Molecular Characterization of bla NDM -Carrying IncX3 Plasmids: bla NDM-16b Likely Emerged from a Mutation of bla NDM-5 on IncX3 Plasmid.

Dissemination of bla NDM , which is carried on the IncX3 plasmid, among Enterobacterales has been reported worldwide. In particular, bla NDM-5 -carrying IncX3 plasmids can spread among several hosts, facilitating their dissemination. Other variants, such as bla NDM-17 -, bla NDM-19 -, bla NDM-20 -, bla NDM-21 -, and bla NDM-33 -carrying IncX3 plasmids, have also been reported. Here, we characterized, using whole-genome sequencing (WGS), a bla NDM-16b -carrying IncX3 plasmid harbored by Escherichia coli strain TA8571, which was isolated from a urine specimen of a hospital inpatient in Tokyo, Japan. The bla NDM-16b differed in sequence from bla NDM-5 (C > T at site 698, resulting in an Ala233Val substitution). This bla NDM-16b -carrying IncX3 plasmid (pTMTA8571-1) is 46,161 bp in length and transferred via conjugation. Transconjugants showed high resistance to β-lactam antimicrobials (except for aztreonam). Because pTMTA8571-1, which carries the Tn 125- related region containing bla NDM and conjugative transfer genes, was similar to the previously reported IncX3 plasmids, we performed phylogenetic analysis based on the sequence of 34 shared genes in 142 bla NDM -carrying IncX3 plasmids (22,846/46,923 bp). Comparative analysis of the shared genes revealed short branches on the phylogenetic tree (average of 1.08 nucleotide substitutions per shared genes), but each bla NDM variant was divided into separate groups, and the structure of the tree correlated with the flowchart of bla NDM nucleotide substitutions. The bla NDM -carrying IncX3 plasmids may thereby have evolved from the same ancestral plasmid with subsequent mutation of the bla NDM . Therefore, pTMTA8571-1 likely emerged from a bla NDM-5 -carrying IncX3 plasmid. This study suggested that the spread of bla NDM -carrying IncX3 plasmids may be a hotbed for the emergence of novel variants of bla NDM . IMPORTANCE bla NDM -carrying IncX3 plasmids have been reported worldwide. Harbored bla NDM variants were mainly bla NDM-5 , but there were also rare variants like bla NDM-17 , bla NDM-19 , bla NDM-20 , bla NDM-21 , and bla NDM-33 , including bla NDM-16b detected in this study. For these plasmids, previous reports analyzed whole genomes or parts of sequences among a small number of samples, whereas, in this study, we performed an analysis of 142 bla NDM -carrying IncX3 plasmids detected around the world. The results showed that regardless of the bla NDM variants, bla NDM -carrying IncX3 plasmids harbored highly similar shared genes. Because these plasmids already spread worldwide may be a hotbed for the emergence of rare or novel variants of bla NDM , increased attention should be paid to bla NDM -carrying IncX3 plasmids in the future.