| In recent years, with the rapid development of livestock scale in Liaoning Province, a large number of antibiotics have been widely used in the prevention of animal diseases, which lead to drug-resistance of Escherichia coli a wide spread. In order to clarify the causes and trends of drug-resistance of Escherichia coli, and establish prevention methods of drug-resistance’s generation to solve bacterial drug resistance, the detection and homologous analysis of major resistant genes of chicken-sourced and human-sourced Escherichia coli were carried out, and the transmitting law of their resistant genes were further analyzed and verified. Those tests will provide scientific evidence for the rational use of medicine and prevention and control of Escherichia coli disease in human and animals. 1. Trace detection of drug resistance of chicken-sourced Escherichia coli.From 2012 to 2015, 218 chicken-derived Escherichia coli were isolated and obtained from a large-scale of broiler chickens farm in Liaoning province, which were measured on 14 kinds of veterinary antimicrobial drugs and 16 kinds of human use of antimicrobial drugs by trace dilution method, and determined with CLSI Enterobacter decision criteria. The results showed that chicken-derived Escherichia coli had obvious resistance to veterinary antimicrobial drugs(Ampicillin, Enrofloxacin, Trimethoprim-sulfamethoxazole, Sulfafurazole, Tetracycline) and human use of antimicrobial drugs(Piperacillin, Ticarcillin, Ciprofloxacin). The average resistant rates were more than 60%, and its resistance generally showed an increasing trend. Analyzing and comparing the resistance test results of isolated chicken-derived Escherichia coli in four years and the MIC value of wild type strains reported by EUCAST, they showed the same resistant range, but MIC values of Spectinomycin, Ampicillin, Enrofloxacin, Trimethoprim-sulfamethoxazole and other drugs moved to the right obviously, indicaty that Escherichia coli increased resistance. Drug varieties with high resistance rate with increasing strength, such as Tetracycline, Florfenicol and others were the long-term used drugs in farms, indicating that the generation of bacterial resistance is closely related to long-term use. 2. Detection and homologous analysis of resistance genes from chicken-sourced and human-sourced Escherichia coli.The isolated chicken-sourced and human-sourced Escherichia coli were tested by judging susceptibility of 14 kinds of veterinary and 16 human use of with antibiotic drugs. The results showed that Escherichia coli are resistant to 10 kinds of veterinary antimicrobial drugs and 6 kinds of human use of antibacterial drugs. 10 main related genes of 6 kinds of antimicrobial drugs of a higher common resistance rates of isolated strains were detected, and the results showed chicken-sourced and human-sourced Escherichia coli commonly carried seven species of resistant gene, including that the detection of rate of gyrA and gyrB reached 100%; resistant gene carried commonly by nucleotide homologous analysis showed that homology of gyrB, sul2, TEM were 100%, 99.55% and 99.38%, respectively, and homology of mcr-1, floR, gyrA reached 90%, 98.23%, 97.75%, 93.33%, respectively. Studies showed that the chicken-sourced and human-sourced Escherichia coli from a broiler farm commonly produced an apparent resistance to veterinary and human use of antimicrobial drugs, and part of related resistance genes were highly homologous, indicating that bacterial resistance and its related genes may spread in chicken-sourced and human-sourced Escherichia coli in a broiler farm. 3. Comparative Analysis of homology and resistance of drug-resistant strains of Escherichia coli from different sources.Using pulsed field gel electrophoresis to identify the molecular typing of isolated and obtained chicken-sourced Escherichia coli, the resistant strains’ genotypes of different sources were compared to analyze genetic correlation to prove whether the strains with the same kind of resistant genes spread among chickens, humans and other different individuals, and then were compared with the apparent resistance spectrum type of Escherichia coli. The results showed that there were exactly the same PFGE patterns among strains from the animals, breeders and different individuals in a same broiler farm. Illustraty that strain carrying the same kind of resistant genes could spread on horizontal direction in broilers, breeders and different individuals. Compared to PFGE typing and susceptibility results of Escherichia coli from different sources, part of the same PFGE typing of strains didn’t have the same resistant spectral shape, but part of the different PFGE typing of strains might have the same type of resistant spectral shape. Resistant genes were complex and complicated. The same type of cloning strains may obtain or lose resistant plasmids in the process of proliferation and spread, which resulted in the change of resisitance. Different cloning strains may spread resistance on horizontal direction through resistant plasmids. 4. Adapter test of Escherichia coli with resistant genes.To confirm whether the horizontal transmission of resistance of Escherichia coli occurred, plasmid transformation test was conducted on isolated resistant strains. Treating strains successfully converted plasmid as affording strains. Escherichia coli J53 was regarded as a recipient strain, to determine whether positive plasmids propagated interactively, and to detect resistance, resistance genes and PFGE profile type of zygotes. The results showed that zygotes detected resistant genes(mcr-1, floR, sul2). Zygotes tested resistance produced resistance to the corresponding drugs(Polymyxin, Florfenicol and Sulfisoxazole), indicating that resistant genes can spread in horizontal direction through plasmids-mediated among different sources of Escherichia coli strains, and then spread drug-resistance. The study showed that if Escherichia coli from chicken were highly resistant to drugs, it may be possible to transmit to human-sourced Escherichia coli in the breeding process. |
