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

Abstract

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. IMPORTANCE IncX3 plasmids harboring bla NDM-5 play a major role in the spread of carbapenem resistance in Asia, particularly in China, in clinical and environmental settings. In this study, we present that Enterobacterales isolates carrying IncX3 plasmids harboring bla NDM-5 have been disseminated in Japan, where their identification was previously rare. In addition, bla NDM-16b , a single-nucleotide variant of bla NDM-5 , was found to be carried by an identical IncX3 plasmid. A comparative sequence analysis revealed that the bla NDM-16b gene emerged from a single nucleotide substitution on an IncX3 plasmid harboring bla NDM-5 . The bla NDM-16b gene did not confer elevated carbapenem resistance compared to bla NDM-5 in our assay using transformants carrying the plasmid harboring either of these genes, although the A233V substitution was reported to confer stability to the enzyme in ion-depleted conditions. Nevertheless, vigilance regarding the emergence of novel variants is required.