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Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin.

ABSTRACT: Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae. Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of col^R in K. pneumoniae are largely due to chromosomal mutations in two-component regulators, although a plasmid-mediated col^R mechanism has recently been uncovered. However, the effects of intrinsic colistin resistance are yet to be characterized on a whole-genome level. Here, we used a genomics-based approach to understand the mechanisms of adaptive col^R acquisition in K. pneumoniae. In controlled directed-evolution experiments we observed two distinct paths to colistin resistance acquisition. Whole genome sequencing identified mutations in two colistin resistance genes: in the known col^R regulator phoQ which became fixed in the population and resulted in a single amino acid change, and unstable minority variants in the recently described two-component sensor crrB. Through RNAseq and microscopy, we reveal the broad range of effects that colistin exposure has on the cell. This study is the first to use genomics to identify a population of minority variants with mutations in a col^R gene in K. pneumoniae.

SUBMITTER: Cain AK

PROVIDER: S-EPMC6026146 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin.

Cain Amy K AK Boinett Christine J CJ Barquist Lars L Dordel Janina J Fookes Maria M Mayho Matthew M Ellington Matthew J MJ Goulding David D Pickard Derek D Wick Ryan R RR Holt Kathryn E KE Parkhill Julian J Thomson Nicholas R NR

Scientific reports 20180629 1

Colistin remains one of the few antibiotics effective against multi-drug resistant (MDR) hospital pathogens, such as Klebsiella pneumoniae. Yet resistance to this last-line drug is rapidly increasing. Characterized mechanisms of col<sup>R</sup> in K. pneumoniae are largely due to chromosomal mutations in two-component regulators, although a plasmid-mediated col<sup>R</sup> mechanism has recently been uncovered. However, the effects of intrinsic colistin resistance are yet to be characterized on ...[more]

PMID: 29959380

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Project description:Klebsiella pneumoniae is emerging as an important nosocomial pathogen due to its rapidly increasing multidrug resistance, which has led to a renewed interest in polymyxin antibiotics, such as colistin, as antibiotics of last resort. However, heteroresistance (i.e., the presence of a subpopulation of resistant bacteria in an otherwise susceptible culture) may hamper the effectiveness of colistin treatment in patients. In a previous study, we showed that colistin resistance among extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates emerged after the introduction of selective digestive tract decontamination (SDD) in an intensive care unit (ICU). In this study, we investigated heteroresistance to colistin among ESBL-producing K. pneumoniae isolates by using population analysis profiles (PAPs). We used whole-genome sequencing (WGS) to identify the mutations that were associated with the emergence of colistin resistance in these K. pneumoniae isolates. We found five heteroresistant subpopulations, with colistin MICs ranging from 8 to 64 mg/liter, which were derived from five clonally related, colistin-susceptible clinical isolates. WGS revealed the presence of mutations in the lpxM, mgrB, phoQ, and yciM genes in colistin-resistant K. pneumoniae isolates. In two strains, mgrB was inactivated by an IS3-like or ISKpn14 insertion sequence element. Complementation in trans with the wild-type mgrB gene resulted in these strains reverting to colistin susceptibility. The MICs for colistin-susceptible strains increased 2- to 4-fold in the presence of the mutated phoQ, lpxM, and yciM alleles. In conclusion, the present study indicates that heteroresistant K. pneumoniae subpopulations may be selected for upon exposure to colistin. Mutations in mgrB and phoQ have previously been associated with colistin resistance, but we provide experimental evidence for roles of mutations in the yciM and lpxM genes in the emergence of colistin resistance in K. pneumoniae.

Project description:BackgroundBacteriophages (phages) are one of the most promising alternatives to traditional antibiotic therapies, especially against multidrug-resistant bacteria. Klebsiella pneumoniae is considered to be an opportunistic pathogen that can cause life-threatening infections. Thus, this study aims at the characterization of a novel isolated phage vB_Kpn_ZC2 (ZCKP2, for short).MethodsThe phage ZCKP2 was isolated from sewage water by using the clinical isolate KP/08 as a host strain. The isolated bacteriophage was purified and amplified, followed by testing of its molecular weight using Pulse-Field Gel Electrophoresis (PFGE), transmission electron microscopy, antibacterial activity against a panel of other Klebsiella pneumoniae hosts, stability studies, and whole genome sequencing.ResultsPhage ZCKP2 belongs morphologically to siphoviruses as indicated from the Transmission Electron Microscopy microgram. The Pulsed Field Gel Electrophoresis and the phage sequencing estimated the phage genome size of 48.2 kbp. Moreover, the absence of lysogeny-related genes, antibiotic resistance genes, and virulence genes in the annotated genome suggests that phage ZCKP2 is safe for therapeutic use. Genome-based taxonomic analysis indicates that phage ZCKP2 represents a new family that has not been formally rated yet. In addition, phage ZCKP2 preserved high stability at different temperatures and pH values (-20 - 70 °C and pH 4 - 9). For the antibacterial activity, phage ZCKP2 maintained consistent clear zones on KP/08 bacteria along with other hosts, in addition to effective bacterial killing over time at different MOIs (0.1, 1, and 10). Also, the genome annotation predicted antibacterial lytic enzymes. Furthermore, the topology of class II holins was predicted in some putative proteins with dual transmembrane domains that contribute significantly to antibacterial activity. Phage ZCKP2 characterization demonstrates safety and efficiency against multidrug-resistant K. pneumoniae, hence ZCKP2 is a good candidate for further in vivo and phage therapy clinical applications.

Project description:Global increases in the use of carbapenems have resulted in several strains of Gram-negative bacteria acquiring carbapenem resistance, thereby limiting treatment options. Klebsiella pneumoniae is a common carbapenem-resistant pathogenic bacterium that is widely studied to identify novel antibiotic resistance mechanisms and drug targets. Antibiotic-resistant clinical isolates generally harbor many genetic alterations, and the identification of responsible mutations would provide insights into the molecular mechanisms of antibiotic resistance. We propose a method to prioritize mutated genes responsible for antibiotic resistance on the basis of expression changes in their local subnetworks, hypothesizing that mutated genes that show significant expression changes among the corresponding functionally associated genes are more likely to be involved in the carbapenem resistance. For network-based gene prioritization, we developed KlebNet (www.inetbio.org/klebnet), a genome-scale cofunctional network of K. pneumoniae genes. Using KlebNet, we reconstructed the functional modules for carbapenem resistance and virulence and identified the functional association between antibiotic resistance and virulence. Using complementation assays with the top candidate genes, we were able to validate a novel gene that negatively regulated carbapenem resistance and four novel genes that positively regulated virulence in Galleria mellonella larvae. Therefore, our study demonstrated the feasibility of network-based identification of genes required for antibiotic resistance and virulence of human-pathogenic bacteria.IMPORTANCEKlebsiella pneumoniae is a major bacterial pathogen that causes pneumonia and urinary tract infections in human. K. pneumoniae infections are treated with carbapenem, but carbapenem-resistant K. pneumoniae has been spreading worldwide. We are able to identify antimicrobial-resistant genes among mutated genes of the antibiotic-resistant clinical isolates. However, they usually harbor many mutated genes, including those that cause weak or neutral functional effects. Therefore, we need to prioritize the mutated genes to identify the more likely candidates for the follow-up functional analysis. For this study, we present a functional network of K. pneumoniae genes and propose a network-based method of prioritizing the mutated genes of the resistant clinical isolates. We also reconstructed the network-based functional modules for carbapenem resistance and virulence and retrieved the functional association between antibiotic resistance and virulence. This study demonstrated the feasibility of network-based analysis of clinical genomics data for the study of K. pneumoniae infection.

Dataset Information

Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin.

Publications

Morphological, genomic and transcriptomic responses of Klebsiella pneumoniae to the last-line antibiotic colistin.

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