| In recent years,an old antibiotic(colistin) was now re-introduced to treat multidrug-resistant(MDR)bacterialinfection,due to the increasing prevalence ofmultidrug-resistant(MDR)bacteria;especially these bacteriaoften carried carbapenem resistance genes.Unfortunately,colistin resistance phenomenon was becoming more and more serious. The polymyxin resistance was considered to be caused by chromosome mutation,sinceplasmid-mediated colistin resistance genemcr-1was reported,mcr-1 was now becoming a hot research.In this study,we detected the mcr-1 genefrom Escherichia coli(E.coli) isolates which isolated from a livestock farm of shanghai which was firstreportedto carry mcr-1 gene,in order to study the prevalence and transfer mechanism of mcr-1gene,so as to provide a theoretical basis for controlling the spread of mcr-1 gene.A total of 47 E.coli were collected from swine in a livestock farm of shanghai in this study.PCR amplification and sequencing were conducted to detectmcr-1 gene.The resultshowed that mcr-1gene were detected in all non-susceptibleisolates,the percentage of mcr-1 was 57.4 %(27/47).This result indicated that the colistin resistance was mainly caused by mcr-1 in this livestock farm.Antimicrobial susceptibility test of the mcr-1-carryingisolates was performed by the agar dilution method. Results showed thatthe resistance rate to ampicillin was 77.8%(21/27), the resistance rate to cefotaximewas 33.3%(9/27), the resistance rate to fosfomycin was 25.9%(7/27),theresistance rate tochloramphenicol, florfenicoland trimethoprim-sulfamethoxazole were 100%, indicating that all strains were showing varying degrees of multidrug resistance.All positive isolates were susceptible to amikacin and imipenem.Pulsed field gel electrophoresis(PFGE) was carried out to analyze clonal relationship of mcr-1-positive isolates.We can find that 26 isolates were successed by PFGE, one was failed, 19 PFGE types(A-S) were detectedin these 26 isolates, indicating that the majority mcr-1-positive isolates done not exist clone relationship.Conjugation experiments were performed for 27 mcr-1-carrying E. coli isolates to investigate the transferability of mcr-1 gene. The results showed that the colistin resistance genemcr-1 from 22 E.coli isolates were successfully transferred by conjugation,of which,2 E.coli isolates obtained twodifferent transconjugants,while 5 E.coli isolates were failed by conjugation in every test.Replicon typing was conducted for 24 transconjugants. The results show that ninetransconjugants were Inc X4 plasmids, sixwere Inc HI2 plasmids, four were Inc I2 plasmids, two were Inc F plasmids, the otherthree were Inc Y, Inc HI2 and Inc Y,F: 2 A- B: 53 and Inc X4 plasmid. PCR was performed to detectother resistant genes from transconjugants.TheblaCTX-M-14, fos A3, oqx ABandflo R gene were detected in five Inc HI2 plasmids, one Inc HI2 plasmidcarried blaCTX-M-14, fos A3 andflo R, whileblaCTX-M-15,fos A3 and flo Rwere detected in one Inc HI2 plasmid, which demonstrated that these type plasmid usuallyco-transfermcr-1 gene with blaCTX-M, fos A3, oqx AB and flo R.The genetic context surrounding the mcr-1 gene was investigated by PCR mapping and sequencing, and the three different genetic environment of mcr-1were determined.Insertion sequence ISApl1 was only detected in theupstream of mcr-1 gene in seven isolates,mcr-1 genewas flanked by two ISApl1 elements in six isolates, while ISApl1 was not detectedin another fourteen isolates.S1 pulsed-field gel electrophoresis(PFGE) and Southern blotting were conducted for 24 transconjugants and 5 original bacteria which carried mcr-1 gene.The results showed that the size of Inc X4 plasmid was approximately 28.8~33.3kb,Inc I2 plasmid was approximately 54.7~78.2kb, Inc Y plasmid wasapproximately 78.2kb, Inc HI2 plasmid was approximately 216.9~244.4 kb, F29 plasmid was about 54.7kb, F53 plasmid size was about 138.9kb.Of the five original bacteria which were failed by conjugation experiment, the mcr-1 resistance gene of one isolate was located in chromosome, the others were located in plasmid, the plasmid size of one isolate was approximately 28.8~33.3kb, one was about 244.4~310.1kb,and anothertwo isolates were approximately 78.2~104.5kb.The results of this study showed that mcr-1 gene was widely disseminated in this livestock farm, mainly through Inc HI2, Inc X4 and Inc I2 plasmid. The Inc HI2 plasmid which carried mcr-1usuallyco-transfer mcr-1 withblaCTX-M, fos A3,oqx AB and flo Rgene. Therefore,we should pay enough attention on this new-found colistin gene mcr-1. |
PDF Full Text Request