| With intensive farming gradually becoming the main farming mode in China,the extensive use of veterinary antibiotics in the process of farming,especially the irrational use,has led to the aggravation of bacterial resistance.The detection rate of drug-resistant bacteria with commonly used clinical antibiotics such asβ-lactams,tetracyclines,sulfonamides,quinolones,amido alcohols,aminoglycosides,polymyxin is high.In order to understand the production,distribution,transmission of drug-resistant indicator Escherichia coli and the influencing factors of drug resistance changes in the process of broiler breeding,this study conducted four times of incoming and outgoing chicken sampling in two broiler farms from July 2019 to September 2020.The samples included cloacal samples of breeder and commercial chicken,environmental samples of commercial chicken farms(ground swabs,cages/bedding,water samples)The management system,the use of antibiotics and disinfection and epidemic prevention were investigated.The purpose of this study was to understand the changes of drug resistance of Escherichia coli from the aspects of animals,environment,people,feeding management and the use of antibiotics in the feeding process,and to analyze the characteristics of drug resistance changes,so as to provide reference for controlling bacterial drug resistance and ensuring healthy breeding.In this study,macconkey medium and biochemical experiments were used to isolate and identify E.coli.The drug-resistant phenotype of E.coli was determined by broth dilution method.The drug-resistant genes were detected by liquid chip method.The transmission and distribution of E.coli in the breeding link were detected by MLST(multilocus sequence typing).The main results are as follows:1.Establishment of 17 drug resistance gene detection methods based on liquid chip technologySpecific primers and liquid chip probes were designed for 17 kinds of drug-resistant genes,and the corresponding positive plasmids were constructed.The optimal conditions for system one(9-fold)hybridization were 30 min and 20℃,and the optimal conditions for system two(8-fold)hybridization were 35 min and 30℃.The two systems had good specificity,and the minimum effective detection amounts were 10~3～10~5 and 10~2～10~6respectively.This method was in good agreement with PCR method It has a good coincidence rate.2.Determination of resistance phenotype of E.coliAt the time of entering chickens,the E.coli carried by chickens in H-farm and J-farm were resistant to at least one kind of antimicrobial agent,the multiple drug resistance rates were96.0%and 87.2%respectively,and the high resistant strains accounted for more than 60.0%and 25.6%respectively;meropenem and myxin resistant bacteria were detected in chickens in air and feed of H-farm respectively,and these two kinds of drug-resistant bacteria also appeared in environmental samples at the time of chicken exporting,indicating that the drug-resistant bacteria in the initial stage had spread in the feeding cycle.There were drug-resistant bacteria in the environment of the two farms at the beginning,J-farm feed and ground resistance rates were 90.9%and 66.7%,and drug-resistant bacteria were detected in other environmental samples.The proportion of drug-resistant bacteria in the environment of H-farm was 100%,and they were resistant to 14 kinds of drugs in different degrees.The overall drug resistance rate of chickens was higher than that of chickens,especially the kinds of drugs used during the breeding period.The drug resistance rate of all kinds of samples of H-farm increased by 18.9-66.7%,while that in J-farm also increased by 9.2-90.9%.After one feeding cycle,the multiple drug resistance rate of H-farm increased from 90.00%to 96.59%,while J-farm decreased from 84.71%to 77.46%.It is probable that the antibiotics used in J-farm were less than those used in H-farm,contributing to the drug resistance rate of other drugs decreased.The drug resistance characteristics of J-farm breeders and chickens were highly similar,and the drug resistance rate of most drugs was slightly higher than that of chickens.3.Distribution and molecular epidemiology of drug resistance genes in Escherichia coli before and after cultureOnly mcr-1 was not detected in H-FARM,and the detection rate of quinolones and tetracyclines was high.bla NDM-1 was detected in both incoming and outgoing chicken samples;bla SHV-1,blacmy-1,bla NDM-1 and mcr-1 were not detected in J-farm,and the detection rate of quinolones,tetracyclines and glucosamines was high.In addition,after a feeding cycle,the detection rate of all kinds of drug resistance genes increased significantly.H-farm 73 strains and J-farm 100 strains of E.coli were classified by MLST,and 34 and52 kinds of sequence types were obtained,respectively.ST 48(11/73,15.06%)was the dominant type of H-farm,while ST 155(12/100,12%)was the dominant type of J-farm.The dominant strains came from commercial chicken samples,environmental samples and human samples from commercial chicken farms and breeding chicken samples.Conclusion:(1)Compared with the common PCR detection technology,liquid chip technology has the advantages of high throughput,high sensitivity and good repeatability in the detection of drug resistance genes,which can achieve the effect of rapid detection.(2)The use of antibiotics in the feeding process can improve the tolerance of E.coli to the corresponding kinds of drugs.(3)The initial amounts of bacteria(including drug-resistant bacteria)in the commercial chicken itself and the breeding environment will proliferate in the feeding cycle,which will lead to the wide distribution of drug-resistant bacteria and the spread of drug-resistant genes,and increase the overall drug resistance of the flo R.Good feeding management is beneficial to slow down the increase of drug resistance.(4)The drug resistance of E.coli in breeder chickens will have a great impact on the drug resistance of commercial chickens,while the influence of breeders on the drug resistance of E.coli in farms is relatively low. |
PDF Full Text Request