Study On The Conditions Of Transfer Of OqxAB Gene Mediated By Plamid Between Escherichia Coli And The Phenotype And Genotype Of Its Transconjugant

Escherichia coli,commonly known as E.coli,is one of the important food borne opportunistic pathogens in inexperimentinal flora,which has brought huge econo MIC losses to the global aquaculture industry.Oqx AB efflux pump is one of the important components of RND(resistance nodulation cell division)efflux pump family.It has efflux effect on many kinds of antibiotics including enrofloxacin,ciprofloxacin,olaquindox and chloramphenicol,metalions and some quaternary ammonium salt disinfectants.It is one of the important transfer resistance mechanisms of E.coli.However,there are few basic researches on the transfer conditions of plasmid mediated oqx AB gene between E.coli and the changes of stability,growth and drug resistance phenotype of transconjugants and the correlation analysis of MICs.Based on the above situation,we studied the effects of enrofloxacin,olaquindox and chloramphenicol on the transfer of oqx AB gene between E.coli C26 and E.coli C600 isolated from chickens with positive oqx AB gene as donor and Receptor strains,respectively.The plasmid sequence analysis of E.coli C26-600(M1)and Transcriptome of E.coli C600 and C26-600(M1)were carried out,aim to investigate the effects of different concentrations of antibiotics on the ability of the transfer of oqx AB gene between E.coli,and the stability regulation,growth ability and resistance phenotype of transconjugants after transfer.1.Investigation of oqxAB gene prevalence and bacterial phenotype in Escherichia coliIn 2019,200 clinical chicken-derived Escherichia coli isolated from Anhui and Jiangxi regions were experimented for oqx AB gene: 32 of these Escherichia coli with positive oqx AB gene were experimented for drug susceptibility to 16 antibacterial drugs,according to the CLSI document M100 for drug resistance determination.The results showed that the positive rate of oqx AB gene in 200 strains of clinical Escherichia coli was18.5%(37/200);among 32 strains of oqx AB gene positive Escherichia coli,the highest drug resistance rate was ampicillin,spectinomycin,tetracycline,florfenib and sulfisoxazole,all reached more than 85%;and the several drugs with resistance rates below 20% were trimethoprim,ceftazidime,meropenem,colistin and methaquine.At the same time,the oqx AB gene-positive Escherichia coli is often multi-drug resistant,with 5,10,and 12 resistant strains reaching 84%(27/32),37%(12/32)and 9%(3/32)respectively.2.Effect of different conditions on oqx AB gene transfer in E.coliEscherichia coli C600 was used as the recipient bacteria for the conjugative transfer experiment,and E.coli C15,C16,C26 and C27 were used as the donor bacteria for the pre-conjugative transfer experiment.According to the results of preliminary experiments,Escherichia coli C26 was selected as the donor bacteria for the conjugative transfer experiment.The conjugative transfer experiment uses 3 kinds of antibacterial inducers(enrofloxacin,olaquindox,chloramphenicol),and 6 kinds of induction concentration(0MIC,1/32 MIC,1/16 MIC,1/8 MIC,1/4 MIC,1/2 MIC)and the conjugation ratio of donor to recipient bacteria [(108 CFU/m L:105 CFU/m L),(105 CFU/m L: 105 CFU/m L),(102 CFU/m L:105 CFU/m L)] are the basic conditions.The induction results of the three antibacterial drugs showed that the transfer of oqx AB gene in Escherichia coli was least regulated by chloramphenicol,enrofloxacin and Olaquindox.The results of induction at different concentrations showed that the conjugation rate of the three antibacterial drugs increased first and then decreased with the increase of the induced concentration,and the conjugation rate was the highest at 1/8MIC~1/4MIC.The induction results of different donor and recipient bacteria conjugation ratios showed that when the donor and recipient bacteria ratios gradually decreased from 1000:1,1:1,and 1:1000,the conjugation rate gradually decreased.3.Analysis of stability,resistance phenotype and growth ability of transconjugant10 strains of transconjugant(M1~M10)were cultured in anti-LB broth(except for streptomycin),and the ratio of resistant bacteria in the transconjugant broth was determined;the drug-resistant phenotype change of the transconjugant M1 was detected;the transconjugant M1 was determined Growth curve.The results of verification of the stability of the transconjugant showed that the ratio of resistant bacteria ranged from 24.81% to 94.81% after 10 days of passage in the non-resistant LB broth(except for streptomycin).The transconjugant M1(69.57%)was used as the follow-up experiment strain.The results of the drug sensitivity experiment of the transconjugant M1 showed that compared with the recipient bacteria,the transconjugant is more resistant to ampicillin,gentamicin,tetracycline,florfenicol,compound trimethoprim,ceftazidime,enrofloxacin,and ofloxacin The resistance levels of10 antimicrobials including olaquindox,olaquindox,and chloramphenicol have increased by more than 16 times,and the resistance levels of spectinomycin,ceftiofur and apramycin have also increased by more than 4 times;compared with the donor The resistance levels of bacteria and transconjugant to ceftazidime,apramycin and ofloxacin are also increased by 4 times or more.The results of the growth curve determination of the transconjugant M1 showed that the growth rate was inhibited compared with the recipient bacteria,and the final concentration of the bacteria was similar to that of the donor bacteria.4.Genome sequencing analysis of transconjugant plasmidsIn this study,plasmid sequencing analysis was performed on E.coli C26-600(M1)in the conjugative and transfer assay.The results indicated that the plasmid type of E.coli C26-600(M1)is Inc X,with a size of 138,728 bp.The plasmid-mediated drug resistance background in E.coli C26-600(M1)includes not only quinolones(oqx A/oqx B),but also?-lactams(bla TEM-1,bla TXM-55),aminoglycosides((aad A5),chloramphenicols(cm LA,flo R,cat B3),quaternary ammonium salts(qac E11),phosphomycin(fos A3),and rifampin(arr-3).5.Comparative transcriptome sequencing analysis of transconjugant and recipient bacteriaIn the conjugative transfer experiment,the recipient bacteria Escherichia coli C600 and the conjugant Escherichia coli C26-600(M1)were analyzed by comparative transcriptome sequencing.Differentially expressed genes related to multidrug efflux pumps were screened out,including RND family(acr F,acr R)and MFS family(emr Y,mdt G,emr D,mdf A).Differentially expressed genes related to adaptation and stability mainly include hip A,hip B and umu D,which are involved in maintaining the stability of the plasmid in the adaptor E.coli C26-600(M1).The differentially expressed genes related to the toxin-antitoxin(Toxin-Antitoxin,TA)system mainly include yaf N,hok E,hok B,hok D,mqs R,rel B and mqs A.This project is the first time to study the effects of antibacterial substrate,concentration of antibacterial agents and different conjugation ratio of bacteria on oqx AB gene transfer in E.coli.It is confirmed that different types and concentrations of antibacterial agents and different conjugation ratio of donor and recipient bacteria will affect the transfer of oqx AB gene in E.coli.This study has important theoretical significance for the risk assessment of clinical oqx AB gene resistance transmission and the development of clinical drug resistance prevention and control measures.