Antimicrobial-resistant Mechanism And Homology Analysis Of Escherichia Coli To Polymyxin

Objective:1.To understand the clinical resistance of carbapenems and ESBLs-producing Escherichia coli to common antibiotics,and to screen for more sensitive antibiotics to guide clinical anti-infective treatment.2.To explore the resistance mechanism of Escherichia coli to polymyxin,and to analyze the homology of polymyxin-resistant Escherichia coli,and to know whether there are clones in this area.Methods:From May 2016 to October 2018,90 cases of carbapenem-resistant and extended-spectrum?-lactamase-producing Escherichia coli isolated from the Second Affiliated Hospital of Nanchang University were collected,and the same part of the same patient was excluded.The Vitek 2 Compact automatic microbiological analyzer was used for bacterial identification and drug susceptibility testing.The multi-drug resistant strains were tested for phenotype by modified carbapenemase inactivation test and EDTA synergistic test.Using the E-test method,90 strains of Escherichia coli were tested for polymyxin susceptibility,and a total of 3 strains of polymyxin resistant strains were screened.PCR amplification of carbapenemase,ESBLs,quinolone resistance genes and plasmid-mediated and chromosome-mediated polymyxin resistance genes,sequencing positive amplification products to determine their genotype;plasmid-binding assay to detect the transfer characteristics of drug-resistant genes.The clonal type of mcr-1 positive strain was analyzed by multilocus sequence typing?MLST?,and the eBURST software was used for affinity analysis to confirm whether the same clonal spread existed in the region;S1nuclease-Pulsed Field Gel Electrophoresis?S1-PFGE?and plasmid sequencing were used to analyze plasmid characteristics of the strains carrying the mcr-1 gene.Results:Among 90 strains of Escherichia coli,ESBLs and carbapenem-resistant strains were 78?86.7%?and 12?13.3%?,respectively.90 samples were mainly composed of38.9%?35/90?blood and 33.3%?30/90?urine;and mainly from hepatobiliary surgery17.8%?16/90?,followed by urinary surgery 15.6%?14/90?and comprehensive ICU14.4%?13/90?.The strains had the highest resistance rates to ampicillin,cefazolin and ceftriaxone,with the proportions of 88.9%,87.8%and 85.6%,respectively,and the lowest resistance to tigecycline,amikacin and polymyxin.Three strains of polymyxin-resistant Escherichia coli?numbered N408,N433,N980?were detected,and the MIC values were all greater than 2?g/ml.The positive rate of the mCIM test and the EDTA synergistic test were both 28.6%;mCIM and EDTA synergistic tests of N408 were positive,while N433 and N980 were opposite.PCR amplification and sequencing analysis showed that all three strains carried the polymyxin resistance gene mcr-1.In addition,N980 carried bla_TEM-1,bla_CTX-M-9,pmrA and phoP resistance genes,and N433 carried qnrB,pmrA,phoP,phoQ and mgrB resistance genes,N408carries pmrA and phoP resistance genes.No other carbapenem-resistant genes such as OXA,NDM,KPC,SHV,and IMP were detected in the three strains.Through the conjugation test,the three strains transmitted the mcr-1 gene to Escherichia coli J53competent bacteria,the zygote was resistant to polymyxin.The zygote was amplified by PCR and sequenced to carry the mcr-1 gene.MLST of 3 strains carrying mcr-1gene were successfully classified,N408 and N980 were ST453,N433 was ST10,and eBURST software analysis showed that both ST453 and ST10 belonged to CC3 clone complex.The S1-PFGE results showed that the mcr-1 gene-positive Escherichia coli carried plasmids of different sizes.Conclusion:The clinical isolates of Escherichia coli are more resistant and multi-drug resistant,and have the highest resistance rate to ampicillin,cefazolin and ceftriaxone,and the lowest resistance to tigecycline,amikacin and polymyxin.Plasmid-mediated mcr-1 gene and chromosomal gene mutation are the main mechanisms of Escherichia coli resistance to polymyxin,and the mcr-1 gene can be disseminated horizontally by plasmid.Carrying multiple drug resistance genes such as?-lactams,quinolones and mcr-1 genes is the multidrug-resistant mechanism of Escherichia coli.By genetic correlation analysis of 3 strains of Escherichia coli carrying mcr-1 gene,two of them were of the same ST type,and all three experimental strains belonged to the CC3clone complex,which may have certain clonal correlation.