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Phenotypic and genotypic typing of Salmonella entericaserovar Enteritidis isolates from poultry farms in TunisiaGuedda Int...
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Phenotypic and genotypic typing of Salmonella enterica serovar Enteritidis isolates from poultry farms in Tunisia


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Phenotypic and genotypic typing of Salmonella enterica serovar Enteritidis isolates from poultry farms in Tunisia

  1. 1. Phenotypic and genotypic typing of Salmonella entericaserovar Enteritidis isolates from poultry farms in TunisiaGuedda Intissar1, Abbassi Mohamed Salah1, Debya Rafika1, Chebbi Chokri1, Mami Hela1, Ben Hassen Assia2, Hammami Salah1Institute of Veterinary Research of TunisiaInstitute of Veterinary Research of Tunisia11, National Bone Marrow Transplantation Centre, National Bone Marrow Transplantation Centre22INTRODUCTIONINTRODUCTIONSalmonella enterica serotype Enteritidis is a common cause of salmonellosis among humans and animals in Tunisia and in many other countries. The main sourcesof these infections have been meats, poultry eggs and eggs products. Previous studies have reported that poultry can become contaminated with S. enteritidis at thefarm 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 toinvestigate the occurrence of Salmonella spp. in the environments of poultry farms in Tunisia, as well as to characterize strains of serovar Enteritidis by plasmidanalysis, pulsed field gel electrophoresis (PFGE) and antibiotic sensitivity testing.Materials and methodsMaterials and methods-Samples collection and bacterial isolates : Two hundred forty five samples(faeces, water and environmental swabs) were taken at eight poultry farmslocated in different geographical areas of Tunisia. Samples were processedaccording to international norms for Salmonella ISO 6579, 2002 and AnnexD, 2006.- Serotyping Salmonella: Serotyping was performed by the slideagglutination method with the use of antiserum (BioRad, France, Marnes lacoquette).-Antibiotic susceptibilities:Disk-agar diffusion method (CA-SFM guidelines).- Isolation of plasmid DNA: For Salmonella serovar Enteritidis, plasmidDNA was isolated by the alkaline lysis method (7). The approximatemolecular sizes of plasmids were determined by comparison with plasmids ofknown 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 restrictionendonuclease (Amersham Bio-sciences, Orsay, France) were performed asdescribed previously (2). Electrophoresis was performed with CHEF DR IIIsystem (BioRad laboratories, Richmond, CA).Fig.1. Pulsed field gel electrophoresis (PFGE) patterns for XbaI digested genomic DNA of S. enteritidis strains obtainedfrom Tunisian poultry farms. Lane 1: Lambda ladder marker for PFGE, lanes 2- 14: PFGE patterns for S. enteritidis strains.DiscussionDiscussion-Phenotypic typing of Salmonella isolates showed that S. enteritidis was theprevalent serotype with 16/21 strains. This result is in accordance with recentstudy by Ben Aissa et al. (2007) confirming that S. enteritidis is the mostfrequently isolated serotype from animal origin (especially poultry) in Tunisiaduring the last decade.- Employment of antibiogram typing is not a very useful tool for subtypingS. enteritidis strains, because the majority of isolates were susceptible to allantibiotic tests.- It has been showed that the majority of Salmonella enteritidis strains carry aserospecific virulence plasmid of ca.54 kb (3,5). Concordantly, a single 54 kbplasmid was also common in our S. enteritidis isolates being detected in 12out of 16 strains. Poppe et al. (1993) suggested that possession of a singleplasmid is not very discriminatory for serotype Enteritidis.- The majority of S. enteritidis isolates (12/16) belonged to genotype X1 asrevealed by XbaI-PFGE patterns (6). These clonal strains were found in fourfarms located in different geographical areas. This is an agreement with astudy by Liebana et al. (2001) in which the majority of S. enteritidis strainsisolated from English poultry farms belonged to a single XbaI-PFGE pattern.ConclusionConclusionIn conclusion, the combined use of phenotypic and genotypic methodsindicate the occurrence of a particle S. enteritidis clone in different poultryfarms in Tunisia. Among this prevalent clone many strains were resistant to atleast one antibiotic. It would be interesting to investigate more strains frommore locations to confirm the clonality of Tunisian isolates and to determinethe extent of antimicrobial resistance strains.ResultsResults* Twenty one Salmonella isolates were collected from the eight poultry farms,16 were identified as Salmonella serovar Enteritidis. The other non S. Enteritidisisolates were: S. typhimurium (2 strains), S. scharzengrund (2 strains) and S.braenderup (one strain).* Seven strains were susceptible to all tested antimicrobials. Ten S. enteritidisexhibited resistance to at least one antimicrobial. Salmonella serovar Enteritidisisolates displayed resistance most often to ticarcillin and amoxicillin and weresusceptible to third-generation cephalosporin (cefotaxime and ceftazidim) andazetronam.* Four different plasmid profiles were generated among the 16 serotypeEnteritidis isolates (P1 to P4). Three were plasmid-free and 13 isolates possessedone to three plasmids with molecular sizes ranging from ca.2 to ca.54 Kb. Themost prevalent plasmid profile was P4, found in ten out of 16 isolates, containingonly the large plasmid (ca.54 Kb).* XbaI-PFGE analysis of 16 isolates generated two profiles containing 9 to 12DNA fragments. The most prevalent type was X1 found in 12/16 of the isolatesand was detected in four farms. The second subtype X2 was detected in one farmand contained 4 isolates (Fig.1).ReferencesReferences1- Ben Aissa, R., Al-Gallas, N., Troudi, H., Belhadj, N., Belhadj, A., 2007. Trends in Salmonella entericaserotypes 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. JClin Microbiol. 39 (10), 3609-3616. 3- Nauerby, B., Pedersen, K., Dietz, H., Madsen, M., 2000. Comparisonof Danish Isolates of Salmonella enterica Serovar Enteritidis PT9a and PT11 from Hedgehogs (Erinaceuseuropaeus) 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 Salmonellaenteritidis strains. Can. J Vet Res. 57, 176-184. 5- Rychlik, D., Hradecka G.H., 2006. Distribution andfunction 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 chromosomalDNA 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-levelgentamicin resistant Enterococcus faecium isolates from different continents. Antimicrob Agents Chemother.37, 681-684.