Whole-Genomic Analysis of NDM-5-Producing Enterobacteriaceae Recovered from an Urban River in China

Purpose: Three NDM-5-producing Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Citrobacter braakii, one each) were isolated during a screening study for the presence of carbapenemase-producing Enterobacteriaceae (CPE) strains in urban rivers in China. The aim of the present study was to characterize these NDM-5-producing isolates by using whole-genome analysis. Methods: In vitro susceptibility testing was performed using the broth microdilution method. Conjugation assay was carried out to investigate the transferability of bla(NDM-5)-harboring plasmids. Whole-genome sequencing was performed using an Illumina HiSeq combined with the PacBio RSII system. The genetic characteristics of the bla(NDM-5)- harboring plasmids were analyzed. Antimicrobial resistance genes and virulence genes were identified from the genome sequences. Phylogenetic analysis was performed based on core genome. Results: Antimicrobial susceptibility testing showed that all three isolates were resistant to carbapenems, cephalosporins, quinolones, and aminoglycosides, and susceptible to colistin. Whole-genome sequencing showed that each isolate carried multiple antibiotic resistance genes mediating multidrug resistance, and harbored numerous virulence genes, some of which were located on plasmids. In these isolates, bla(NDM-5) was carried by an IncX3 plasmid in K. pneumoniae and C. braakii, and on an IncR/IncX1 plasmid in E. coli. Conjugation experiments showed that these bla(NDM-5)-haboring plasmids were successfully transferred to E. coli J53. Phylogenetic analysis revealed that E. coli SCLZR49 was present in a cluster with isolates of different origin, K. pneumoniae SCLZR50 was mainly clustered with clinical isolates, and C. braakii SCLZR53 had closely genetic relationship with environmental isolates. Conclusion: This study revealed contamination of the urban river ecosystems by clinically significant carbapenemase gene bla(NDM-5), raising the possibility of plasmid transmission into the environmental from humans and highlighting the need for a constant surveillance of CPE in the environment under the "One-Health" perspective.