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

Abstract

NDM-5 carbapenemase was mainly identified in Escherichia coli, while the rapid transmission of bla_NDM-5 among Enterobacteriaceae has raised serious public attention. This study identified 14 NDM-5-producing Klebsiella pneumoniae isolates from 107 carbapenem-resistant K. pneumoniae isolates, recovered from blood, urine, and normally sterile body fluids of pediatric patients from January 2016 to December 2018. All NDM-5-producing isolates were highly resistant to β-lactams, while tigecycline and polymyxin B exhibited excellent antimicrobial activity. These 14 strains belonged to 9 different sequence types (STs) and displayed various pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they were not clonally related. S1-PFGE followed by Southern blotting showed that the bla_NDM-5 gene was located on an ∼46-kb IncX3 plasmid in all strains. All bla_NDM-5-carrying plasmids were successfully transferred into recipient E. coli J53. PCR-based sequencing demonstrated that all of the bla_NDM-5-carrying plasmids shared highly similar backbones, with nucleotide sequence identity of >99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 bla_NDM-5-carrying plasmids, with dynamic changes observed only in bla_NDM-5-surrounding elements. Interestingly, the IncX3 bla_NDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% bla_NDM-5-carrying plasmid loss after coculture with linoleic acid for 5 days. These results showed that the IncX3 plasmid facilitated the dissemination of bla_NDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniaeIMPORTANCE 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 bla_NDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of bla_NDM-5 among K. pneumoniae strains 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 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 bla_NDM-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 bla_NDM-5-carrying plasmids.

Keywords: IncX3 plasmid; K. pneumoniae; NDM-5; conjugal transfer; plasmid stability.

FIG 1

Comparative analysis of pSCK27-NDM5 with other reference IncX3 bla_NDM-5-carrying plasmids. The type IV secretion system (T4SS) and bla_NDM-5 genetic elements are indicated. The circular map was created by the use of the BLAST Ring Image Generator (BRIG). Concentric rings represent the similarity between pSCK27-NDM5 in the inner ring and other reference sequences in the outer rings. The nine reference IncX3 plasmid sequences were obtained from GenBank and were listed with plasmid name (GenBank accession number, bacterial host, country of detection): pP768-NDM5 (MF547510, E. coli, China), pEC463-NDM5 (MG545911, E. coli, China), pK725-NDM5 (MK450348, K. pneumoniae, China), pMGF008 (NEWC01000014, K. quasipneumoniae, Malaysia), pMTC948 (MH349095, E. coli, China), pNDM5_025943 (CP027204, E. coli, China), pTBCZ-NDM5 (MH107030, K. pneumoniae, China), pZHDC40 (KY041843, E. coli, China) and pNJ-NDM5 (KX447767, E. coli, United States).

FIG 2

IncX3 bla_NDM-5 plasmid loss assay in strains K2-7, K4-2, and K6-2. (A) Bacterial growth curve of strains in LB broth with or without linoleic acid. OD 600nm, optical density at 600 nm. (B) Bacterial conjugation was monitored through liquid-mating conjugation assay in the presence of 2.5 mM or 5 mM linoleic acid. “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 < 0.05, one-way analysis of variance (ANOVA) with Bonferroni correction.