BackgroundThe aim of this study was to elucidate the molecular epidemiology of carbapenem non-susceptible Enterobacteriaceae(CNSE) isolated in the Eastern region of Heilongjiang Province, China, and the mechanism of carbapenem resistance.MethodsA total of 53 CNSE isolates were collected in a grade-3 hospital in Heilongjiang province. Sensitivity to antibiotics was determined using the VITEK-2 Compact automatic system. The modified Hodge test (MHT) and modified carbapenem inactivation test (mCIM) were performed for phenotypic identification. Beta-lactamases gene were detected by Polymerase chain reaction(PCR) and DNA sequencing. The transfer of blaNDM and blaKPC was investigated through conjugation experiment. The clinical data of patients were retrospectively reviewed. Homology of Carbapenem-resistant Klebsiella pneumoniae(CRKP) was conducted by multilocus sequence typing (MLST).ResultsCNSE were highly resistant to the majority of antimicrobial agents. The resistance rate was 100% for first, third, fourth generation cephalosporins and enzyme inhibitor compounds. Gentamicin and tobramycin recorded a resistance rate higher than 80%. Less than 30% resistance was detected for amikacin and levofloxacin. Among CNSE 52(98.1%) and 48(90.6%) of CNSE were positive for mCIM and MHT respectively. There were 42 positive blaKPC genes, three blaNDM-1 genes, three blaNDM-5 genes, one blaNDM-7 gene, and six blaIMP-4 genes. Most isolates harbored multiple drug resistance gene, especially as related to extended-spectrum-β-lactamases, blaSHV, blaTEM and blaCTX-M-15 genes.The resistant gene was transferred into recipient Escherichia coli J53 through conjugation in 21.3% (10/47) of the strains. MLST revealed that ST76 (n = 36) was the most predominant clone, followed by ST896, ST323 and ST11. A new one ST 2946 was identity by this study.ConclusionThe carbapenem resistance phenomenon is alarming and blaKPC-2 is the main resistant gene of CNSE in our hospital. This is the first report of an outbreak caused by blaKPC-2 positive K. pneumoniae ST76 in the Eastern region of Heilongjiang Province, China. Relevant departments should implement infection control and prevention measures to avoid further dissemination of the multi drug-resistant bacteria (MDR).

Carbapenem-resistant Enterobacter cloacae complex (CRECC) has increasingly emerged as a major cause of healthcare-associated infections, with colistin being one of the last-resort antibiotics of treatment. Mobile colistin resistance (mcr)-9 is a member of a growing family of mcr genes and has been reported to be an inducible gene encoding an acquired phosphoethanolamine transferase. Here, we collected 24 ECC strains from Chongqing, China from 2018 to 2021. Subsequently, antibiotic resistance genes and the transmission dynamics of the strains were determined by PCR, whole-genome sequencing, and bioinformatic analysis. The mcr-9 was identified in IncHI2/2A or IncHI2/2A + IncN plasmids from six CRECC strains and was co-located with blaNDM-1 or blaIMP-4 in 2/6 plasmids. The genetic environment of mcr-9.1 was composed of IS903B-mcr-9.1-wbuC-IS26 in the five mcr-9.1-harboring-plasmid, but IS1B was located downstream of mcr-9.2 in the pECL414-1 sequence. We also found that the pNDM-068001 plasmid carrying mcr-9.1 could be a hybrid plasmid, formed by a Tn6360-like blaNDM-1 region inserted into an mcr-9.1-positive IncHI2/2A plasmid. A conjugation assay showed that plasmids mediated the co-dissemination of mcr-9 and metallo-β-lactamase (MBL) genes. In addition, we performed induction assays with sub-inhibitory concentrations of colistin and found an increase in the relative expression levels of the mcr-9.2, qseC, and qseB genes, as well as an increase in the minimum inhibitory concentration values of colistin in the CRECC414 strain. These findings provide a basis for studying the regulatory mechanisms of mcr-9 expression and highlight the importance of effective monitoring to assess the prevalence of MBL and mcr-9 co-existing plasmids.