The Transmission Mechanism Of Bla_NDM-1 In Carbapenem-resistant Enterobacter Cloacae Isolates

Objectives: 10 NDM-1-producing Enterobacter cloacae isolates which were screened in the early stage of the study were detected the presence of integron and insert sequence common regions 1 (ISCR1) to understand the distribution of them in carbpenem-resistance Enterobacter cloacae. Multilocus-sequence typing (MLST),pulsed field gel electrophoresis (PFGE), conjugation experiment and southern blot were performed to analyze the molecular epidemiology of NDM-1-producing E.cloacae and determine whether blaNDM-1 was transferable by plasmids, then for providing a theoretical basis for formulating preventive and control measures.Methods: ? A total of 10 NDM-1-producing E.cloacae isolates were collected from the First Affiliated Hospital of Kunming Medical University during June 2012 and January 2016. Identification and antibiotic susceptibility testing were perfomed by VITEK 2 system.? Integrons and ISCR1 mobile elements were determined by PCR and sequencing, then analyzing the relationship between blaNDM-1 gene and the two mobile elements.?Clone relationships were analyzed by using pulsed field gel electrophoresis (PFGE) and multilocus-sequence typing (MLST). ? Conjugation experiment and southern blot hybridization were performed to analyze the plasmid which mediated the spread of blaNDM-1 gene.Results: ? 10 E.cloacae isolates and transconjugants were exhibited similar resistant patterns to carbapenems,cephalosporins and penicillins, but reverse patterns to monobactams,tetracyclines,fluoroquinolones,aminoglycosides, donors were resistant to these antibiotics, while conjugants were sensitive. ? 80%(8/10) of the NDM-1-producing isolates harbored class 1 integrons,10%(1/10) strains carried class 2 integrons and 50%(5/10) isolates carried ISCR1.The prevalence of class 1,2 integron variable regions was 70% and 10%, respectively. None of the isolates harbored class 3 integrons and ISCR variable regions. The integron variable regions harbored the genes which encoded resistance to aminolycosides(aadA2,aadA5,aadB,aac(6')-Ib-cr) and sulfamethoxazole/trimethoprim(dfrA17,dfrA12,dfrA15).? 6 E.cloacae isolates exhibited highly similar PFGE patterns,belong to the same clone and MLST assigned them to the sequence type 74,the remaining four strains belonged to ST754,ST175 and ST 182,respectively.There were two strains shared the same sequence type (ST182). ?All of the blaNDM-1 plasmids were successfully transferred into E.coli 600 by conjugation. A blaNDM-1 plasmid of ?33.3kb was identified by S1-PFGE/Southem blot in nine E.cloacae isolates and another blaNDM-1 plasmid of ?50kb was identified in one E. cloacae isolate(T3).Conclusions :? In terms of susceptibility phenotype, NDM-1-producing E.cloacae isolates often exhibited multiple drug resistance (MDR).? Although integrons and ISCR1 didn't carry the blaNDM-1 gene , the integrons harbored the gene cassettes which encoded resistance to aminolycosides and sulfonamides, thus integrons were the basis for the formation of multiple drug-resistant bacteria in this region.? Homology analysis showed that NDM-1-producing Enterobacter cloacae had a small clone spread in our hospital.? Plasmid was the most important mobile element that mediated the horizontal transfer of blaNDM-1 gene.