Dissemination and Stability of the blaNDM-5-Carrying IncX3-Type Plasmid among Multiclonal Klebsiella pneumoniae Isolates

The emergence and spread of New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 blaNDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of blaNDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 blaNDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 blaNDM-5-carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 blaNDM-5-carrying plasmids.

fore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 bla NDM-5carrying plasmids. KEYWORDS IncX3 plasmid, K. pneumoniae, NDM-5, conjugal transfer, plasmid stability N DM (New Delhi metallo-␤-lactamase) carbapenemase is an important type of carbapenemase with the ability to hydrolyze almost all ␤-lactams, and 24 NDM variants (NDM-1 to NDM-24) have been identified to date (1). The NDM-5 carbapenemase differs from NDM-1 by only two amino acid substitutions (Val88Leu and Met154Leu) and shows increased resistance to carbapenems and expanded-spectrum cephalosporins (2). NDM-5 has been reported all over the world, but it has been identified mainly in Escherichia coli (3,4). Recently, clonal dissemination of NDM-5producing Klebsiella pneumoniae strains was reported in children (5), suggesting the rapid transmission of bla NDM-5 among Enterobacteriaceae. Here, we present the dissemination of the IncX3 bla NDM-5 -carrying plasmid among multiclonal K. pneumoniae strains in children, and the stability of IncX3 bla NDM-5 -carrying plasmid within K. pneumoniae was also investigated.
The IncX3 plasmid facilitated the dissemination of BLA NDM-5. S1-PFGE and Southern blot analysis demonstrated that all of the NDM-5-producing K. pneumoniae isolates possessed 2 to 3 plasmids and that the bla NDM-5 genes were all located on plasmids with similar sizes (ϳ46 kb) (Fig. S2). The bla NDM-5 -carrying plasmids of all 14 K. pneumoniae isolates could be transferred into recipient E. coli strain J53, at a frequency of 3.5 ϫ 10 Ϫ4 to 6.6 ϫ 10 Ϫ4 transconjugants per donor cell. The transconjugants exhibited significantly increased resistance to carbapenems compared with E. coli J53 (see Table S2 in the supplemental material). All bla NDM-5 -carrying plasmids were classified as IncX3 type through PCR-based replicon typing (9). The genetic relatedness among bla NDM-5 -carrying plasmids from different strains was determined through PCR-based sequencing for bla NDM-5 surrounding elements and the type IV secretion system (T4SS) (10). All of the bla NDM-5 -carrying plasmids shared highly similar backbones, including bla NDM-5 genetic elements and T4SS, with nucleotide sequence identity of Ͼ99%.
K. pneumoniae strain K2-7 was randomly selected, and plasmid DNA (pSCK27-NDM5) from the corresponding E. coli J53 transconjugant was extracted using a Qiagen Plasmid midi kit (Qiagen, Hilden, Germany) and was sequenced with an Illumina MiSeq system (Illumina, CA, USA). The reads were assembled de novo into contigs using SPAdes 3.9.0, and gaps were closed through PCR and Sanger sequencing. Comparative analysis of the representative fully sequenced IncX3 bla NDM-5 -carrying plasmids was performed to assess the genetic context of the bla NDM-5 gene. The functional genes were identical across IncX3 bla NDM-5 -carrying plasmids, with all plasmids carrying genes for replication (repB), partitioning (parA and parB), and conjugative transfer (virB1, virB2, virB3 /4, virB5, virB6, virB8, virB9, virB10, virB11, and virD4) (Fig. 1). Structural differences resulting from potential insertions or deletions were observed only on bla NDM-5 genetic surrounding elements, which could also be regarded as the variable region of the IncX3 plasmids. The variable region on IncX3 plasmids is highly dynamic, and it is unclear if these differences have any effect on the expression of bla NDM-5 .
Conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniae. The stability of the IncX3 bla NDM-5 -carrying plasmid within K. pneumoniae was investigated, and three NDM-5-producing strains (K2-7, K4-2, and K6-2) were randomly selected. The proportion of the bacterial population that retained the bla NDM-5carrying plasmid was determined over a period of 5 days (11). Bacteria were subcultured into antibiotic-free Luria-Bertani (LB) broth at a dilution of 1 in 1,000 daily. In order to investigate the impact of conjugal transfer on the stability of bla NDM-5 -carrying plasmid, the conjugation inhibitor linoleic acid was added to LB broth at final concentrations of 2.5 and 5 mM. The culture was diluted each day, and each dilution was plated on LB agar and incubated overnight at 37°C. A total of 100 colonies were randomly collected from all dilutions and spotted on LB plates in the presence and absence of meropenem. Plasmid retention was calculated by comparing the number of colonies on the LB agar plate containing meropenem with that on pure LB agar.
The IncX3 bla NDM-5 -carrying plasmids showed strong stability in clinical isolates, without apparent plasmid loss after serial subculture for 5 days (Fig. 2). It seems that reducing antibiotic use alone is likely insufficient for reversing resistance. However, after the conjugation inhibitor linoleic acid was added, a gradually increase in the level of bla NDM-5 -carrying plasmid loss could be observed in all three strains (Fig. 2). Linoleic acid targets type IV secretion traffic ATPase VirB11, and addition of linoleic acid can significantly decrease the conjugation efficiency of several plasmid groups (12,13). Linoleic acid significantly decreased bla NDM-5 -plasmid conjugation efficiency but did not exert any tremendous effect on bacterial growth (Fig. 2). These strains displayed 10% to 15% bla NDM-5 -carrying plasmid loss after coculture with linoleic acid for 5 days, indicating that conjugal transfer contributed significantly to the persistence of IncX3 bla NDM-5 -carrying plasmid. Plasmid conjugation is mediated by T4SS, and T4SS is conserved among all epidemic IncX3 bla NDM-5 -carrying plasmids (14). Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugationassisted persistence of IncX3 bla NDM-5 -carrying plasmid.
It is commonly believed that a plasmid-free bacterial host can compete successfully with bacterial cells harboring plasmids, due to the fitness costs of plasmid carriage (15).  (Continued on next page) However, recent studies indicated that significant changes in chromosomal and epidemic resistance plasmid gene expression may have allowed K. pneumoniae to ameliorate the associated fitness costs of plasmid carriage (11). Though the plasmid loss assay in this study lasted for 5 days, the results do not mean that coculture of IncX3 plasmid-free and plasmid-harboring K. pneumoniae for relatively long periods would definitely lead to an increased level of plasmid loss in bacterial populations. In addition, 3% to 5% plasmid loss was still observed in clinical strains after 1 day of culture with 5 mM linoleic acid, suggesting that inhibition of conjugal transfer is likely to promote IncX3 bla NDM-5 -carrying plasmid loss from K. pneumoniae.  "Conjugation efficiency" refers to the relative conjugation frequencies of the IncX3 bla NDM-5 -carrying plasmids after adding the conjugation inhibitor linoleic acid. (C) IncX3 bla NDM-5 plasmid stability in strains cultured with or without linoleic acid. Bacteria were subcultured into fresh LB broth without antibiotics at a dilution of 1 in 1,000 daily for 5 days. The experiment was repeated on three separate occasions, and error bars represent standard deviations. *, P Ͻ 0.05, one-way analysis of variance (ANOVA) with Bonferroni correction.
In summary, this study presented the dissemination of highly similar 46-kb IncX3 bla NDM-5 -carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of bla NDM-5 among K. pneumoniae via the IncX3 plasmid. Moreover, the IncX3 bla NDM-5 -carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and conjugal transfer contributed significantly to plasmid maintenance within K. pneumoniae.
All procedures in this study that involved human participants were performed in accordance with the ethical standards of the Institutional Review Board Ethics Committee of Shanghai Children's Medical Center. For this type of retrospective study, formal consent is not required.
Data availability. The complete sequence of plasmid pSCK27-NDM5 was submitted to the GenBank database under accession number MT663954.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.