The Molecular Characteristics,transmission Mechanism And Genetic Environment Of Intestinal NDM-producing Escherichia Coli Isolates

BackgroundEscherichia coli is a common bacterium in the gut of humans and animals.It is part of the normal flora and was first discovered in a stool sample of healthy infants in 1885.When the body's resistance is reduced,or the intestinal mucosa is damaged,E.coli can cause bloodstream infection by breaking through the intestinal mucosal barrier.Meanwhile,it also can cause urinary tract,respiratory tract and other infections.Among the pathogens that cause nosocomial infections,E.coli is the most common.Carbapenems are commonly used as the effective drugs for the treatment of infections caused by multidrug resistant strains,however,in recent years,with the widespread use of antibiotics,more and more carbapenem-resistant Enterobacteriaceae?CRE?isolates have been discovered among China,posing a serious threat to the clinic.The production of NDM-type carbapenemase is one of their main mechanisms of resistance.Studies have shown that CRE were multi-drug resistant or even pan-drug resistant.Meanwhile,blaNDM gene can be located on chromosomes and plasmids.And the horizontal transfer of the blaNDM gene among different plasmids can be mediated by IS26 element.The transfer plasmids can promote the spread of the blaNDM gene among various Enterobacteriaceae isolates,which ultimately leads to a significant increase in the rate and prevalence of CRE isolation.CRE colonization in the intestine is an important source of carbapenem resistance gene diffusion in hospital.At present,NDM-producing E.coli isolates have been detected in stool samples from different regions of China,more seriously,a pan-resistant E.coli isolate with bla_NDM-5 and mcr-1 genes was found.Since colistin is often used as the last barrier to treat infections caused by CRE isolates,the presence and spread of mcr-1 gene in NDM-producing E.coli isolates leads to a reduction in treatment efficiency,which has attracted people's attention.However,there is no large-scale screening of the intestinal NDM-producing E.coli isolates,and its molecular characteristics,transmission mechanism and genetic environment are not clear.Therefore,in order to better provide scientific basis for the clinical treatment of infections caused by NDM-producing E.coli isolates and control the spread of bla_NDMDM and mcr-1 genes,this study collected 1,115 non-repetitive stool samples from patients in the First Affiliated Hospital of Zhejiang University,and screened for the intestinal NDM-producing E.coli isolates.We investigated the homology of isolates through pulsed-field gel electrophoresis?PFGE?method and the multilocus sequence typing?MLST?results obtained by whole genome sequencing.In vitro susceptibility test was used to determine the resistance characterization.And the resistance genes were found by ResFinder.S1 nuclease-PFGE,Southern blotting,PCR-based replicon typing?PBRT?,Plasmidfinder,Blast and transconjugation experiments were performed for clarify the mechanism of dissemination.Finally,the genetic environment of drug resistance genes was generated through RAST and Easyfig.Method1.CRE isolates were initially screened from 1115 non-repetitive stool samples,which were collected from patients at the First Affiliated Hospital of Zhejiang University,through MAC selection medium containing 2?g/ml meropenem.Then MALDI-TOF MS and PCR amplification techniques were used for identification of the strains,common carbapenem resistance genes(bla_NDM,bla_KPC,bla _IMP,bla _VIM and bla_OXA-48)and colistin resistance gene?mcr-1?.Finally,the 24 NDM-producing E.coli isolates were identified through the 16s rRNA detection technology,PCR amplification and gene sequencing analysis technology.2.The PFGE was first used to analyze the homology of the strains and the duplicate strains were eliminated.Then the whole genome of remaining strains were sequenced by the second and third generation sequencing techniques,and the ST classification was detected by MLST software.Finally,the PFGE and MLST results were used to analysis the homology of these isolates.3.Agar dilution and broth microdilution methods were used to determine the resistance characterization.4.The drug resistance genes were described by ResFinder.5.S1 nuclease-PFGE,Southern blotting,PBRT,Plasmafinder,Blast and transconjugation experiments were performed for the plasmid characterization.6.The genetic environment of the drug resistance gene was presented by RAST and Easyfig software.Result1.From January 2016 to June 2018,a total of 24 E.coli isolates containing bla_NDMDM were identified in 1115 non-repetitive stool samples,with 17,4,2 and 1 carrying bla_NDM-5,bla_NDM-1,bla_NDM-13 and bla_NDM-9,respectively.9 NDM-producing E.coli isolates carried the mcr-1 gene.2.PFGE analysis of 25 NDM-producing E.coli isolates revealed 18 different clonal types.Among them,L704 and L724,L935 and L938 were separated from the same patient and had the same clonal type,therefore,in the following studies,L724 and L938were excluded,a total of 22 isolates were included in the study.MLST typing of the 23NDM-producing E.coli isolates revealed 13 different STs,and ST167 was the most common ST type.3.All of the 22 isolates were intermediate or resistant to amoxicillin/clavulanate,piperacillin/tazobactam,cefotaxime,ceftazidime,cefpirome,cefepime,meropenem,imipenem,ertapenem and levofloxacin.8 isolates showed resistance to colistin.4.All of the 22 isolates carried a variety of drug resistance genes,including carbapenems,?-lactams,colistin,macrolides,quinolones,tigecycline,fosfomycin,sulphonamides,aminoglycosides,phenicols,trimethoprims and rifampicin resistance genes,and mcr-1 gene was detected in L704,L743,L889,L906,L930,L932 and L935.5.The bla_NDM gene was located on self-transfer plasmids,with six plasmid replication types and different molecular sizes?⁴0kb to³00kb?.Among them,⁴0 kb IncX3 was the major type.There were 13 types of gene environment around the bla_NDM gene,but bla_NDM-ble_MBL-trpF-dsbC was a conserved sequence.6.The mcr-1 gene was located on the⁶0 kb IncI2 plasmid.The surrounding genetic context of mcr-1 was the same,with an order of topB-hha-pap2-mcr-1-nikB-nikA-traL.Conclusion1.The intestinal NDM-producing E.coli isolates are multi-drug resistant and even pan-drug resistant,clinical monitoring of such strains should be strengthened.2.The ST types of the isolates are highly diverse and the bla_NDMDM genes are located on plasmids,with various plasmid replication types.The ST167 and self-transferred IncX3plasmids are the key factors for promoting the spread of the bla_NDM gene.At the same time,a variety of genetic environments of bla_NDM are found and bla_NDM-ble_MBL-trpF-dsbC is the conserved structure sequence.3.Self-transferred IncI2 plasmids mediate the dissemination of the mcr-1 gene.The surrounding genetic context of mcr-1 is the same,with an order of topB-hha-pap2-mcr-1-nikB-nikA-traL.