| Escherichia coli and Enterococcus are commensal bacteria in human and animal intestinal, which are regarded as indicator organism for antimicrobial resistance in the community and farms. Tibetan pig originated in Tibet region, which is free-ranging in its natural environment with adequate water and food, and no antimicrobials are used for therapy or growth promotion.Researches on antimicrobial resistance characteristics and mechanisms of E. coli&Enterococcus from Tibetan pigs have public significance in the field of veterinary and human medicine.A total of232fecal samples were collected from Tibetan pigs in2012. The animals were grazed with extensive management in a semi-mountainous region, closed to Jue Mugou, Linzhi County, Tibet. Importantly, antimicrobials were not used as either therapeutic agents or as growth promoters in these animals. A total of129E. coli isolates and84Enterococcus isolates were identified from the fecal samples. The isolation rates for E. coli and Enterococcus were55.6%and36.2%, respectively. Among the84Enterococcus isolates, four different species were identified:E. faecium (45/84,53.6%), E.faecalis (31/84,36.9%), E. hirae (5/84,6.0%), and E. mundtii (3/84,3.6%). E. mundtii from pigs were isolated for the first time. The disk diffusion and the broth microdilution methods were used to determine the susceptibility of of E. coli isolates to11antimicrobial agents. The result indicated that all129E. coli isolates were susceptible to amoxicillin/clavulanic acid. For other β-lactam antibiotics, including ceftiofur and ampicillin, the frequency of resistance was less than30%(10.9%and27.9%, respectively). Less than10%of E. coli isolates were resistant to ciprofloxacin (7.8%), gentamicin (7.0%), or spectinomycin (2.3%). Approximately20%of the isolates showed resistance to sulfamethoxazole/trimethoprim (19.4%), nalidixic acid (19.4%), or streptomycin (16.3%), and the resistant rate to florfenicol was27.9%. Approximately40%of the isolates were resistant to tetracycline (40.4%), which was the highest rate of resistance among all of the tested antimicrobial agents. Furthermore, there are32strains,24.8%of the E. coli isolates were resistant to more than three antimicrobial agents.Enterococcus isolates were tested by the disk diffusion method, the broth microdilution method, and the agar screening method for their susceptibility to13antimicrobial agents. The results showed that none of84Enterococcus isolates were resistant to amoxicillin/clavulanic acid or vancomycin, but more than60%of the isolates were resistant to oxacillin, clindamycin, or tetracycline (92.8%,82.1%, and64.3%, respectively), and the rate of resistance to erythromycin was nearly50%(48.8%), due to the natural resistance of Enterococcus to oxacillin, clindamycin and erythromycin. And17.9%of the isolates were resistant to florfenicol. However, Enterococcus isolates showed low rates of resistance to some of the other tested antimicrobials, including:penicillin (6.0%), ciprofloxacin (3.6%), levofloxacin (1.2%), and ampicillin (1.2%). In addition, in tests for high-level resistance to streptomycin and gentamicin, only one Enterococcus isolate tested was positive in each of the respective assays. Except for tetracycline, erythromycin and penicillin, there is no significant (P＜0.05) difference to susceptibility to other antimicrobials among four different species Enterococcus isolates.For the129E. coli isolates and84Enterococcus isolates, the PCR results revealed that there were11strains harboring blTEM and blaCTX-M,1strain harboring qnrB,9strains harboring qnrD,7strains harboring qnrS,31strains harboring floR,3strains harboring cfr from E. coli isolates, respectively. Many other kinds of resistant genes, such as blaSHV、CMY-1、CMY-2、blaoxA-2、blaoxA-10、qnrA、qnrC、 qepA、aac(6’)-Ib、qxA and oqxB were failed to detected in E. coli isolates. The co-harboring of PMQR and β-lactamase determinants was found among E. coli isolates. In the Enterococcus isolates, the florfenicol resistance gene, fexA,fexB, and cfr, were detected in12,2, and3strains, respectively. There were2florfenicol-resistant Enterococcus isolates were failed to detect resistant genes, such as cfr,fexA and fexB. The sequencing result showed that all the blaTEM were TEM-1, for the blCTX-M,8strains were CTX-M-14, and3strains were CTX-M-15, all the qnrS were qnrSl.The E. coli isolates with resistant genes were analyzed by pulsed-field gel electrophoresis. The results showed that there was a predominant clone prevalence existed in the E. coli isolates which harbors β-Lactamase genes, PMQR determinants or florfenicol resistant genes, indicating that the related antibiotic resistance maybe attributable to colony spread in E. coli from Tibetan pigs. But PFGE analysis of the florfenicol resistance isolates in Enterococcus showed great genomic diversity with different patterns, which indicated that most of florfenicol resistant genes were transmitted through horizontal transfer among Enterococci from Tibetan pigs.In conclusion, the investigation showed that a small number of isolates from Tibetan pigs were resistant to common antimicrobial agents, except for tetracycline. The prevalence rate of antibiotic resistant gene was lower than other regions in China. However, the horizontal transfer is the main way to the spread of antibiotic resistant genes among the isolates from Tibetan pigs, which suggested that strict control of the use of antibacterial drugs in Tibetan pigs should be applied to strengthen the management of feeding. It should be also important to avoid irrelevant persons and other animals getting in touch with Tibetan pigs in the farm. |
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