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Comparative metabolomics revealed key pathways associated with the synergistic killing of multidrug-resistant Klebsiella pneumoniae by a bacteriophage-polymyxin combination.

ABSTRACT: Resistance to the last-line polymyxins is emerging in multidrug-resistant Klebsiella pneumoniae and phage therapy is a promising alternative. However, phage monotherapy often rapidly causes resistance and few studies have examined antibiotic-phage combinations against K. pneumoniae. Here, we investigated the combination of polymyxin B with a novel phage pK8 against an mcr-1-carrying polymyxin-resistant clinical isolate Kp II-503 (polymyxin B MIC, 8 mg/L). The phage genome was sequenced and bacterial metabolomes were analysed at 4 and 24 h following the treatment with polymyxin B (16 mg/L), phage pK8 (10² PFU/mL) and their combination. Minimal metabolic changes across 24 h were observed with polymyxin B alone; whereas a significant inhibition of the citrate cycle, pentose phosphate pathway, amino acid and nucleotide metabolism occurred with the phage-polymyxin combination at both 4 and 24 h, but with phage alone only at 4 h. The development of resistance to phage alone was associated with enhanced membrane lipid and decreased amino acid biosynthesis in Kp II-503. Notably, cAMP, cGMP and cCMP were significantly enriched (3.1-6.6 log₂fold) by phage alone and the combination only at 4 h. This is the first systems pharmacology study to investigate the enhanced bacterial killing by polymyxin-phage combination and provides important mechanistic information on phage killing, resistance and antibiotic-phage combination in K. pneumoniae.

SUBMITTER: Han ML

PROVIDER: S-EPMC8760530 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Comparative metabolomics revealed key pathways associated with the synergistic killing of multidrug-resistant Klebsiella pneumoniae by a bacteriophage-polymyxin combination.

Han Mei-Ling ML Nang Sue C SC Lin Yu-Wei YW Zhu Yan Y Yu Heidi H HH Wickremasinghe Hasini H Barlow Christopher K CK Creek Darren J DJ Crawford Simon S Rao Gauri G Dai Chongshan C Barr Jeremy J JJ Chan Kim K Turner Schooley Robert R Velkov Tony T Li Jian J

Computational and structural biotechnology journal 20220106

Resistance to the last-line polymyxins is emerging in multidrug-resistant Klebsiella pneumoniae and phage therapy is a promising alternative. However, phage monotherapy often rapidly causes resistance and few studies have examined antibiotic-phage combinations against K. pneumoniae. Here, we investigated the combination of polymyxin B with a novel phage pK8 against an mcr-1-carrying polymyxin-resistant clinical isolate Kp II-503 (polymyxin B MIC, 8 mg/L). The phage genome wa ...[more]

PMID: 35070170

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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:Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.

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Comparative metabolomics revealed key pathways associated with the synergistic killing of multidrug-resistant Klebsiella pneumoniae by a bacteriophage-polymyxin combination.

Publications

Comparative metabolomics revealed key pathways associated with the synergistic killing of multidrug-resistant <i>Klebsiella pneumoniae</i> by a bacteriophage-polymyxin combination.

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