| The livestock and poultry breeding industry has gradually changed from decentralized breeding to intensive breeding in China.Antibiotics,as growth promoters and antibiotics for the treatment of bacterial diseases,are widely used in livestock and poultry breeding industry.The unreasonable use of antibiotics leads to the enhancement of bacterial antibiotics resistance and the spread of antibiotics resistance genes,which brings harm to public health.Enterococcus faecalis is one of the main pathogens causing human or animal infectious diseases,and oxazolidinone antibiotics are the "last line of defense" for the treatment of vancomycin-resistant Enterococci,so it is of great health significance to monitor their antibiotics resistance.The purpose of this study was to investigate the microflora structure and antibiotic resistance of microorganisms in internal swine farms in Zhejiang Province,and to explore the horizontal transformation mechanism of plasmid-mediated oxazolidinone resistance gene optrA in Enterococcus faecalis.The main research results are as follows:1.Fecal samples of swine were collected from 15 pig farms in Hangzhou,Ningbo,Jinhua,Quzhou and Wenzhou of Zhejiang Province.The structure of microflora,antibiotic resistance,abundance and potential host of antibiotics resistance genes,the differences of microflora and ARGS among cities were analyzed.In terms of microbial structure distribution,bacteria are the main microorganisms in pig farms in Zhejiang Province,accounting for more than 90%.At the genus level,Enterococcus,Psychrobacter and Escherichia coli are the main genus.Non-metric multidimensional scaling(NMDS)shows that there are some similarities in the distribution of sample species in the same region.In terms of antibiotic resistance,176 antibiotic resistance genes corresponding to 70 antibiotics were matched,and 71 drug resistance genes were shared in samples from five cities.The three most abundant drug resistance genes are chloramphenicol resistance gene cml,sulfonamide resistance gene sul2 and bacitracin resistance gene baca.There were significant differences in the abundance of macrolide resistance gene cml,multidrug resistance gene mexf,tetracycline resistance gene tet K,aminoglycoside resistance gene aph3 ib and trimethoprim resistance gene dfra17 among the five cities.Through the contribution analysis of species and antibiotics resistance genes,it was found that Acinetobacter was the potential host of sulfonamides resistance gene sul2,while Escherichia coli,Enterococcus,Staphylococcus and Cryophilus were the main potential hosts of bacitracin resistance gene baca.The correlation between Enterococcus and antibiotic resistance genes was analyzed by constructing a network co-occurrence map.Enterococcus was negatively correlated with sulfonamides resistance gene sul and macrolide resistance gene cml,and positively correlated with bacitracin resistance gene baca,tetracycline resistance gene tet,and efflux pump resistance gene acr.2.Antibiotics sensitivity test of MIC and PCR technique were used to explore the phenotypic composition and genotypic distribution of antibiotics resistance of Enterococcus faecalis,so as to evaluate the level and difference of antibiotics resistance of Enterococcus faecalis in pig farms in Zhejiang Province.406 strains of Enterococci were detected from 545 fecal samples in Zhejiang Province.The total isolation rate was74.50%,of which 226 strains were Enterococcus faecalis.Antibiotics sensitivity test of MIC showed that the resistance rate of Enterococcus faecalis to nearly 57%(8 to 14)of commonly used antibiotics was more than 50%,and the highest resistance rate was pleuromulins antibiotic pleuromulin,which was 98.23%.The resistance rates of tilmicaoxin,erythromycin,tetracycline and florfenicol were all higher than 80%,and the resistance rate of oxazolidinone antibiotics linezolid was 38.49%.The proportion of multi-drug-resistant bacteria(3R-14R)is as high as 98%.Enterococci from different regions are mainly 7R-10 R,accounting for more than 50%.The overall antibiotics resistance in Hangzhou,Quzhou and Jinhua was more serious than that in the coastal cities of Ningbo and Wenzhou.Oxazolidinone resistance genes were mainly optrA,and the detection rate was 71.68%.The detection rate of poxt A was 7.08%,and no cfr and cfr(B)genes were detected.the detection rate of optrA in Hangzhou was significantly higher than that in Ning Bo.The main phenicols resistance genes are optrA and fex A,and optrA and fex A often co-exist in the strains.3.The transformation mechanism of optrA gene resistant to oxazolidinone in porcine Enterococcus faecalis was studied by means of Multilocus sequence typing,conjugation transfer,replicon typing and plasmid sequencing.Multilocus sequence typing test showed that optrA resistance genes were transmitted horizontally mainly by horizontal gene transfer in Enterococcus faecalis.The conjugation transfer experiment showed that the optrA gene of some strains was located on the plasmid and could be transmitted.The plasmid replicon typing experiment showed that the Rep A＿N type plasmid was the main type carrying optrA resistance gene.The plasmid p HZ318-optrA contains two foreign insertion regions,both of which are multidrug resistance regions(MDR).Through the analysis of the flanking sequence and genetic environment of optrA resistance gene in p HZ318-optrA plasmid,it was found that optrA and fex A drug resistance genes coexisted in IS1216E-fex A-optrA-ferr-erm(A)-IS1216 E transposon,and there was a partial complete sequence of Tn554 transposon downstream,which endowed the strain with resistance to macrolides and streptomycin.Only one insertion sequence ISVlu1 was found in p NB304-optrA,and no other moving elements such as transposons were found.To sum up,the microflora structure of swine farms in Hangzhou is more complex than that in Ningbo,and the species of antibiotics and resistance genes are more abundant.Enterococcus is one of the dominant bacteria in swine farms and the potential host of resistance genes such as baca.The detection rate of optrA in Hangzhou area was significantly higher than that in Ningbo area,and optrA often coexisted with fex A.Resistance gene optrA in Enterococcus faecalis can be transmitted horizontally among strains through mobile genetic elements such as IS1216 E and Tn554,mediated by Rep A＿N type plasmids.The stability of Rep A-N plasmid p HZ318-optrA in Enterococcus faecalis was better than that of Rep3 plasmid p NB304-optrA,with more mobile elements,and the transmission ability is stronger.The results of this study provide a reference basis for the rational use of antibiotics in the pig industry,and provide scientific guidance for slowing down the development of multi-antibiotic resistant Enterococci and exploring new antibiotics resistance control strategies. |
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