Sample collection
Samples (n= 2657) were collected from 18 farms and one cattle slaughterhouse in Tacheng, Bole, Yili, Wujiaqu, Changji, Wulumuqi and Akesu in Xinjiang of China between October 2012 and March 2017, including 1155 fresh feces, 1236 rectal swabs, 110 feed, 108 water and 48 carcass swabs (Table 2).
Bacterial isolate
Each 1 g or 1 ml sample (feces/feed/water) were aseptically added to 9 ml of trypticase soya broth (TSB) containing 20 mg/l novobiocin and were incubated for 6-8 h at 37 °C. A rectal swab was transferred into a separate tube containing 2 ml nutrient broth and cultured at 37 °C for 24 h [21]. One carcass swab was put into a stomacher bag and added 500 ml of modified trypticase soya broth containing 8 mg/l novobiocin. Each sponge was mixed in the stomacher bag for 2 min and then incubated for 20 h at 37 °C [22]. This was streaked out onto Sorbitol MacConkey agar supplemented with 0.01mg/l cefixime and 0.5mg/l potassium tellurite (Haibo, Qingdao,China) (CT-SMAC) and incubated for one day at 37 °C. One or more pale colonies were individually selected as presumptive E. coli O157 per sample. The prevalence of E. coli O157:H7 was assessed via polymerase chain reaction (PCR) (rfbE and fliC genes [23]) (Table 3). The positive isolates were each inoculated into separate TSB and incubated for one day at 37 °C, from which glycerol stock was made and then stored at -80 °C for further analysis.
Virulence analysis of isolates
DNA extraction
DNA was extracted by boiling the isolates. Each colony was inoculated on CT-SMAC and incubated for 16 h at 37 °C to obtain fresh colony. Several colonies were selected and suspended separately in 200 μl of sterile distilled water in 1.5ml eppendorf tubes. The suspensions were then boiled at 95 °C for 10 min in a water bath. After centrifuging at 12000 rpm for 10 min, the supernatant containing the template DNA was transferred into 1.5ml Eppendorf tubes without nuclease and were stored at -20 °C until use.
Determination of virulence genes by PCR
To characterize the virulence genes, amplification products of stx1, stx2, eae, hly and tccP genes were used, which encode for Stx1, Stx2 toxins, intimin, enterohemolysin, and tir couple cytoskeleton protein respectively. The primers, conditions and references cited are listed in Table 3. Amplification of the targeted gene used EX Taq (TaKaRa, Dalian, China) with the following PCR program: 94 ℃ for 4 min, 30 cycles of denaturation at 94 ℃ for 30 s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 30 s, with a final extension at 72 ℃ for 10 min. Adjust annealing temperature according to primer Tm value (Table 3). The PCR amplicons (10 μl) were subjected to electrophoresis on a 1.2% agarose gel in 1× TAE buffer at 115 V for 30 min, and stained with SYBR Green (Fermentas, Germany).
Antimicrobial susceptibility tests
The susceptibility of to antibiotics was tested using the Kirby-Bauer disc diffusion technique. Antibiotic discs obtained from OXOID, UK, including ampicillin (AMP), piperacillin (PIP), cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), aztreonam (ATM), ampicillin-sulbactam (SAM), piperacillin- tazobactam (TZP), amoxicillin-clavulanic acid (AMC), gentamicin (GEN), amikacin (AMI), streptomycin (STR), trimethoprim-sulfamethylisoxazole (SXT), chloramphenicol (CHL), levofloxacin (LEV), ciprofloxacin (CIP), tetracycline (TET), and polymyxin B (PB) [26]. E. coli ATCC25922 was used as a quality control strain in the susceptibility tests. The ESBLs-producing isolates were determined by double-disk synergy tests according to CLSI [26].
Detection of antibiotic resistance genes
The following resistance determinants were investigated by PCR: blaCTX-M (the CTX-M-type genes were detected using universal primers blaCTX-M-U [27], and the entire CTX-M-type genes were amplified using the primers blaCTX-M-1G [27], blaCTX-M-2G [28] or blaCTX-M-9G [29]), blaTEM [30], and blaSHV [30] which encode β-lactamases; chloramphenicol (cmlA1 [31]) efflux pumps; sulfonamide resistance gene (sul1 [32]); and the tetA [33], tetE [33], and tetG [33] tetracycline efflux pumps. Primer for the different genes are listed in (Table 3). Purified PCR products were sequenced. The DNA sequences and deduced amino acid sequences were compared with sequences reported in GenBank to confirm the subtypes of the β-lactamase gene.
Conjugation experiments and plasmid analysis
Sodium azide-resistant E. coli J53 was used as a recipient and conjugated to a blaCTX-M-producing isolate by filtration. Transconjugants were selected on Mac Conkey agar containing cefotaxime or ceftazidime (4 μg/ml) and sodium azide (200 μg/ml). ESBLs and antibiotic susceptibility were also tested in selected transconjugants, and the presence of bla genes was determined using PCR as described above. The resistance plasmids carried by transconjugants were typed by using PCR-based replicon typing [34].
Epidemiological typing
All available isolates were characterized by pulsed field gel electrophoresis (PFGE) using the CHEF-MAP-PER System (Bio-Rad Laboratories, Hercules, CA, USA) as described by Gautom [35]. Briefly, chromosomal DNA of E. coli O157:H7 isolate was isolated and the inserts were digested with XbaI (TaKaRa Dalian, China) for 16 h at 37 ℃. The electrophoresis was performed at 6.0 V/cm for 18.5 h with an angle of 120º at 14℃. The pulse time was increased from 0.5 to 60 s. The Salmonella serotype Braenderup H9812 (ATCC BAA-664) was chosen as the molecular weight marker. Gels were then stained in ethidium bromide (1.0 mg/L). The results were interpreted according to the criteria of Tenover et al. [36].