Molecular Epidemiology Of Mobile Colistin Resistance Gene Mcr-1 And Comparative Evaluation Of Different Susceptibility Testing Methods Of Polymyxin

Objective1.The prevalence of mobile colistin resistance gene mcr-1 was investigated in the1598 Enterobacteriaceae strains collected from 2017 to 2020 in the First Affiliated Hospital,Fujian Medical University.And further to study the epidemiological characteristics and antibiotic resistance spectrum of mcr-1-positive isolates,in order to provide the reference basis for clinical prevention and control of the transmission and prevalence of plasmid-mediated colistin resistance gene mcr-1.2.Using broth microdilution as the gold standard,the performance of four susceptibility testing methods were evaluated,to provide a valuable reference for choosing an optimal antimicrobial susceptibility testing method in the laboratory.Methods1.The mcr-1 gene was detected by polymerase chain reaction in the 1598 strains of Enterobacteriaceae.And the carbapenemase genotypes of all strains carrying mcr-1gene were screened by polymerase chain reaction;the minimum inhibitory concentrations of polymyxin and ceftazidime-avibactam were detected by BMD;the antimicrobial susceptibility testing of commonly used antibiotics were carried out by Vitek-2 Compact system.Clonal relatedness of the mcr-1 carrying isolates was analysed by pulsed-field gel electrophoresis.2.Eighty-eight nonduplicate clinical Enterobacteriaceae isolates,of which six strains were positive for mcr-1,were subjected to test the minimal inhibitory concentration（MIC）of polymyxins using the following methods:colistin broth disk elution,Vitek-2^TM,BD Phoenix^TM,commercial broth microdilution and reference broth microdilution.With BMD as reference,essential agreement（EA）,categorical agreement（CA）,very major errors（VME）and major errors（ME）of polymyxins for different methods were analysed.Kappa consistency test,paired Chi-square test and Spearman rank correlation test were used to analyze the consistency between the four antimicrobial susceptibility testing methods and the gold standard.Results1.We identified 1598 isolates of Enterobacteriaceae（including 735 strains of Escherichia coli,742 strains of Klebsiella pneumoniae,33 strains of Enterobacter cloacae,24 strains of Enterobacter aerogenes,23 strains of Citrobacter freundii and41 strains of other species of bacteria）,and only four E.coli isolates possessed mcr-1gene.The carrying rate of mcr-1 gene was 0.25%,and the carrying rate of mcr-1gene in Escherichia coli was 0.54%.All 4 mcr-1 carrying E.coli isolates exhibited resisitance to polymyxins（MIC:4～8μg/ml）.And they also showed resistance to penicillins,cephalosporinsⅢandⅣ,monobactams,quinolones,folate pathway antagonists and tetracyclines.However they showed susceptibility to carbapenems,aminoglycosides,cefoperazone-sulbactam and tegacycline.Only one of the four mcr-1carrying isolates also carried carbapenem resistance gene NDM,and it was resistant to ceftazidime-avibactam.PFGE showed that 4 mcr-1-bearing E.coli isolates belonged to different genotypes.2.The results of BMD showed that among 88 strains of Enterobacteriaceae,7 strains were resistant to polymyxin and 81 strains were sensitive to polymyxin,the MIC values of polymyxin-resistant strains were between 4μg/ml and 16μg/ml.Taking broth microdilution as reference,the EA of colistin broth disk elution,Vitek-2^TM,BD Phoenix^TM,commercial broth microdilution were 94.32%,92.05%,90.90%and96.59%,respectively.And the CA were 100%for each method.No very major error（VME）and major error（ME）were recorded for four methods.The consistency between four susceptibility testing methods and the gold standard was positive（kappa values 1,p<0.001,Mc Nemar test p=1 and Spearman’s r>0.5,p<0.05）.Conclusions1.Our data indicated a low level of prevalence of mcr-1-positive Enterobacteriaceae isolates（0.25%）and Escherichia coli isolates（0.54%）in our hospital.All the mcr-1-positive E.coli isolates were not only resistant to polymyxin,but also resistant to a variety of common clinical antibiotics.And all were multidrug-resistant bacteria.Only one mcr-1-bearinng E.coli isolate co-harbored the carbapenem resistance gene NDM.The results of PFGE suggested that 4 mcr-1-positive isolates were non-homologous and there has been no outbreak.2.In the present work,four susceptibility testing methods all met the standards recommended jointly by the CLSI and EUCAST,of which the performance of the commercial broth microdilution and CBDE fared relatively well.Thus,these four methods could be routinely used in clinical microbiology laboratory of our hospital for colistin and polymyxin B susceptibility testing.