Epidemiological Study Of Plasmid-mediated Colistin Resistance Gene Mcr-1 In Intestinal Tract From Healthy Population Of Zhejiang Province

OBJECTIVE: To investigate the current situation of colistin resistance and the prevalence of plasmid-mediated colistin resistance gene mcr-1 in Escherichia coli isolates from healthy population in Zhejiang Province;to further characterize the mcr-1-carrying E.coli isolates,and illuminate the spreading mechanism of mcr-1 among E.coli isolates,finally to evaluate the risk of mcr-1 transmission in clinical practice and provide scientific evidence for the prevention and control of mcr-1 transmission.METHODS: A total of 1011 stool samples from healthy population throughout Zhejiang Province were collected and submitted to broth enrichment method for culture of bacteria;PCR were directly used for detection of mcr-1;For mcr-1-carrying isolates,minimum inhibitory concentration(MIC)was determined by agar dilution method,resistance to cephalosporin and quinolones was confirmed by PCR;Transconjugation assay was conducted to characterize mcr-1-carrying plasmid;Pulsed field gel electrophoresis(PFGE)and multilocus sequence typing analysis were performed for homogeneity analysis in addition to molecular typing.RESULTS: Among the 1011 stool samples from healthy population,127 samples were positive for colistin resistance gene mcr-1 with a detection rate of 12.6%,for 2 samples,mcr-1-positive E.coli and Klebsiella pneumoniae isolates were simultaneously isolated.Of the 129 mcr-1-carrying isolates,116 were E.coli.The resistance rates of mcr-1-positive E.coli isolates to the third generation of cephalosporin were relatively higher as compared with the forth generation of cephalosporin(resistance rates of 5% for cefepime);Of those isolates,35.5% were resistant to ciprofloxacin;As for other antimicrobial drugs,the resistance rates were low,and none of carbapenem-resistant isolates was isolated.Thirty-three out of 129 isolates were identified to produce Extended Spectrum Beta-Lactamases(ESBLs)with detection rate of 25.6%,additionally,20 was positive for bla TEM-1 and 1 was positive for bla SHV-1;furthermore,14 isolates were positive for plasmid-mediated quinolone resistance genes,as 7 positive for qnrS1,and 2 positive for qnrS2 and 5 with acc(6')-Ib-cr mutation.Seventy-seven transconjugants were successfully obtained by transconjugation assay,in addition to mcr-1,bla CTX-M and plasmid-mediated quinolone resistance genes were also identified in those transconjugants,demonstrating the transferability of resistance gene-carrying plasmid.A total of 67 sequence types were identified by MLST analysis with ST10 being the most prevalent one(18/116,15.5),besides,8 sequence types 13.8% were found to be closely related to ST 10 with difference of one allele,namely CC10 clone complex.As for PFGE patterns,no homogeneity were presented among those isolates,except for 2 highly homologous isolates from Hangzhou and another 2 from Jiaxing.CONCLUSIONS: 1.A high detection rate of mcr-1 was observed in isolates from intestinal tract among healthy population of Zhejiang Province as high as 12.6%;90.0% of mcr-1-positive isolates belonged to E.coli,making it to be the biggest repositories of colistin resistance gene.2.mcr-1-positive isolates remained high susceptibility to commonly used antimicrobial drugs except for cephalosporin and quinolone with slightly higher resistance rate,none of carbapenem-resistant mcr-1-positive strain was isolated.3.As confirmed by transconjugation assay,colistin resistance gene mcr-1 was mostly located on transferable plasmid,which can also carry bla CTX-M-1/ bla CTX-M-9 group genes and quinolone resistance genes such as qnrS1,contributing to the transmission of resistance among different strains.Two strains of Escherichia coli were detected simultaneously with mcr-1 and a novel chimeric recombinant gene bla CTX-M-64,which was the first time found in the intestinal tract of Escherichia coli.4.High heterogeneity was observed among the majority of mcr-1-carrying E.coli isolates as demonstrated by PFGE and MLST analysis,with CC10 complex being the most prevalent,counting for 29.3%(34/116).Except for 2 highly homologous isolates from Hangzhou and another 2 from Jiaxing,clonal transmission of mcr-1-positive E.coli isolates was not observed.