Resistance Transmission Mechanism And Epidemiological Analysis Of Enterobacter Cloacae Carrying NDM-1 Gene

Background:With the widespread use of carbapenem in recent years,the prevalence of carbapenem-resistant Enterobacteriaceae（CRE）has shown a global trend.Unlike the species distribution of CRE in the United States,Europe and South Asia（mainly caused by E.coli and K.pneumoniae）,Enterobacter cloacae was predominant among CRE isolates in our region from 2011 to 2015.Moreover,E.cloacae has strong and complex genetic components,which can be colonized and survived in different metabolic environments,thus making the treatment and prevention for this speices are more difficult than other nosocomial pathogens.Recent reports have confirmed that carbapenemases are considered as one of the important mechanisms of carbapenem resistance in E.cloacae,among which NDM-1（New Delhi Metallo-beta-Lactamase-1）is the most concerned.Notably,isolates carrying NDM gene can exhibit superior resistance and hydrolyze most all the beta-lactamase antibiotics.Moreover,most of these strains are located in the plasmids or genetic elements that play an important role in the rapid transmission in different species.Presently,previous researches on NDM-1 was mostly focused on E.coli（CREO）and K.pneumoniae（CRKP）,but relatively uncommon in E.cloacae.Moreover,the prevalence,transmission mechanism and risk factors for NDM-1 positive E.cloacae infections are still unclear.Therefore,our study was initiated to explore into the following aspects.1.To describe the prevalence and resistance mechanism of clinical CR-ECL isolates collected between 2012 and 2016;2.To analyze the transmission mechanism of NDM-1 in clinical CR-ECL isolates;3.To study the genetic context of NDM-1 and IMP-8 in E.cloacae;4.To examine the clonal relatedness and report the first outbreak of NDM-1 producing E.cloacae in China;5.To evaluate the risk factors and clinical outcomes of NDM-1 positive E.cloacae infections in hospitalized patients.Methods:1.E.cloacae isolates were collected successively for approximately 4 years and identified using the VITEK2 compact.Additionally,microbroth dilution method was used to determine the MICs of CR-ECL isolates.2.Resistance mechanisms of carbapenem resistant E.cloacae were further analyzed by PCR amplification of resistance determinants and porin genes as well as efflux pump inhibition test.3.The metallase phenotype was verified by E-test slip in all NDM-1 positive strains.4.Position of NDM-1 gene was preliminarily determined by plasmid conjugation and transformation assays,and transferability of NDM-1 gene was also evaluated in our study.5.Susceptibility of transconjugants and transformants with NDM-1 was evaluated by microbroth dilution method,and PCR was further performed to amplify their resistance determinants.6.All NDM-1-encoding plasmids were characterized for the incompatibility groups by using the replicon typing method.7.PCR mapping was conducted to analyze the surrounding environment of NDM-1 and IMP-8 genes,and further to make the comparison annotation.8.DiversiLab was used to analyze the clonal relatedness of NDM-1 isolates,and multi-sequence locus typing（MLST）was used to analyze the sequence types（STs）of these isolates.9.We conducted a retrospective case–control study to evaluate the risk factors and clinical outcomes for the isolation of NDM-1-producing E.cloacae.Results:1.138 CR-ECL strains were isolated in our region from 2012 to 2016.All these strains were resistant to ertapenem（ETP）,and highly sensitive to imipenem（IMP）and meropenem（MEM）.Among which,20 strains carried NDM-1.2.Metal enzyme phenotypes of 20 strains carrying NDM-1 were positive,and NDM-1 was confirmed to be located on the plasmid.3.All isolates carrying NDM-1 plasmid were successfully introduced into transconjugants and transformants.However,we found that conjugation and transformation obtained NDM did not show the corresponding resistant phenotypes compared with the donor isolates.4.PCR-based replicon typing revealed that most of NDM-1 plasmids belonged to the incompatibility group of IncFIIA type.5.One strain co-expression of IMP-8 and NDM-1 was found,which showed the superior multidrug resistance,and only displayed sensitive to colistin and tigecycline.PCR assays confirmed that bla_NDM-1 along with bla_CTX-M-9,aac（6’）-Ib-cr and aac（6’）-Ib were successfully transferred by both conjugation and transformation,however,bla_IMP-8,bla_TEM,and qnrS were only obtained via transformation assay.6.Genetic environment analysis revealed that all NDM-carrying plasmids had a highly conserved sequence,bla_NDM-1-ble_MBL-trpF-tat,which was similar not only to the Acinetobacter isolated in China,but also to the E.cloacae and K.pneumoniae from Australia.We speculate that this sequence may be one of the main genetic contexts for the rapid spread of NDM gene among different bacterial species and geographic regions.In addition,transposon elements IS26,intI1 and tniC can promote the prevelance and transmission of IMP-8.7.MLST showed that ST88 was the most common clone type of NDM-1 isolates（6/20,30.0%）,followed by ST93（5/20,25.0%）and ST78（2/20,10.0%）.8.DiversiLab analysis showed that 20 NDM-1-producing E.cloacae isolates could be divided into 15 different groups.Notably,four of them were isolated from respiratory department and belonged to the same clone type ST88,showing a nosocomial outbreak and causing two deaths of them.9.Risk factor analysis revealed that transferring from another hospital and prior antibiotic use of carbapenems were independent risk factors for infection of NDM-1-positive E.cloacae,and most of these patients had poor clinical outcomes.Conclusion:1.NDM-1-positive E.cloacae showed strong resistance to multiple antibiotics,and had the complex genetic backgrounds,as well as often accompanied by some other resistance genes,thus leading to the stronger bacterial resistance.2.CR-ECL isolates with NDM-1 were mostly located on the plasmids,and IncFIIA was the most common plasmid type of our region.3.The highly conserved regions(bla _NDM-1-ble_MBL-trpF-tat)surrounding bla_NDM-1 gene could promote the widespread of NDM-1 among different bacterial species and geographic regions,while the IS26,intI1,and tniC could be involved in the spread of IMP-8.4.Transferring from another hospital and prior antibiotic use of carbapenems were independent risk factors for infection of NDM-1-positive E.cloacae.