Epidemiological, molecular characterization and antibiotic resistance of Salmonella enterica serovars isolated from chicken farms in Egypt

This study was conducted in 41 broiler flocks located in Kafr El-Sheikh Province in Delta Egypt. Twenty flocks of 1-week-old birds and 21 flocks of 5-week-old birds were investigated. The observed clinical symptoms were observed and recorded (Table 1). Five living morbid birds from each flock were randomly selected and humanly sacrificed. At necropsy, sections of liver and intestinal wall plus contents were collected aseptically and processed for Salmonella isolation. From the same bird bile was aspirated from the gall bladder. Wetted cotton swabs in bacteriological transport media were used to collect samples from each specimen. Collected swabs and tissue samples were immediately frozen on ice and stored at −20 °C for further investigation within 5 h. Each tissue sample and swabs were inoculated in 10 ml selenite F broth (Oxoid, UK) and incubated at 37 °C overnight. A loopful of inoculated broth was streaked on selective Salmonella Shigella (SS) agar (Oxoid, UK) and incubated at 37 °C overnight. The suspected colony was sub-cultured on Xylose lysine deoxycholate (XLD) agar (Oxoid, UK) and on brilliant green (BG) agar (Oxoid, UK) and incubated at 37 °C for 16–18 h. The suspected colonies were collected for further biochemical identification using API 20E (BioMérieux, Marcy-l’Étoile, France).

The identified bacterial cultures were cultivated on SS agar and inoculated on Luria–Bertani (LB) broth (Oxoid, UK) and incubated at 37 °C overnight. The DNA was extracted from bacterial cultures on broth using Qiagen DNA extraction kit (Qiagen, UK) according to the manufacturer’s instructions.

In order to make a rapid and definite diagnosis of Salmonella, PCR was conducted using primers to detect the gene marker for S. enterica invA [32], sdfI primers specific for detection of S. enterica serovars Enteritidis [33], and Typh, Sal and fliC specific primers for serovar S. Typhimurium [34, 35] (Table 2).

invA positive strains were tested for the presence of the sefA gene, which encodes for SEF14 fimbriae that can be detected in S. enterica serovar Enteritidis strains and will also be present in the poultry-associated serotype S. Gallinarum.

In order to detect the zoonotic potential of our isolated strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium we screened for the presence of the sopE gene [36].

The PCR reaction was geared to a previously described protocol for Salmonella [32‐36]. Conserved forward and reverse primers (Eurofins, Japan) were used to generate the target amplicon (Table 1). The PCR cycling conditions were carried out as the following: initial denaturation at 94 °C for 5 min. Thirty cycles of amplification were run for 5 s, at 94 °C, 10 s at 68 °C and 20 s at 72 °C, with the final extension continuing at 72 °C for 7 min. Different annealing temperatures were used as described in Table 1. Five microliter aliquots of reaction mixture were electrophoresed through 1.5% agarose gels (Nippongene, Japan).

The class one integrons PCR fragments were purified from the agarose gel using Nucleospin Gel Extraction Kit (Macherey–Nagel, Germany) and sequenced (Genome centre—Gifu University, Japan). The sequencing results were analysed using BLAST webpage (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

The antimicrobial susceptibility testing was performed using the Kirby–Bauer disc diffusion test [37] at the Clinical Veterinary Microbiology Laboratory of the Royal Dick School of Veterinary Study, University of Edinburgh. Briefly, one colony from the SS agar plate of each strain was picked up and streaked onto Mueller–Hinton blood agar (Oxoid, UK) and incubated at 37 °C overnight. Bacterial colonies were suspended in 0.9% NaCl to obtain a McFarland turbidity of 0.5 (Dr. Lange, photometer CADAS 30, Berlin, Germany) that containing about 1–2  ×  10⁸ colony forming units (CFU)/ml of Escherichia coli strain American Type Culture Collection (ATCC) 25922. Approximately, 300 μl of the saline suspension was spread onto the surface of a Mueller–Hinton agar plate (Oxoid, UK) using a sterile swab. The antimicrobial discs (Oxoid, UK) of six clinically used antibiotics that are used in the Egyptian poultry production (tetracycline 30 μg, ampicillin 10 μg, sulfamethoxazole/trimethoprim 25 μg, gentamicin 10 μg, streptomycin 25 μg and chloramphenicol 30 μg) were distributed onto the surface of the Mueller–Hinton agar plates using a Multi-disc dispenser (Oxoid, UK). The plates were incubated at 37 °C overnight. The diameters of the inhibited zones were measured using sliding callipers and interpreted using standard break points according to the method described by The European Committee on Antimicrobial Susceptibility Testing [38] (Table 3).

The gene associated with antibiotic resistance was tested in isolated Salmonella strains. Isolates were screened for the presence of 18 genes known to be associated with resistance to the seven tested antibiotics (Table 4).

The mortality rate associated with Salmonella infection and the rate of S. enterica serovar Typhimurium isolation from internal organs were analysed by the student t test [39].