergmann, S. B/P/76 Chemaly, M. M. C/P/183, D/P/187, D/P/190 Bergstrøm, A. B/P/182 Chen, J. A/O/04, A/P/36 Bernardi, T. D/P/145 Chen, Y. A/P/16 Berrang, M. E. D/P/126 Chenal-Francisque, V. A/O/06, C/P/100, D/O/46 Berzins, A. D/P/155 Chiarini, E. D/P/134 Besse, N. G. C/P/103 Chopade, N. A. E/P/179 Bhosle, S. N. C/P/107, C/P/108 Christensen, B. B. B/P/182 Bianchi, D. M C/P/123 Civera, T. C/P/123, C/P/124 Bielecka, M.K. C/O/24 Clark, A. D/O/38 Bitar, A. P. B/P/58, B/P/59 Cocolin, L. A/P/32, A/P/38, D/P/150, D/P/164 Blatter, S. D/P/131 Colaneri, C. A/P/01 Block, C. S. C/O/29 Comaposada, J. D/P/136 Boelaert, F. C/O/32, D/P/170 Commeau, N. D/P/154 Boone, R. C/P/94 Corbett, D. B/P/63 Boor, K. J. A/O/13, A/P/31 Corde, Y. B/P/47 Borges, S. A/P/30, D/P/163 Cormica, M. G. C/P/110 Borza, A. C/P/119, C/P/120 Cornu, M. D/O/40, D/P/154 Boscher, E. A/P/04 Correia, C. D/P/188, D/P/189, D/P/191 Bosley, J. C/P/119 Cortesi, M. L. D/P/129 Botello-Morte, L. A/P/43 Cossart, P. B/O/20 Bottero, M. T. C/P/124 Costas, B. D/O/37 Boucheix, C. B/O/20 Courvalin, P. A/P/01, C/P/90 Bover-Cid, S. D/O/43 Courtillon, C. C/P/183, D/P/187

Couture, H. C/P/97 Fernandes, L. P. D/P/140 Crerar, S. E/O/49 Fernandez Guerrero, M. L. B/P/75 Cross, L. C/P/92 Fernandez-Prieto, L. A/P/00, B/O/19 Cummins, J. B/P/62 Ferreira, I. D/P/188, D/P/189 Cunha, C. I. D/P/189, D/P/191 Ferreira, M.A.S.S. A/P/42, D/P/198 Czuprynski, C. B/P/60 Ferreira, P. B/O/23 D’Orazio, V. A/P/43 Ferreira, R.B. A/O/02 da Silva Felício, M.T C/O/32 Ferreira, V. A/P/30, D/P/160 da Silva, M. B/O/16 Ferreira-Dias, S. D/P/153 Dal Bello, B. D/P/150 Ferrini, G. D/P/136 Dalgaard, P. D/O44 Fertikov, V. C/P/96 Dalmasso, A. C/P/124 Filiatrault, M. J. A/O/13 D’Amico, D. C/P/112 Fletcher, G. B/P/53 Daneshvar, M. I. A/P/33 Flieger, A. A/P/03 Daniel, P. D/P/171 Foglini, M. C/P/93 Danielsson-Tham, M.-L. C/O/30, C/P/122 Forster, B. M. B/P/58 Datta, A. A/P/26, B/P/67 Fortes, E. D. A/P/33, D/P/144 David, C. B/P/54 Foster, T. D/P/159 Davis, A. D/P/159 Fox, E. D/P/130 D’Costa, D. C/P/106, C/P/108 Fraccalvieri, R. A/P/37 De Cesare, A. C/P/116 Franco, B. D. G. M. C/P/118, D/P/134, de Courseulles, E. D/P/192 D/P/135, D/P/139 De Martinis, E. C. D/P/148 Frank, J. D/P/126 De Valk, H. C/O/31, C/P/100 Fravalo, P. C/P/183, D/P/187, D/P/190 Dear, P. C/P/85 Freitag, N. B/P/56 Decastelli, L. C/P/123 Freitag, N. E. B/O/22 DeLappe, N. C/P/110 Frewer, L.J. E/PL/14 Dell’Era, S. A/O/07 Furtado, D. N. D/P/135 Den Bakker, H.C. A/P/33, D/P/144 Furtado, R. D/P/188, D/P/189, D/P/191 Deng, X. A/O/08 Fuss, A. A/O/11 Desai, M. C/P/92 Gaedt Kastbjerg, V. A/P/23, B/P/72 Deschamps, J. D/P/142 Gahan, C.G. B/P/61, B/P/62, B/P/69 Destro, M. T. D/P/134, D/P/135 Galander, S. A/P/03 Desvaux, M. A/P/08, D/P/142, D/P/145 Gallagher, D. C/O/28 Desvaux, M. D/P/142 García-del Portillo, F. A/P/43 Dhuri, R. B. C/P/108 Garg, S. D/P/157 di Febo, T. C/P/185 Garland, C. D. D/O/38 di Giannatale, E. C/P/186, D/P/196, D/P/197 Garriga, M. D/O/43, D/P/136 di Pasquale, F. C/P/101, D/P/151 Garry, P. D/P/154 Disson, O. C/O/24 Garvey, P. C/P/110 Doijad, S. C/P/107, C/P/108, D/P/157 Gendel, S. C/P/97 Dolci, P. D/P/164 George, C. D/P/171 Donaldson, J. R. B/P/48 Gião, M. S. D/P/127 Donnelly, C. C/P/112 Gibbs, P. C/P/115, D/P/160 Dorey, M. C/P/119, C/P/120 Gibson, H. D/P/193 Douey, D. C/P/119, C/P/120 Gillespie, I. C/O/26, C/O/27, C/P/91, E/P/178 Doux, C. D/P/192 Gillespie, I. A. C/O/33,C/P/98 Dowd, G. B/P/69 Gilmartin, N. C/P/99 Duarte Cruz, C. B/P/53 Gilmour, M. A/P/29, C/PL/08 Dumas, E. A/P/08 Glenn, S. B/P/54, D/P/128 Dusch, V. D/O/46, C/P/100 Gloria, L. C/P/122 Dussurget, O. B/P/55 Gnanou-Besse, N. D/P/192 Ebersbach, T. B/P/182 Goebel, W. B/O/18 Egorova, I. C/P/95, C/P/96 Gopal, S. A/O/11 Einar Granum, P. E/P/181 Górgolas, M. B/P/75 Eiriz, E. A/P/45 Gormley, F. E/P/178 Eisenreich, W. B/O/18 Gotz, A. B/O/18 Elemam M. M. C/P/88 Gouin, E. B/O/20 Eleni, S. A/P/12 Goulão, M. M. A/P/42 Eliav, H. C/O/29 Goulet, V. C/O/31, D/O/46, C/P/100 Ells, T. C. A/P/40 Gounadaki, A. S. D/P/172 Eloranta, K. C/P/120 Graham, S. A/P/29 Endres, J. D/P/148 Gram, L. A/P/23, B/P/72 Engeljohn, D. D/PL/11 Granier, S. A. A/P/01 Ermolaeva, S. A/P/34, B/O/15, B/P/73 Grant, K. C/O/26, C/O/27 Eugster, M. R. A/O/10, A/P/28 Grant, K. C/O/33, C/P/85, C/P/91, C/P/92, Eusébio, C. A/P/44 C/P/98, E/P/178 Eylert, E. B/O/18 Grassi, M. A. C/P/123 Fablet, A. D/P/147 Graves, L. C/O/36, A/P/33 Faith, N. B/P/60 Grépinet, O. B/P/47 Faith, N. G. B/P/74 Groelly, J. D/P/145 Faleiro, M. L. A/P/10 Guevara, R. E. C/P/86, C/P/89, E/O/47 Fall, P.A. D/P/137 Guillet, C. C/O/24 Fang, W. A/O/04, A/P/36 Gunjal, P. A/P/06 Farber, J. A/P/09, C/PL/08, C/P/97, D/P/143 Haahr Kallipolitis, B. A/P/24 Fávero, L.M. D/P/139 Haase, J. A/P/27 Favretti, M. C/P/84 Hagen, N. A/O/11 Felício, T. D/P/170 Hain, T. B/P/77, B/P/78 Félix, B. C/P/82 Hain, T. E/P/179

Halbedel, S. A/P/03 Koshy, A. A/O/03 Halberg Larsen, M. A/P/15 Kotwal, S. C/P/117 Handan Baysal, A D/P/162 Koutsoumanis, K. D/O/37 Hara, K. C/P/119 Kreft, J. A/O/11 Hayes, R. D/P/193 Krishnendu, M. B/P/77 Hearty, S. C/P/99 Kurkure, N. V. A/P/07, B/P/49, C/O/25 Heavin, S. A/O/09, A/P/13 Kusumoto, A. C/P/121 Hébert, K. D/P/143 Lad, M. D/P/159 Hebraud, M. D/P/145 Ladely, S. D/P/126 Hébraud, M. A/P/08, D/P/142 Laksanalamai, P. A/P/26 Heir, E. D/P/161 Laksanalamai, P. B/P/67 Helsel, L. O. A/P/33 Landgraf,M. D/P/135 Henriques, A. B/P/65 Langsrud, S. D/P/161, E/P/181 Herrera, J.J.R. A/P/45 Lardeux, A,-L. D/O/40, D/P/192 Hershko-Klement, A. C/O/29 Larios, O. A/P/29 Herve-Bazin, M. C/P/90 Larsen, M. H. A/P/23, D/P/168 Higuchi, Y. A/P/35 Larsson, J.T. C/O/34 Hill, C. B/PL/06, B/P/50, B/P/69 Latorre, L. A/P/37, C/P/87, C/P/116 Hill, D. D/P/193 Laurent, E. C/P/100, D/O/46 Hof, H. A/P/27 Lawrence, M. L. B/P/48 Hogg, T. C/P/105, C/P/113, D/P/160, D/P/165 Le Bouquin, S. D/P/190 Holch, A. B/P/72 Le Monnier, A. C/O/24, C/P/90 Holck, A. D/P/141 Le Nôtre, Y. C/P/183, D/P/187, D/P/190 Holman, D. B. A/P/40 Leclercq, A. A/O/06, C/O/24, C/P/90, Hormazábal, J.C. D/PL/12 C/P/100, D/O/46 Hunt, K. D/P/130 Lecuit, M. A/O/06, B/PL/03, C/O/24, C/P/83, Huszczynski, G. C/P/119, C/P/120 C/P/90, D/O/46 Huwiler, S. A/P/28 Lee Chang, K. J. A/O/03 Hyytia-Trees, E. C/O/36 Lee, B. A/P/29 Iannetti, L. D/P/194 Leisner, J.J. A/P/15 Ida, M. A/P/35 Leite, I. F. C/P/115 Igimi, S. A/P/18, A/P/22 Lelli, R. C/P/185 Imhof, R. D/O/41 Lemos Vaz-Velho, M D/P/169 Ingmer, H. A/P/15, A/P/23, D/P/132 Lengeling, A. B/P/76 Ioannis, D. A/P/12, A/P/19 Li, Z. A/O/08 Iugovaz, I. C/P/94 Licht, T. R. B/P/182 Jablonska, J. B/P/81 Lienenklaus, S. B/P/81 Jacobsen, E. E/P/181 Lindbäck, T. B/P/79 Jakabi, M. C/P/118 Lindström, M. A/P/11 Javanmardi, F. D/P/133 Little, C. C/P/91, E/P/178 Jean, A. C/P/97 Little, C. L. C/O/33, C/P/98 Jen, C. B/P/54 Liu, J. C/P/119 Jenö, P. A/O/02 Lobacz, A. D/O/45 Jiang, L. A/P/36 Loessner, M. J. A/O/01, A/O/07, A/O/10, A/P/28 Jofré, A. D/P/136 Lombard, B. D/P/146 Johansson, J. A/O/12, A/P/46 Lomonaco, S. C/P/123, C/P/124 Johnson, D. D/P/156 Loose, M. B/P/78 Join-Lambert, O. C/O/24 Lopes, J.F. C/P/115 Jordan, K. D/O/42, D/P/130, D/P/172 Lopez, J. A/O/06 Joyce, S. A. B/P/61, B/P/69 Lopez-Valladares, G. C/O/30 Jusdado, J. J. B/P/75 Lortholary, O. C/O/24 Kai, A. A/P/35 Lourenço, A. A. D/P/167 Kalebar, S. C/P/106, C/P/107, C/P/108, E/P/179 Louro, P. D/P/153 Kallipolitis, B. A/P/02 Luber, P. C/PL/10 Kalorey, D. R. C/P/106 Luchansky, J. B. B/P/74 Kalorey, D. R. A/P/06, A/P/07, B/P/49, Luciani, M. C/P/185 C/O/25, C/P/117 MacDonald, D. C/PL/08 Kaminskaya, A. A/P/34 Machado, H. D/P/167 Kanarata, S. C/P/109 Maciag, P.C. B/O/17 Kaneko, S. A/P/35 Madarame, H. B/P/52 Kantikova, M. D/P/152 Madrazo-Toca, F. A/P/00, B/O/19 Karatzas, K. A/O/09 Magalhães, R. A/P/30, A/P/44, C/P/105, Kathariou, S. B/P/74 C/P/111, C/P/113, D/P/166, D/P/165 Kawamoto, K. B/O/16, C/P/121 Magnani, M. C/P/93 Kearney, A. A/P/29 Mahmoodi,S. M. M. D/P/133 Keevil, C. W. D/P/127 Maia, C. D/P/188, D/P/189, D/P/191 Keich, U. A/O/13 Maia, C. H. A/P/42 Kent, H. A/P/29 Maia, R. L. E/O/48 Kerouanton, A. B/P/47 Mailles, A. C/P/83 Kiil Nielsen, P. A/P/02, A/P/24 Makela, P. C/O/32 Kim, J. B/P/74 Makela, P. D/P/170 King, L. C/O/31, C/P/100 Makino, S.I . B/O/16,C/P/121 Klumpp, J. A/O/01 Maklon, K. C/P/121 Knabel, S. J. C/O/35, C/P/124 Malik, S.V. S. C/O/25, C/P/106 Knoll, L. B/P/60 Mamzer-Bruneel, M.F. C/O/24 Knudsen, G. B/P/182 Manfreda, G. C/P/116 Kolbasov, D. C/P/95, C/P/96 Marault, M. C/P/82 Korikanthimath, V. S. C/O/25 Marceddu, M. D/P/174 Korkeala H. A/P/11, D/P/155 Marfoglia, C. C/P/184, D/P/197

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Rowlands, R. E. G. C/P/118, D/P/139 Teixeira, A. P. B/P/47 Rubinstein, E. B/O/20 Teixeira, P. A/P/30, A/P/44, C/P/105, C/P/111, Saá Ibusquiza, P. A/P/45 C/P/113, C/P/115, D/P/158, D/P/160, D/P/163, Sabol, A. C/O/36 D/P/165, D/P/166, E/O/48 Sahaghian, R. B/P/74 Teixeira, P. D/P/176 Sahu, S. B/P/67 Témoin, S. B/P/47 Salah, S. D/O/46 Tenenhaus-Aziza, F. D/O/42 Salem, S. C/P/100 Tham, M. D. C/P/117 Salini, R. D/P/197 Tham, T. N. B/O/20 Salvat, G. D/P/147 Tham, W. C/O/30, C/P/117 Salzberg, S. L. A/O/08 Thierry-Bled, F. D/O/46 Santagada, G. A/P/37 Thomaz, M. R. S. D/P/140 Santos, I. A/P/30, A/P/44, C/P/111, C/P/113, Todorov, S. D/P/134, D/P/135, D/P/169 D/P/166, D/P/165 Tola, S. C/P/87 Santos, M. I. A/P/42, D/P/188, D/P/189, D/P/191 Tonucci, F. C/P/93 Santos, S. D/P/188, D/P/189 Saraiva, M. D/P/188 Toquin, M. -T. C/P/, D/P/, D/P/ Sauders, B.D. A/P/33 Torres, R. B/P/75 Savelli, C. C/PL/08 Torresi, M. C/P/184, D/P/196 Scattolini,S. D/P/195 Tortorello, M. L. A/O/08 Schechner, V. C/O/29 Tran, C. A/O/06 Scherer, S. A/P/25 Tresse, O. D/O/39 Schirmer, B. C. D/P/141 Trigo, M.J. A/O/02 Schittler, L. D/P/138 Trottier, Y. C/P/94 Schlech, W. B/P/80 Trout-Yakel, K. A/P/29 Schlech, W.F. C/PL/07 Truelstrup Hansen, L. A/P/40 Schughart, K. B/P/76 Tyler, S. A/P/29 Schuler, S. B/P/54 Ueland, Ø. E/P/181 Schuppler, M. A/O/07 Umap, S. A/P/07 Schvartzman, S. D/O/42, D/P/172 Upham, J. C/P/119, C/P/120 Scortti, M. C/O/24 Utratna, M. A/P/41 Scott, V. N. A/P/16 Vaikousi, H. D/O/37 Seavey, M. M. B/O/17 Vaillant, V. C/O/31, C/P/83, C/P/100 Secic, I. B/P/79 Valinsky, L. C/O/29 Selyaninov, Y. C/P/95 van der Veen, S. A/O/05 Semprini, P. C/P/185, C/P/186, D/P/196 Van Domselaar, G. A/P/29 Serio, A. C/P/101, D/P/151 Van Stelten, A. A/P/16 Serraino, A. D/P/194 Vandevelde, M. B/P/52 Seuberlich, T. B/P/52 Varfolomeev, A. A/P/34 Sewell, D. B/O/17 Vasantrao Kurkure, N. A/P/06 Shama, G. D/P/128 Vázquez, D. A/P/45 Shaw, I. A/P/41 Shimojima, Y. A/P/35 Vazquez-Boland, J. C/O/24 Silins I. D/P/155 Vaz-Velho, M. D/P/134 Silva, A. D/P/153 Velge, P. B/P/47 Silva, J. A/P/44, D/P/158, D/P/163 Verran, J. D/P/132 Silva, W. P. D/P/138 Vidić, B. D/P/177 Silveira Naleiro, E. A/P/05 Vignaud, M. C/P/82 Simpson, J. M. A/P/16 Virgilio, S. C/P/87 Skandamis, P.N. D/P/172 Vishal Singh, P. C/O/30 Skovager, A. D/P/132 Vongkamjan, K. D/P/144, D/P/160 Škuntova, O. D/P/152 Voronin, M. C/P/95 Slepkov, E. R. B/P/58, B/P/59 Wadge, A. E/PL/13 Smith, M. A. B/O/14 Waidmann, M. S. B/P/50, B/P/51, D/P/148 Solodova, E. B/P/81 Walton, J. D/P/193 Somervuo, P. A/P/11 Wang, J. B/P/63 Somov, G.P. B/P/73 Wang, W. A/O/13 Sonegaonkar, A. A/P/07 Warke, S. A/P/07, B/P/49 Sorvillo, F. C/P/86 Weiss, S. B/P/81 Sousa, S. B/O/23, B/P/66, B/P/68 Whitehead, K. D/P/132 Souza, V. M. D/P/140 Whiting, R. C. A/P/16 Sperandii, A. C/P/102 Wiedmann, M. A/PL/02, A/O/13, A/P/16, Sperandii, A. F. D/P/195 A/P/31, A/P/33, D/P/160, D/P/144 Stabler, R. A/P/05 Wilhelm, T. C/P/122 Stahl, J.-P. C/P/83 Williams, D. B/O/14 Steigerwalt, A. G. A/P/33 Winkelströter, L. K. D/P/148 Stephan, R. A/O/01, D/P/131 Wong, H. T. L. A/P/39 Stessl, B. C/P/104 Stoll, R. A/O/11, B/O/18 Wren, B.W. A/P/05 Stollewerk, K. D/P/136 Xayarath, B. B/P/57 Sukhapesnaa, J. C/P/109 Yurov, D. A/P/34 Sun, Q. A/O/13 Zahle Andersen, A. A/P/24 Suzuki, H. A/P/18, A/P/22 Zaytseva, E.A. B/P/73 Swain, B. K. C/O/25 Zeiman, E. A/O/10 Swaminathan, B. A/P/33 Zeppa, G. D/P/150 Switt, A. M. D/P/144, D/P/160 Zhang, W. A/O/08 Tam Dao, T. C/P/82, D/P/146 Zuliani, V. D/P/154 Tamburro, M. C/P/85 Zurbriggen, A. B/P/52 Tasara, T. A/O/01, D/P/131 Zweifel, C. D/P/131

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