Genomic characterization of plasmids harboring blaNDM-1, blaNDM-5, and blaNDM-7 carbapenemase alleles in clinical Klebsiella pneumoniae in Pakistan

Abstract

Klebsiella pneumoniae is notorious for causing healthcare-associated infections, which become more complicated by the acquisition of bla_NDM genes via mobile genetic elements. Although Pakistan is a well-established hot spot of bla_NDM-positive K. pneumoniae, detailed molecular descriptions of bla_NDM-carrying plasmids are scarce. Seven K. pneumoniae isolates harboring bla_NDM were recovered from clinical sample sources during a 6 month period and tested for antimicrobial susceptibility. A long-read approach was used for whole-genome sequencing to obtain circularized plasmids and chromosomes for typing, annotation, and comparative analysis. The isolates were susceptible to colistin and tigecycline only among the tested antibiotics. We identified five sequence types (STs): ST11, ST16, ST716, ST464, and ST2856. Notably, three strains possessed the hypervirulent capsule KL2, while five were classified as O locus type O2a. Evidence of genetic diversity was further highlighted by the presence of four IncC plasmids harboring bla_NDM-1, two IncX3 plasmids harboring bla_NDM-5, and a single hybrid IncFIB/IncHI1B plasmid harboring bla_NDM-7. These plasmids also carried additional antimicrobial resistance (AMR) genes conferring resistance to aminoglycosides, cephalosporins, and fluoroquinolones. We identified the plasmidome of the K. pneumoniae isolates and characterized the New Delhi metallo-beta-lactamase (NDM)-carrying plasmids. Genetic analysis confirmed the presence of bla_NDM-1 and bla_NDM-5 on broad host range plasmids and bla_NDM-7 in a previously unreported hybrid plasmid backbone. We emphasized the critical role of plasmids in spreading bla_NDM in the clinical setting in Pakistan. Hence, we stressed the urgent need for enhanced surveillance, not least in low-middle income countries, infection control measures, and adherence to the "Access," "Watch," and "Reserve" guidelines in antibiotics use.

Importance: Infections caused by NDM-producing Klebsiella pneumoniae are a significant challenge to treat and represent a crucial health burden in low- and middle-income countries (LMICs). Most of the bla_NDM are located on plasmids that promote horizontal gene transfer. However, there is a lack of comprehensive information on the genetic context of the NDM-carrying plasmids in Pakistan. This study presents a detailed analysis of seven NDM-plasmids in clinical K. pneumoniae isolates, shedding light on their high-risk sequence types and multiple resistance determinants. We also describe the plasmid-bearing NDM alleles (bla_NDM-1, bla_NDM-5, and bla_NDM-7). Notably, we are the first to report bla_NDM-7 on the hybrid IncFIB/IncHI1B backbone in Pakistan, a plasmid that has rarely been reported previously globally. Understanding the plasmid genomic landscape is paramount to comprehensively understanding the AMR scenario in this LMIC.

Keywords: CR-KP; NDM; antibiotic resistance; carbapenemases; mobile genetic elements; plasmids.

Fig 1

Source of specimens, patients’ demographics, clinical information, characterization of STs, capsule typing, and ARGs. Blue, green, and red circles indicate the K-locus; yellow, purple, and black stars indicate the O-locus; and red squares are the ARGs.

Fig 2

Schematic representation of NDM-harboring plasmids in K. pneumoniae. The map of (A) pKPP-1-IncC plasmid harboring bla_NDM-1, (B) pKPP-6 IncX3 plasmid harboring bla_NDM-5, and (C) pKPP-3-hybrid IncFIB/IncHI1B plasmid harboring bla_NDM-7. Red arrows in the inner and external rings depict the ARGs, while purple arrows show the various insertion elements (ISs) and functional proteins.

Fig 3

Comparative analysis of the genetic context of bla_NDM alleles. (A) Genetic context with bla_{NDM-1, -5, -7} alleles in red arrows, additional ARGs in blue arrows, ISs in yellow arrows, and proteins in gray. The sequences KPP-1, KPP-4, KPP-2, KPP-5, KPP-6, and KPP-7 are all identical. (B) Comparative analysis of the genetic context of bla_{NDM-1, -5, -7} alleles with previously published sequences (CP115151.1, CP050164.1, CP132611.1, CP091474.2, and CP093504.1).