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.

often coexists with many other resistance determinants (1).bla NDM genes are rapidly spreading, particularly in Asian countries, and have been detected in various sources outside clinical settings, such as sewage, livestock, and even food samples from South, East, and Southeast Asian countries (2)(3)(4)(5), indicating their presence in the community.One of the major vehicles for the gene is the IncX3-type plasmid, which is highly conjugative and broadly disseminated, particularly in China.In Japan, identifica tion of the bla NDM gene is still rare, and bla NDM-1 and bla NDM-5 have been sporadically reported in clinical isolates since their first isolation (6,7).In this study, we report the isolation and comparative analysis of IncX3-type plasmids harboring bla NDM-5 and bla NDM-16b , a single nucleotide variant of bla NDM-5 , in a Japan Antimicrobial Resistant Bacterial Surveillance (JARBS) conducted during 2019-2020 (8).
Comparative sequence analysis of these IncX3 plasmids, six from our surveillance and pTMTA8571-1, showed that they were highly identical and differed by minimum single nucleotide substitutions, even though they originated from different hospitals and were identified in various STs or species.Differences among them were classified into four categories: substitutions at either of the two sites on the IS26 transposase coding region, the presence or absence of ISAba125, and a substitution on the bla NDM gene.The two bla NDM-5 -carrying plasmids, pJBBDAGF-19-0019 and pJBBEABG-19-0024, were identical, and the pJBBEABG-19-0025 plasmid differed from these two plasmids by a single nucleotide on the IS26 coding sequence.pJBBEABG-19-0025 differed from another bla NDM-5 -carrying plasmid from Klebsiella aerogenes, pJARBSGNR_440044-19-0003, by another nucleotide in the IS26 sequence.pJARBS-GNR_23029-19-0094 lacked ISAba125 compared to pJARBSGNR_440044-19-0003, as previously reported (9).Notably, bla NDM -16b -carrying plasmids were identical to one of those carrying bla NDM-5 except for the bla NDM gene.pJBBDACG-19-0070 and pTMTA8571-1 were completely identi cal to plasmids pJBBDAGF-19-0019 and pJBBEABG-19-0025, respectively, except for a substitution on bla NDM that yielded an NDM A233V substitution.Identical plasmids were also identified in the public databases.We searched for plasmid sequences similar to our plasmids using NCBI BLAST and included 40 sequences in our analysis.We found 17 and 12 sequences completely identical to pJBBDAGF-19-0019 and pJBBEABG-19-0025, respectively, which were isolated from China, South Korea, and the United Arab Emirates before our surveillance period (Fig. 1; Table S1).Several plasmid sequences with single nucleotide substitutions compared to these plasmids were also deposited, mainly from China, indicating that these identical plasmids were disseminated in these Asian countries.
As shown above, the bla NDM-16b -carrying plasmid pJBBDACG-19-0070 and the bla NDM-5 -carrying plasmid pJBBDAGF-19-0019 were identical except for a single nucleotide substitution on bla NDM , which allowed us to conduct a comparative analysis between bla NDM-5 and bla NDM-16b on the same IncX3 plasmid background.Plasmid DNA was extracted from isolates carrying these two plasmids and introduced into the E. coli strain HST08 using electroporation.The size and location of the bla NDM gene in the transformants were confirmed by S1-nuclease pulsedfield gel electrophoresis, followed by Southern hybridization with a digoxigenin-labeled (Roche, Burgess Hill, United Kingdom) bla NDM specific probe (11).The minimum inhibitory concentrations (MICs) of the transformants and their parental strains were determined using broth microdilution for meropenem and imipenem and by MicroScan WalkAway for the other β-lactams.The results were comparable between the transformants, and both the IncX3-bla NDM-5 and IncX3-bla NDM-16b plasmids conferred the same level of resistance to the β-lactams tested on the susceptible parental strain.In this study, we showed that highly similar IncX3 plasmids harboring bla NDM-5 were disseminated in Japan and its neighboring countries.Amino acid substitutions at 110th and 235th on IS26 transposase were the major difference among these plasmids.It is unclear whether these substitutions have occurred once each or several times independently during the spread of these plasmids.The 110th amino acid is located in the catalytic domain of the transposase, and leucine is well conserved at this position in IS6/IS26 family transposases (12).The effect and significance of substitution at this residue by isoleucine are unclear, although the substitution apparently does not affect the hydrophobic characteristics at this position.Reports of IncX3 plasmids with bla NDM-16b are still rare, even though their sequence is identical to those carrying bla NDM-5 , suggesting that bla NDM-16b recently emerged from bla NDM-5 on the IncX3 plasmid.Our MIC assay showed that NDM-16b and NDM-5 confer comparable resistance against meropenem on the host bacterium.The result is consistent with a previous report in which the assay was performed using bacteria harboring the bla NDM genes cloned into a commercially available vector (13).Meanwhile, an alanine-233 to valine substitution on NDM-5 is known to confer stability to the enzyme under zinc-limited conditions (13,14).Therefore, it could be beneficial for bacteria to produce NDM-16b rather than NDM-5 to survive iron-depleted conditions, such as at infection sites.Given that this is the case, an organism probably obtained a single nucleotide substitution causing the alanine-233 to valine mutation, that is, bla NDM-5 to bla NDM-16b evolution, but not vice versa, during its spread in clinical settings or under antimicrobial exposure.Notably, bla NDM-16b was found in a relatively highly multidrug-resistant strain, E. coli ST746.Collectively, our analysis supported the notion that bla NDM-16b emerged from IncX3 plasmids harboring bla NDM-5 , which was recently suggested by another research group (10).In summary, we showed that highly identical IncX3 plasmids harboring bla NDM-5 are spreading throughout Japan.These plasmids were disseminated in neighboring countries, mainly China, suggesting that Japanese IncX3 plasmids originated in these countries.Although sequence variation among these plasmids was highly limited, one of them occurred in the bla NDM-5 gene, yielding a new variant, bla NDM-16b .This substitution did not confer elevated resistance to the host bacterium, as we showed in this study; however, such a substitution might occur at other nucleic acid positions and increase the enzymatic activity of the encoded NDM.Thus, the emergence of novel variants is conceivable, and continued vigilance should be exercised to prevent their further spread.

FIG 1
FIG 1 Relationship between the IncX3 plasmids carrying bla NDM-5 or bla NDM-16b .Plasmids obtained during our surveillance are underlined, and those marked with an asterisk were obtained in this study.SNS denotes single nucleotide substitutions on IS26, bla NDM , or at any position of the plasmid if there are no specifications.The 110th and 235th amino acid residues of IS26 transposase (trp), bla NDM genes, and the presence of ISAba125 insertions are shown in the figure.Graphs show the number and year of isolation of identical plasmids deposited in GenBank from other countries.Red, China; blue, South Korea; green, the United Arab Emirates (UAE).

TABLE 1
Antimicrobial susceptibility patterns of NDM-producing isolates and transformants a The minimum inhibitory concentration (MIC) was determined using the broth microdilution method.ObservationMicrobiology Spectrum November/December 2023 Volume 11 Issue 6 10.1128/spectrum.02167-234