From March to October in 2015, a total of 1122 samples were isolated from 7 slaughterhouses (chicken meat, n=150; slaughtering environment of chicken, n=109; pork, n=42; slaughtering environment of pork, n=136) and 5 supermarkets (chicken, n=92; retail environment of chicken, n=350; pork, n=58; retail environment of pork, n=175), in 12 districts of Chongqing, China. Meat mentioned above was from unpacked fresh meat; slaughtering environment including wash water, knives, floor, feces, apparatus, containers, tables, carcasses, and blood; retail environment including chopping boards, ice, knives, floor, containers, wash water, and tables. All collected samples were stored in an icebox and transported to a laboratory within 2 hours of collection for immediate processing and then held in a refrigerator at 4°C.
Isolation and serotyping
After a pre-enrichment step of each samples in 10 mL sterile buffered peptone water (BPW) and incubated overnight at 37°C. 0.2mL of each pre-enriched suspensions were added into 2 ml of Rappaport-Vassiliadis enrichment Broth (RVB) and 2 ml of Tetrathionate broth (TTB) respectively, then incubated at 42°C for 24 h. One loopful of each RVB and TTB culture was then streaked onto Xylose Lysine Tergitol 4 (XLT-4) agar plates, which were incubated at 37°C for 24 to 48 h. Among suspected colonies, only one was picked up from a plate and confirmed by specific gene through Polymerase Chain Reaction (PCR) of Salmonella using assays. Each isolate was serotyped by slide agglutination based on the Kauffmann-White-Le Minor Scheme .
Antimicrobial susceptibility testing
The standard Kirby-Bauer disk diffusion method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2010) was carried out to test antimicrobial susceptibility of the Salmonella isolates to 13 categories of antimicrobials (Hangzhou Microbial Reagent., Ltd.): ampicillin (AMP 10 μg), cefoperazone (CFP 75 μg), piperacillin (PRL 100 μg), tetracycline (TE 30 μg), ceftazidime (CAZ 30 μg), doxycycline (DOX 30 μg), ceftriaxone (CRO 30 μg), minocycline (MH 30 μg), norfloxacin (NOR 10 μg), sulfamethoxazole (SXT 1.25 μg), ofloxacin (OFX 5 μg), chloramphenicol (C 30 μg) and ciprofloxacin (CIP 5 μg). Escherichia coli ATCC 25922 was invoked as the control organism. According to the CLSI, the isolates were considered to be susceptible, intermediate, or resistant. Salmonella isolates resistant to three or more antimicrobial classes were defined as MDR isolates.
Antimicrobial resistance genes
All of the drug-resistant Salmonella isolates were examined for the presence of resistance genes, including β-lactams (blaTEM, blaCTX-M, and blaOXA), tetracycline (tet(A), tet(B)and tet(C)), florfenicol (floR), and sulfonamide (sul1, sul2 and sul3). All of the genes were identified by PCR amplification. The primer sequences and predicted sizes for PCR amplification of different resistance genes from Salmonella were listed in Table 1. PCR amplification was performed using a DNA Thermal Cycler (Life Technologies, USA) with the following conditions: 95℃ for 10 min; 30 cycles of 94℃ for 45 s, 55-70℃ for 50 s, 72℃ for 50 s; and 72℃ for 10 min. 2% (w/v) agarose (Biowest, Spain) gel electrophoresis was used to analyze the PCR products.
Multilocus sequence typing
Protocols used for MLST of Salmonella were described online . Seven housekeeping genes were amplified by PCR, including thrA, purE, sucA, hisD, aroC, hemD, and dnaN. PCR products were purified and sequenced by Sanger method, and the alleles and STs were assigned according to the MLST scheme . The unweighted pair group method with arithmetic means analysis (UPGMA) was utilized to infer relationships among the isolates through MEGA7 software .
All statistical analyses were done using SPSS 20.0 (SPSS Inc., Chicago, IL), and the chi-squared test was applied to assess any statistically significant (P<0.05) differences in this study.