Phenotypic and genotypic typing of Salmonella enterica serovar Enteritidis isolates from poultry farms in Tunisia Guedda Intissar1, Abbassi Mohamed Salah1, Debya Rafika1, Chebbi Chokri1, Mami Hela1, Ben Hassen Assia2, Hammami Salah1 Institute of veterinary research of Tunisia1, National Bone Marrow Transplantation Centre2 INTRODUCTION Salmonella enterica serotype Enteritidis is a common cause of salmonellosis among humans and animals in Tunisia and in many other countries. The main sources of these infections have been meats, poultry eggs and eggs products. Previous studies have reported that poultry can become contaminated with S. enteritidis at the farm level. These organisms can be transported to the production facility and can become a source of contamination for the final products. The aim of this study is to investigate the occurrence of Salmonella spp. in the environments of poultry farms in Tunisia, as well as to characterize strains of serovar Enteritidis by plasmid analysis, pulsed field gel electrophoresis (PFGE) and antibiotic sensitivity testing. Materials and methods Samples collection and bacterial isolates : Two hundred forty five samples (faeces, water and environmental swabs) were taken at eight poultry farms located in different geographical areas of Tunisia. Samples were processed according to international norms for Salmonella ISO 6579, 2002 and Annex D, 2006. - Serotyping of Salmonella: Serotyping was performed by the slide agglutination method with the use of antiserum (BioRad, France, Marnes la coquette). -Antibiotic susceptibilities:Disk-agar diffusion method (CA-SFM guidelines). - Isolation of plasmid DNA: For Salmonella serovar Enteritidis, plasmid DNA was isolated by the alkaline lysis method (7). The approximate molecular sizes of plasmids were determined by comparison with plasmids of known size from Escherichia coli V 517 (53, 7, 5.4, 5, 4, 3, 2.6 and 2 kb). Pulsed field gel electrophoresis: For Salmonella serovar Enteritidis, preparation and digestion of genomic DNA using XbaI restriction endonuclease (Amersham Bio-sciences, Orsay, France) were performed as described previously (2). Electrophoresis was performed with CHEF DR III system (BioRad laboratories, Richmond, CA). Results * Twenty one Salmonella isolates were collected from the eight poultry farms, 16 were identified as Salmonella serovar Enteritidis. The other non S. Enteritidis isolates were: S. typhimurium (2 strains), S. scharzengrund (2 strains) and S. braenderup (one strain). * Seven strains were susceptible to all tested antimicrobials. Ten S. enteritidis exhibited resistance to at least one antimicrobial. Salmonella serovar Enteritidis isolates displayed resistance most often to ticarcillin and amoxicillin and were susceptible to third-generation cephalosporin (cefotaxime and ceftazidim) and aztreonam. * Four different plasmid profiles were generated among the 16 serotype Enteritidis isolates (P1 to P4). Three were plasmid-free and 13 isolates possessed one to three plasmids with molecular sizes ranging from ca.2 to ca.54 Kb. The most prevalent plasmid profile was P4, found in ten out of 16 isolates, containing only the large plasmid (ca.54 Kb). * XbaI-PFGE analysis of 16 isolates generated two profiles containing 9 to 12 DNA fragments. The most prevalent type was X1 found in 12/16 of the isolates and was detected in four farms. The second subtype X2 was detected in one farm and contained 4 isolates (Fig.1). Discussion Phenotypic typing of Salmonella isolates showed that S. enteritidis was the prevalent serotype with 16/21 strains. This result is in accordance with recent study by Ben Aissa et al. (2007) confirming that S. enteritidis is the most frequently isolated serotype from animal origin (especially poultry) in Tunisia during the last decade. - Employment of antibiogram typing is not a very useful tool for subtyping S. enteritidis strains, because the majority of isolates were susceptible to all antibiotic tests. - It has been showed that the majority of Salmonella enteritidis strains carry a serospecific virulence plasmid of ca.54 kb (3,5). Concordantly, a single 54 kb plasmid was also common in our S. enteritidis isolates being detected in 12 out of 16 strains. Poppe et al. (1993) suggested that possession of a single plasmid is not very discriminatory for serotype Enteritidis. - The majority of S. enteritidis isolates (12/16) belonged to genotype X1 as revealed by XbaI-PFGE patterns (6). These clonal strains were found in four farms located in different geographical areas. This is an agreement with a study by Liebana et al. (2001) in which the majority of S. enteritidis strains isolated from English poultry farms belonged to a single XbaI-PFGE pattern. Fig.1. Pulsed field gel electrophoresis (PFGE) patterns for XbaI digested genomic DNA of S. enteritidis strains obtained from Tunisian poultry farms. Lane 1: Lambda ladder marker for PFGE, lanes 2- 14: PFGE patterns for S. enteritidis strains. References 1- Ben Aissa, R., Al-Gallas, N., Troudi, H., Belhadj, N., Belhadj, A., 2007. Trends in Salmonella enterica serotypes isolated from human, food, animal, and environment in Tunisia, 1994-2004. J. Infect. 20, 1-16. 2- Liebana, E., Guns, D., Garcia-Miguera, L., Woodward, M.J., Clifton–Hadley, A., Davies, R.H., 2001. Molecular typing of Salmonella serotypes prevalent in animals in England: Assessment of Methodology. J Clin Microbiol. 39 (10), 3609-3616. 3- Nauerby, B., Pedersen, K., Dietz, H., Madsen, M., 2000. Comparison of Danish Isolates of Salmonella enterica Serovar Enteritidis PT9a and PT11 from Hedgehogs (Erinaceus europaeus) and humans by plasmid profiling and pulsed–field gel electrophoresis. J Clin Microbiol. 38 (10), 3631-3635.4- Poppe, C., McFadden, K., Brower, A., Demzuk, W., 1993. Characterisation of Salmonella enteritidis strains. Can. J Vet Res. 57, 176-184. 5- Rychlik, D., Hradecka G.H., 2006. Distribution and function of plasmids in Salmonella enterica. Vet Microbiol. 112, 1-10.6- Tenover, F.C., Arbeit, R.D., Goering, R., Micklesen, P.A., Murray, B.E., Persing, D.H., Swaminathan, B., 1995. Interpreting chromosomal DNA restriction by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol. 33 (9), 2233-2239.7- Woodford, N., Morrison, D., Cookson, B., Gorge, R.C., 1993. Comparison of heigh-level gentamicin resistant Enterococcus faecium isolates from different continents. Antimicrob Agents Chemother. 37, 681-684. Conclusion In conclusion, the combined use of phenotypic and genotypic methods indicate the occurrence of a particle S. enteritidis clone in different poultry farms in Tunisia. Among this prevalent clone many strains were resistant to at least one antibiotic. It would be interesting to investigate more strains from more locations to confirm the clonality of Tunisian isolates and to determine the extent of antimicrobial resistance strains.