This study investigated the occurrence of Salmonella in poultry farms in Tunisia. Samples were collected from eight farms and 21 Salmonella isolates were found, with 16 being Salmonella Enteritidis. Genotypic analysis showed that 12 of the S. Enteritidis isolates belonged to the same pulsotype X1 and were found in four different farms, indicating a dominant clone. Plasmid profiling revealed four profiles among the isolates, with the most common containing a 54kb plasmid. The results demonstrate the presence of a common S. Enteritidis clone in multiple Tunisian poultry farms.

1. 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 TunisiaInstitute of Veterinary Research of Tunisia11
, National Bone Marrow Transplantation Centre, National Bone Marrow Transplantation Centre22
INTRODUCTIONINTRODUCTION
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 methodsMaterials 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 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).
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.
DiscussionDiscussion
-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.
ConclusionConclusion
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.
ResultsResults
* 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
azetronam.
* 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).
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