Genetic and phenotypic characterizations of IncX3 plasmids harboring bla_NDM-5 and bla_NDM-16b in Japan

The spread of the gene encoding the NDM-type carbapenemase, bla (NDM), poses a serious threat to clinical practice and public health. This gene is usually carried by transferable plasmids, which facilitates its rapid spread among various species of Enterobacterales. Plasmids with the IncX3 replicon play a major role in the spread of bla (NDM) in Asian countries. In this study, we conducted genomic sequencing and a comparative analysis of the IncX3-type plasmids harboring bla (NDM-5) and bla (NDM-16b), a single-nucleotide variant of bla (NDM-5), isolated in a national antimicrobial-resistant bacterial genomic surveillance in Japan (Japan Antimicrobial Resistant Bacterial Surveillance), which was carried out during 2019-2020. We compared five plasmids harboring bla (NDM-5) and one plasmid harboring bla (NDM-16b). They were highly identical and differed in several nucleotide substitutions or variations in insertion sequences, although they were carried by different species or genotypes. Notably, bla( NDM-16b)-carrying pJBBDACG-19-0070 was identical to a plasmid carrying bla (NDM-5) except for one nucleotide substitution in the bla( NDM) gene throughout the 46 kb sequences. Transformants carrying these two plasmids showed comparative resistance to carbapenems, indicating that a substitution on the NDM enzyme (Ala233 to Val) did not affect carbapenem resistance, at least under normal experimental conditions. A database search revealed that several identical plasmids carrying bla (NDM-5) were reported from neighboring countries, mainly China, before our surveillance period. Our analysis suggested that the IncX3 plasmid harboring bla (NDM-5 )is spreading in Japan, and bla( NDM-16b) emerged from a single nucleotide substitution on the plasmid, with widespread dissemination in Asian countries.