Interspecies Dissemination of a Mobilizable Plasmid Harboring blaIMP-19 and the Possibility of Horizontal Gene Transfer in a Single Patient

Abstract

Carbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-β-lactamase gene was detected in two different species isolated from a single patient. Metallo-β-lactamase-producing Achromobacter xylosoxidans (KUN4507), non-metallo-β-lactamase-producing Klebsiella pneumoniae (KUN4843), and metallo-β-lactamase-producing K. pneumoniae (KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis of K. pneumoniae isolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboring blaIMP-19 was detected in metallo-β-lactamase-producing A. xylosoxidans and K. pneumoniae Although this plasmid was not self-transferable, the conjugation of this plasmid from A. xylosoxidans to non-metallo-β-lactamase-producing K. pneumoniae was successfully performed. The susceptibility patterns for metallo-β-lactamase-producing K. pneumoniae and the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borne blaIMP-19 from A. xylosoxidans to K. pneumoniae in a single patient.

FIG 1

Clinical course and bacterial isolates. The red circles indicate clinical isolates of metallo-β-lactamase (MBL)-producing Achromobacter xylosoxidans. The black squares indicate clinical isolates of Klebsiella pneumoniae (non-MBL producer). The red square indicates a clinical isolate of MBL-producing K. pneumoniae. The numbers above each isolate indicate the day from surgery (living-donor liver transplantation). The red boxes without shading indicate the sites from which each isolate was isolated. The red boxes with shading indicate the isolates used in this study, the isolation sites of these isolates, and MBL production. The blue boxes with shading indicate the antimicrobial agents, and the length of the boxes indicates the duration of antimicrobial therapy for each agent. There was no treatment between POD 57 and POD 85 because this patient showed no sign of sepsis after isolation of MBL-producing A. xylosoxidans.

FIG 2

Schema of circularized plasmids. The red arrows indicate mobility genes (relaxase gene and type IV secretion system coupling protein [T4CP] gene). The orange arrows indicate mating pair formation (MPF) genes. The aqua arrows indicate establishment genes. The green arrows indicate replication and stability genes. The gray arrows indicate other coding genes. The black arrows indicate hypothetical protein-encoding genes. The innermost ring shows GC content, and the middle ring shows GC skew. (A) pKUN4507_1 detected from Achromobacter xylosoxidans KUN4507 and Klebsiella pneumoniae KUN5033. The plasmid pKUN4507_1 harbors mobility genes and two MPF genes, which indicate a mobilizable plasmid. This plasmid harbored a class 1 integron including bla_IMP-19. (B) pKUN4507_2, another plasmid detected in A. xylosoxidans KUN4507. The plasmid pKUN4507_2 lacks a relaxase gene and many MPF genes and is not considered to be transferable. (C) pKUN4843_1, including a class 1 integron and many transposase genes detected in Klebsiella pneumoniae KUN4843 and KUN5033. (D) pKUN4843_2, detected in K. pneumoniae KUN4843 and KUN5033. This plasmid has almost all of the genes required for conjugative transfer and was considered to be a conjugative plasmid.

FIG 3

SNPs accumulated from the most recent common ancestor (MRCA) of Klebsiella pneumoniae. Two variants of a K. pneumoniae descendant (DESC) occurred in the same patient. One of the two descendant (DESC2) variants was considered to be in close contact with Achromobacter xylosoxidans KUN4507, and the plasmid harboring the MBL gene (pKUN4507_1) was transferred from A. xylosoxidans KUN4507 to DESC2. The red bar indicates the duration of the contact of A. xylosoxidans KUN4507 with DESC2. HPD, highest posterior density; POD, postoperative day.