Dissemination of the <i>bla</i> _NDM-5 Gene via IncX3-Type Plasmid among <i>Enterobacteriaceae</i> in Children

Abstract

The continuous emergence of novel New Delhi metallo-β-lactamase-5 (NDM-5)-producing <i>Enterobacteriaceae</i> isolates is receiving more and more public attention. Twenty-two NDM-5-producing strains were identified from 146 carbapenemase-producing <i>Enterobacteriaceae</i> (CRE) strains isolated from pediatric patients between January and March 2017, indicating that the <i>bla</i>_NDM-5 gene has spread to children. All 22 isolates, including 16 <i>Klebsiella pneumoniae</i> strains, four <i>Klebsiella aerogenes</i> strains, and two <i>Escherichia coli</i> strains, showed significantly high resistance to β-lactam antibiotics (except aztreonam) but remained susceptible to tigecycline and colistin. <i>K. pneumoniae</i> and <i>K. aerogenes</i> strains were respectively defined as homologous clonal isolates by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) results confirmed the genetic relatedness with all <i>K. pneumoniae</i> strains belonging to sequence type (ST) 48. Two <i>E. coli</i> isolates (ST617 and ST1236) were considered genetically unrelated. Twenty-two <i>bla</i>_NDM-5 plasmids were positive for the IncX3 amplicon and showed almost identical profiles after digestion with HindIII and EcoRI. Four representative strains (<i>K. pneumoniae</i> K725, <i>K. aerogenes</i> CR33, <i>E. coli</i> Z214, and <i>E. coli</i> Z244) were selected for further study. Plasmids harboring <i>bla</i>_NDM-5 showed strong stability in both clinical isolates and transconjugants, without apparent plasmid loss after 100 serial generations. S1-PFGE followed by Southern blot analysis demonstrated that the <i>bla</i>_NDM-5 gene was located on an ∼46-kb plasmid. Plasmid sequences of pNDM-K725, pNDM-CR33, and pNDM-Z214 were almost identical but were slightly different from that of pNDM-Z244. Compared with pNDM-Z244, ΔIS<i>Aba125</i> and partial copies of IS<i>3000</i> were missing. The genetic backgrounds of the <i>bla</i>_NDM-5 gene in four strains were slightly different from that of the typical pNDM_MGR194. This study comprehensively characterized the horizontal gene transfer of the <i>bla</i>_NDM-5 gene among different <i>Enterobacteriaceae</i> isolates in pediatric patients, and the IncX3-type plasmid was responsible for the spread.<b>IMPORTANCE</b> The emergence of CRE strains resistant to multiple antibiotics is considered a substantial threat to human health. Therefore, all the efforts to provide a detailed molecular transmission mechanism of specific drug resistance can contribute positively to prevent the further spread of multidrug-resistant bacteria. Although the new superbug harboring <i>bla</i>_NDM-5 has been reported in many countries, it was mostly identified among <i>E. coli</i> strains, and the gene transfer mechanism has not been fully recognized and studied. In this work, we identified 22 <i>bla</i>_NDM-5-positive strains in different species of <i>Enterobacteriaceae</i>, including 16 <i>Klebsiella pneumoniae</i> strains, four <i>Klebsiella aerogenes</i> strains, and two <i>Escherichia coli</i> strains, which indicated the horizontal gene transfer of <i>bla</i>_NDM-5 among <i>Enterobacteriaceae</i> strains in pediatric patients. Moreover, <i>bla</i>_NDM-5 was located on a 46-kb IncX3 plasmid, which is possibly responsible for this widespread horizontal gene transfer. The different genetic contexts of the <i>bla</i>_NDM-5 gene indicated some minor evolutions of the plasmid, based on the complete sequences of the <i>bla</i>_NDM-5 plasmids. These findings are of great significance to understand the transmission mechanism of drug resistance genes, develop anti-infection treatment, and take effective infection control measures.