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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.

SUBMITTER: Pitt ME

PROVIDER: S-EPMC5885010 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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

Pitt Miranda E ME Elliott Alysha G AG Cao Minh Duc MD Ganesamoorthy Devika D Karaiskos Ilias I Giamarellou Helen H Abboud Cely S CS Blaskovich Mark A T MAT Cooper Matthew A MA Coin Lachlan J M LJM

Microbial genomics 20180212 3

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 i ...[more]

PMID: 29431605

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Project description:Resistance to polymyxin antibiotics is increasing. Without new antibiotic classes, combination therapy is often required. We systematically investigated bacterial killing with polymyxin-based combinations against multidrug-resistant (including polymyxin-resistant), carbapenemase-producing Klebsiella pneumoniae Monotherapies and double- and triple-combination therapies were compared to identify the most efficacious treatment using static time-kill studies (24 h, six isolates), an in vitro pharmacokinetic/pharmacodynamic model (IVM; 48 h, two isolates), and the mouse thigh infection model (24 h, six isolates). In static time-kill studies, all monotherapies (polymyxin B, rifampin, amikacin, meropenem, or minocycline) were ineffective. Initial bacterial killing was enhanced with various polymyxin B-containing double combinations; however, substantial regrowth occurred in most cases by 24 h. Most polymyxin B-containing triple combinations provided greater and more sustained killing than double combinations. Standard dosage regimens of polymyxin B (2.5?mg/kg of body weight/day), rifampin (600?mg every 12 h), and amikacin (7.5?mg/kg every 12 h) were simulated in the IVM. Against isolate ATH 16, no viable bacteria were detected across 5 to 25 h with triple therapy, with regrowth to ?2-log10 CFU/ml occurring at 48 h. Against isolate BD 32, rapid initial killing of ?3.5-log10 CFU/ml at 5 h was followed by a slow decline to ?2-log10 CFU/ml at 48 h. In infected mice, polymyxin B monotherapy (60?mg/kg/day) generally was ineffective. With triple therapy (polymyxin B at 60?mg/kg/day, rifampin at 120?mg/kg/day, and amikacin at 300?mg/kg/day), at 24 h there was an ?1.7-log10 CFU/thigh reduction compared to the starting inoculum for all six isolates. Our results demonstrate that the polymyxin B-rifampin-amikacin combination significantly enhanced in vitro and in vivo bacterial killing, providing important information for the optimization of polymyxin-based combinations in patients.

Project description:The ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages. IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

Dataset Information

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

Publications

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

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