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Y. enterocolitica is the third bacterial cause of human enteritis in Europe. The species is divided into six biotypes (BT), BT1A regarded as nonpathogenic and pathogenic biotypes 1B, 2, 3, 4 and 5. Pigs, the principal reservoir for human pathogenic strains, do not develop clinical signs. The BT4 is the most frequently biotype isolated from pig and encountered in human yersiniosis. This study investigated the use of in vitro cultured cells to assess the ability of Y. enterocolitica to adhere and invade pig and human cells. We tested in vitro the adhesion and invasion abilities of a collection of 23 Y. enterocolitica on intestinal pork cells IPEC-J2 and on human intestinal cells Caco-2. The overall profile of adhesion / invasion was different in the both tests. Nevertheless, in the two tests, the BT1A and the BT5 strains, which are rarely isolated from pigs, show a low capacity to adhere and to invade. These strains were clustered in the class 1. The class 2, forming by strains having a greater efficiency of adhesion and/or a greater efficiency of invasion, contained predominantly strains of BT4. The results obtained in this study reflect the ability of BT4 to colonize pigs and the low capacity to BT1A and BT5 to colonize pigs and humans. Introduction Y. enterocolitica is a gram-negative zoonotic enteropathogenic bacterium responsible to yersiniosis, the third most commonly reported zoonosis in Europe (EFSA, 2015). Human yersiniosis is associated with a wide variety of clinical symptoms ranging from mild gastroenteritis to invasive syndromes like terminal ileitis (Bottone, 1999). Infection by Y. enterocolitica is due to consumption of contaminated food or water. Following ingestion, the bacteria colonize the lumen of the intestine and cross the intestinal tissue barrier by invading M cells (Grutzkau et al., 1990, Schulte et al., 2000). The invasion of M cells allow the colonization of the underlying tissues and may results in dissemination of the bacteria to the mesenteric lymph nodes and extra-intestinal sites such as spleen, liver or lungs. All Y. enterocolitica are not pathogenic for human. The species is divided into six biotypes. The biotype 1A generally regarded as nonpathogenic and the pathogenic biotypes BT1B, BT2, BT3, BT4, BT5. In France and most other countries worldwide, BT4 is the most prevalent biotype isolated from humans (69%), followed by BT2 (30%) and BT3 (Savin and Carniel, 2008). Because the principal source of contamination is due to ingestion of raw or undercooked pork products, pigs are considered as the principal reservoir for human pathogenic strain of Y. enterocolitica (Bottone, 1999). Pigs do not develop clinical signs, but they do carry pathogenic Y. enterocolitica in their oral cavity and excrete this bacterium in their feces (Thibodeau et al., 1999). Two study in France revealed that pathogenic biotypes BT2, BT3, BT4 and BT5 have been recovered from slaughtered pigs (Fondrevez et al. 2010, Fondrevez et al. 2014) and that Y. enterocolitica BT1A considered as nonpathogenic have been recovered from meat samples (Esnault et al., 2013). Because Y. enterocolitica is found in the intestine of healthy pigs and because the invasion of intestinal epithelial cells is a major step in Y. enterocolitica infection in human, two models using porcine and human intestinal in vitro cell culture were assessed to profile a collection of 23 strains containing the five biotypes found in France. Safepork 2015 Conference Epidemiology and control of hazards in pork production chain – SAFEPORK One health approach under a concept of farm to fork 109 108 Epidemiology of infection / control and mitigation option of foodborne pathogens and zoonotic diseases in the pork production chain. Epidemiology of infection / control and mitigation option of foodborne pathogens and zoonotic diseases in the pork production chain. Material and Methods Bacterial strains and growth media The characteristics of the 23 strains used in this study are listed in table 1. Table 1. Strains used in this study Strains Biotype and origin N° of strains References E. coli FendA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF) U169, hsdR17(rK mK +), λ– 1 (S.G.N. Grant et al., 1990) Y. enterocolitia BT1A, pork meat 2 (Esnault et al., 2013) BT2, slaughtered pig, tonsil swab 3 (Fondrevez et al., 2010, and 2014) BT3, slaughtered pig, tonsil swab 5 (Fondrevez et al., 2014) BT4, slaughtered pig, tonsil swab 12 (Fondrevez et al., 2010, and 2014) BT5, slaughtered pig, tonsil swab 1 (Fondrevez et al., 2014) Overnight cultures of E. coli and Y. enterocolitica were grown at 30°C in BHI (Brain Heart Infusion medium). Human Caco-2 cells and porcine IPEC-J2 cells were cultured at 37°C in the presence of 5% CO2. Caco-2 cells were grown in DMEM supplemented with 10% FBS, 1% NEAA (Non-Essentiel Amino Acid Cell Cultur Supplement) and 20mM HEPES (hydroxyethyl piperazineethanesulfonic acid). IPEC-J2 were grown in DMEM supplemented with 10% FBS. Cell adhesion and invasion assay For cell adhesion and uptake assays, 1.105 cellules Caco-2 or IPEC-J2 cells were seeded and grown 48 hours in individual wells of 24-well cell culture plates before the addition of bacteria. The overnight cultures of E. coli and Y. enterocolitica bacteria were washed and diluted in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% FBS (Fetal Bovine Serum), to obtain a cellular concentration of 2.10cfu/ml. For the infection, approximately 2.107 bacteria were added to the 2.105 cells and the 24well cell culture plates were incubated at 37°C in the presence of 5% CO2. Three hours post-infection, the cells were washed extensively with PBS (Phosphate Buffered Saline). The total number of adherent bacteria was determined by cell lysis using 0.1% Triton X-100 and plating on bacterial media. Bacterial uptake was assessed by adding 100μg of gentamicin per well. After two hours of incubation. The percentage of bacteria that survived killing was determined after plating on bacterial media. For each strain, the relative level of bacterial adhesion and uptake was determined by calculating the number of CFU relative to the total number of bacteria introduced into cells. The experiments were routinely performed in triplicate. Statistical analysis Statistical analyses were performed by using logiciel R (R Development Core Team, 2015). Hierarchical cluster and Newman-Keuls test were used for clustering the strains and the influence of biotype was evaluated by performing Fisher’s Exact Test for Count Data. Results The adhesion/ invasion profile for 23 Y. enterocolitica belonging to the five biotypes found in France have been determined for two lines of in vitro cultured cells. Because the strains were mainly isolated from pork samples, the adhesion and invasion efficiency were first determined on the intestinal porcine cell line model IPEC-J2 (Figure 1). The strains are clustered in two classes based on their adhesion and invasion capacity. Figure 1: Invasion and adhesion profile of 23 Y. enterocolitica into porcine IPEC-J2 cells. The hierarchical cluster and Newman-Keuls analysis clustered the strain into two classes. The ten strains which were in the class 1 presented an adhesion capacity inferior to 15% and an invasion capacity inferior to 3%. The 13 strains of the class 2 were more adhesive and/or more invasive. Among the 23 strains, 43% of the strains (10/23) are in class 1. This class grouped strains having a low capacity of adhesion and a low capacity of invasion. The class 1 contained the two BT1A strains, the strain of BT5 and 67% (2/3) of strains of BT2, 40% (2/5) BT3 strains and 25% (3/12) BT4 strains. The 13 other strains are clustered in class 2. Although the class 2 contains the majority of BT3 strains (3/5) and the majority of BT4 strains (9/12), no significant influence of the biotype parameter was observed in the composition of the two classes (Fisher’s Exact Test for Count Data; p=0.16). The 23 strains were also tested in vitro on human intestinal Caco-2 model (Figure 2). The clustering of the strains in two classes was based on the adhesion parameter. The first class, characterized by strains possessing an adhesion capacity inferior to 7%, contained 17 strains belonging to the five biotypes. Safepork 2015 Conference Epidemiology and control of hazards in pork production chain – SAFEPORK One health approach under a concept of farm to fork 109 108 Epidemiology of infection / control and mitigation option of foodborne pathogens and zoonotic diseases in the pork production chain. Epidemiology of infection / control and mitigation option of foodborne pathogens and zoonotic diseases in the pork production chain. Material and Methods Bacterial strains and growth media The characteristics of the 23 strains used in this study are listed in table 1. Table 1. Strains used in this study Strains Biotype and origin N° of strains References E. coli FendA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF) U169, hsdR17(rK mK +), λ– 1 (S.G.N. Grant et al., 1990) Y. enterocolitia BT1A, pork meat 2 (Esnault et al., 2013) BT2, slaughtered pig, tonsil swab 3 (Fondrevez et al., 2010, and 2014) BT3, slaughtered pig, tonsil swab 5 (Fondrevez et al., 2014) BT4, slaughtered pig, tonsil swab 12 (Fondrevez et al., 2010, and 2014) BT5, slaughtered pig, tonsil swab 1 (Fondrevez et al., 2014) Overnight cultures of E. coli and Y. enterocolitica were grown at 30°C in BHI (Brain Heart Infusion medium). Human Caco-2 cells and porcine IPEC-J2 cells were cultured at 37°C in the presence of 5% CO2. Caco-2 cells were grown in DMEM supplemented with 10% FBS, 1% NEAA (Non-Essentiel Amino Acid Cell Cultur Supplement) and 20mM HEPES (hydroxyethyl piperazineethanesulfonic acid). IPEC-J2 were grown in DMEM supplemented with 10% FBS. Cell adhesion and invasion assay For cell adhesion and uptake assays, 1.105 cellules Caco-2 or IPEC-J2 cells were seeded and grown 48 hours in indiv

Porcine and Human intestinal cells for profiling the capacity of colonization and infection of the foodborne pathogen Yersinia enterocolitica