Occurrence of Highly Conjugative IncX3 Epidemic Plasmid Carrying bla NDM in Enterobacteriaceae Isolates in Geographically Widespread Areas

Abstract

The emergence of New Delhi metallo-β-lactamase (NDM) in common enterobacterial species is a major concern for healthcare. Early reports have revealed that the spread of NDM involved diverse and heterogeneous plasmids. Recently, the involvement of a rare, IncX3 subtype plasmid has been increasingly recognized. Here, we studied the prevalence of IncX plasmid subtypes in 198 carbapenem-resistant Enterobacteriaceae, originating from a territory-wide active surveillance in Hong Kong in 2016. The complete sequences and biological features of the bla _NDM-carrying plasmids were investigated. A total of 62 NDM-type, 21 OXA-48 type, 14 IMP-type, 8 KPC-type, 4 IMI-type producers, and 89 non-carbapenemase-producers were tested for presence of IncX subtypes. IncX3 (n = 60) was the most common subtype, followed by IncX4 (n = 6) and IncX1 (n = 2). The prevalence of IncX3 subtype in isolates producing NDM, other carbapenemase types and non-carbapenemase producers were 75.8, 21.3, and 3.4%, respectively (P < 0.001). An IncX3 plasmid (size ∼50 kb) was confirmed to carry bla _NDM in 47 isolates of different enterobacterial species. Thirteen IncX3 plasmids originating from six healthcare regions in Hong Kong were completely sequenced. The results showed that the IncX3 plasmids carrying bla _NDM share a high degree of sequence identity with a previously reported plasmid, pNDM-HN380 (GenBank accession JX104760), over the backbone and genetic load regions. A blast search further revealed the occurrence of identical or nearly identical IncX3 plasmids carrying bla _NDM in other part of China, Korea, Myanmar, India, Oman, Kuwait, Italy, and Canada. Two IncX3 carrying bla _NDM were investigated further. Conjugation experiments demonstrated that the IncX3 plasmids could be efficiently transferred to multiple enterobacterial species at frequencies that are comparable or higher than the epidemic IncFII plasmid carrying bla _CTX-M (pHK01). In addition, efficient transfer of the NDM plasmids occurred over a range of temperatures. In conclusion, this study demonstrated the important role played by IncX3 in the dissemination of NDM and the occurrence of pNDM-HN380-like plasmids in geographically widespread areas. The high mobility of IncX3 plasmid across different enterobacterial species highlights the ability of this plasmid replicon to be an important vehicle in worldwide dissemination of NDM.

Keywords: Enterobacteriaceae; antimicrobial resistance epidemiology; carbapenems; molecular epidemiology; resistance plasmid.

FIGURE 1

Analysis of IncX3 plasmid sequences in this study. (A) Comparison of genetic load regions in 13 plasmids harboring bla_NDM with two reference plasmids (pIncX-SHV and pNDM-HN380). (B) Phylogenetic analysis of 49 IncX3 plasmids using the Maximum Likelihood method based on the Tamura-Nei model. These included two reference plasmids, 13 plasmids in this study and 34 plasmids identified in GenBank (last accessed 16 May, 2018). Branches were drawn to scale, with lengths measured in the number of substitutions per site. Plasmids sequenced in the present study are marked with an asterisk. Each plasmid was labeled and color-coded by the geographical origin (blue intensity: high, Asia; moderate, Middle East; low, Europe and North America), host source (pink intensity: high, human; low, animals), and NDM allele (green intensity: high, allele 5; moderate, alleles 1 and 7; low, alleles 4 and 17).

FIGURE 2

Conjugation frequencies of IncX3 and IncFII plasmids to six recipients of different species. The donor E. coli strains for the IncX3 plasmids (pNDM-HK1493 and pNDM-HK396) were CRE1493 and CRE396, respectively, while for the IncFII (pHK01) plasmid was combat2D2. The histograms show means and standard deviations (error bars). Conjugation frequencies were logarithmically transformed prior to statistical analysis.

FIGURE 3

Conjugation frequencies of IncX3 and IncFII plasmids at three temperatures. The donor E. coli strains for the IncX3 plasmids (pNDM-HK1493 and pNDM-HK396) were CRE1493 and CRE396, respectively, while for the IncFII plasmid (pHK01) was combat2D2. The histograms show means and standard deviations (error bars). Conjugation frequencies were logarithmically transformed prior to statistical analysis.