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The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

ABSTRACT: Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n?=?35), imipenem (n?=?1) or ciprofloxacin (n?=?1) in addition to known resistance determinants, collectively termed the "secondary resistome". As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5??g/ml, 4-fold below the susceptibility breakpoint (S???2??g/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial "helper" drugs that restore the efficacy of existing antimicrobials.

SUBMITTER: Jana B

PROVIDER: S-EPMC5309761 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

Jana Bimal B Cain Amy K AK Doerrler William T WT Boinett Christine J CJ Fookes Maria C MC Parkhill Julian J Guardabassi Luca L

Scientific reports 20170215

Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of ...[more]

PMID: 28198411

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Project description:The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most β-lactam antibiotics, in Enterobacteriaceae is of increasing concern. The concurrent spread of resistance against colistin, an antibiotic of last resort, further compounds this challenge further. Whole-genome sequencing (WGS) can play a significant role in the rapid and accurate detection/characterization of existing and emergent resistance determinants, an essential aspect of public health surveillance and response activities to combat the spread of antimicrobial resistant bacteria. In the current study, WGS data was used to characterize the genomic content of antimicrobial resistance genes, including those encoding carbapenemases, in 10 multidrug-resistant Klebsiella pneumoniae isolates from Pakistan. These clinical isolates represented five sequence types: ST11 (n = 3 isolates), ST14 (n = 3), ST15 (n = 1), ST101 (n = 2), and ST307 (n = 1). Resistance profiles against 25 clinically-relevant antimicrobials were determined by broth microdilution; resistant phenotypes were observed for at least 15 of the 25 antibiotics tested in all isolates except one. Specifically, 8/10 isolates were carbapenem-resistant and 7/10 isolates were colistin-resistant. The blaNDM-1 and blaOXA-48 carbapenemase genes were present in 7/10 and 5/10 isolates, respectively; including 2 isolates carrying both genes. No plasmid-mediated determinants for colistin resistance (e.g. mcr) were detected, but disruptions and mutations in chromosomal loci (i.e. mgrB and pmrB) previously reported to confer colistin resistance were observed. A blaOXA-48-carrying IncL/M-type plasmid was found in all blaOXA-48-positive isolates. The application of WGS to molecular epidemiology and surveillance studies, as exemplified here, will provide both a more complete understanding of the global distribution of MDR isolates and a robust surveillance tool useful for detecting emerging threats to public health.

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.

Dataset Information

The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

Publications

The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

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