Role of Chromosome- and/or Plasmid-Located bla(NDM) on the Carbapenem Resistance and the Gene Stability in Escherichia coli

The spread of New Delhi metallo-β-lactamase (NDM)-producing Enterobacterales represents a public health risk. The horizontal transfer of plasmids encoding the NDM gene, bla(NDM), usually mediates its spread to other bacteria within the family. In contrast, Enterobacterales with a chromosome-located bla(NDM) is rarely reported. The phenotypic differences between chromosome- and plasmid-located carbapenemase genes are poorly understood. To determine the significance in terms of the location of drug resistance genes, we examined carbapenemase activity and stability of chromosome- and plasmid-located bla(NDM). Escherichia coli M719 possessing both chromosomes- and plasmid-located bla(NDM) genes was used as a wild-type strain (WT) for the construction of mutants, Δpbla(NDM) and Δcbla(NDM), wherein chromosome- or plasmid-located bla(NDM,) was knocked out, respectively. The mutant Δpbla(NDM) showed lower hydrolyzing activity against imipenem and gene expression than the WT or Δcbla(NDM) mutant. The MICs of both mutant strains were still above the breakpoint of imipenem and meropenem. Moreover, the chromosome-located bla(NDM) gene was stable for at least 30 days in the absence of antimicrobial pressure, whereas the Δcbla(NDM) mutant lost bla(NDM) to 87% at 30 days compared to that of the initial inoculum. Organisms harboring the plasmid-located carbapenemase genes were found to provide a higher level of carbapenem resistance than those with chromosome-located genes. However, the latter organisms with chromosomal carbapenemase genes exhibited more stable carbapenem resistance than did the former ones. In summary, chromosomally located carbapenemase genes require further monitoring and more attention should be paid to them. IMPORTANCE Carbapenem-resistant Enterobacterales (CRE) carrying bla(NDM) have spread worldwide since they were first reported in 2009. Many studies using whole-genome sequencing have identified the genetic structures, plasmid scaffolds of bla(NDM), and mechanisms of spread via horizontal transfer. Chromosome-located bla(NDM) and integration mechanisms from plasmids have rarely been reported, and their significance is not fully understood. Here, we showed that the chromosome-located bla(NDM) was associated with lower levels of carbapenem resistance and carbapenemase activity than the plasmid-located bla(NDM). However, it conferred carbapenem resistance above the breakpoints and the loss of chromosome-located bla(NDM) was not observed in the absence of antibiotic pressure. This study suggests that CRE strains carrying chromosome-located bla(NDM) may persist in clinical and environmental settings for a long period even without antibiotic pressure and need to be monitored along with plasmid-located bla(NDM).