Multifactorial chromosomal variants regulate polymyxin resistance in extensively drug-resistant Klebsiella pneumoniae

Abstract

Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) infections cause high mortality and are disseminating globally. Identifying the genetic basis underpinning resistance allows for rapid diagnosis and treatment. XDR isolates sourced from Greece and Brazil, including 19 polymyxin-resistant and five polymyxin-susceptible strains, were subjected to whole genome sequencing. Seventeen of the 19 polymyxin-resistant isolates harboured variations upstream or within mgrB. The most common mutation identified was an insertion at nucleotide position 75 in mgrB via an ISKpn26-like element in the ST258 lineage and ISKpn13 in one ST11 isolate. Three strains acquired an IS1 element upstream of mgrB and another strain had an ISKpn25 insertion at 133 bp. Other isolates had truncations (C28STOP, Q30STOP) or a missense mutation (D29E) affecting mgrB. Complementation assays revealed all mgrB perturbations contributed to resistance. Missense mutations in phoQ (T281M, G385C) were also found to facilitate resistance. Several variants in phoPQ co-segregating with the ISKpn26-like insertion were identified as potential partial suppressor mutations. Three ST258 samples were found to contain subpopulations with different resistance-conferring mutations, including the ISKpn26-like insertion colonizing with a novel mutation in pmrB (P158R), both confirmed via complementation assays. These findings highlight the broad spectrum of chromosomal modifications which can facilitate and regulate resistance against polymyxins in K. pneumoniae.

K. pneumoniaeST258 genomic variability and bacteriophage susceptibility

“…In our study, we identified three novel capsular variants in ST258 clade 1 due to ISKpn 26 insertion in the cps locus, and responsible for the intra-clade variation in our population which correlated remarkably well with reduced host range for many of the clade 1 infecting phages. ISKpn 26 has been recently implicated in unique recombination events in MDR K. pneumoniae (48) and may be worthwhile tracking. Two of the variants (D2 and D4), where the IS interrupted the wcaJ gene, were found in isolates with reduced capsule production in accordance with studies demonstrating that disruption of this gene has a detrimental effect on capsule content (46,49).…”

Evaluating the Genome and Resistome of Extensively Drug-Resistant Klebsiella pneumoniae using Native DNA and RNA Nanopore Sequencing

“…The ability for ONT to sequence long fragments of DNA has significantly aided the assembly of bacterial genomes and plasmids (16-18). In this study, multiple megaplasmids (≥100 kbp) were identified which were previously unresolved via Illumina sequencing (24). These harboured replicons IncA/C2 or a dual replicon, IncFIIK and IncFIB.…”

“…Clinically acquired XDR K. pneumoniae strains were sourced through the Hygeia General Hospital, Athens, Greece (24). Antimicrobial susceptibility assays (Supplementary Table S1), sequence typing and detection of acquired resistance genes for these isolates have previously been determined (24). Strains were stored at −80°C in 20% (v/v) glycerol and the same stock was used as per the prior study (24).…”

“…Antimicrobial susceptibility assays (Supplementary Table S1), sequence typing and detection of acquired resistance genes for these isolates have previously been determined (24). Strains were stored at −80°C in 20% (v/v) glycerol and the same stock was used as per the prior study (24). When required for extractions, glycerol stocks were grown on lysogeny broth (LB) agar plates and 6 morphologically similar colonies were selected for inoculation.…”

“…Of interest was to compare the potential for RNA sequencing to provide a better correlation to the resistance phenotype than DNA sequencing. These isolates have previously undergone ‘traditional’ whole genome sequencing (Illumina) and antimicrobial susceptibility testing (24). Three strains were selected from this cohort which exhibited resistance to all 24 classes or combinations of antibiotics tested, a high abundance of antibiotic resistance genes (≥26) and differing lineages (ST11 (16_GR_13), ST147 (1_GR_13) and ST258 (2_GR_12)).…”