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Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A.

ABSTRACT: The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by (1)H NMR, using a paired wild type (WT) and the ?lpxM mutant strains B5055 and B5055?lpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055?lpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 values were consistent with polymyxin minimum inhibitory concentration (MIC) values for each strain. Accordingly, polymyxin exposure considerably enhanced the permeability of the B5055?lpxM OM. Analysis of the melting profiles of isolated LPS aggregates suggested that bactericidal polymyxin activity may relate to the acyl chains' phase of the outer membrane (OM). The enhanced polymyxin susceptibility of B5055?lpxM may be attributable to the favorable insertion of polymyxins into the more fluid OM compared with B5055. Molecular models of the polymyxin B-lipid A complex illuminate the key role of the lipid A acyl chains for complexation of polymyxin. The data provide important insight into the molecular basis for the increased polymyxin susceptibility of K. pneumoniae strains with under-acylated lipid A. Under-acylation appears to facilitate the integration of the N-terminal fatty-acyl chain of polymyxin into the OM resulting in an increased susceptibility to its antimicrobial activity/activities.

SUBMITTER: Velkov T

PROVIDER: S-EPMC4242410 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A.

Velkov Tony T Soon Rachel L RL Chong Pei L PL Huang Johnny X JX Cooper Matthew A MA Azad Mohammad A K MA Baker Mark A MA Thompson Philip E PE Roberts Kade K Nation Roger L RL Clements Abigail A Strugnell Richard A RA Li Jian J

Innate immunity 20120924 3

The impact of under-acylation of lipid A on the interaction between Klebsiella pneumoniae LPS and polymyxins B and E was examined with fluorometric and calorimetric methods, and by (1)H NMR, using a paired wild type (WT) and the ΔlpxM mutant strains B5055 and B5055ΔlpxM, which predominantly express LPS with hexa- and penta-acylated lipid A structures respectively. LPS from B5055ΔlpxM displayed a fourfold increased binding affinity for polymyxins B and E compared with the B5055 WT LPS. EC50 value ...[more]

PMID: 23008349

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Project description:Background The use of antibiotic adjuvants is a complementary strategy to the development of new antibiotics. The essential role of the ArnA dehydrogenase domain (ArnA_DH) in the addition of 4-amino-L-arabinose (L-Ara4N) to lipid A makes it a potential target in polymyxin adjuvant design. Purpose This study aimed to identify a dehydrogenase inhibitor that enhances the antibacterial effect of polymyxin B (PB) and to further understand the mechanism of this drug combination. Methods A susceptible K. pneumoniae strain, ATCC13883, was used to screen a dehydrogenase inhibitor library based on 3-(4,5)-dimethylthiazol(-z-y1)-2,5-diphenyltetrazolium bromide (MTT) and chequerboard assays. The protein- and cell-based effects of disulfiram (DSF) on ArnA activity were assessed, and the transcription levels of genes in the arn operon were evaluated by quantitative real-time polymerase chain reaction (qRT–PCR). Lipid A was isolated, and a structural analysis was performed. The cell wall function was evaluated through membrane integrity and bacterial viability assays. The in vivo antibacterial activity was evaluated using a mouse pulmonary infection model. Results We screened a dehydrogenase inhibitor library and found that the anti-alcoholism drug DSF significantly enhanced the antibacterial activity of PB in vitro and in vivo. The protein-based enzyme activity assay showed that DSF exerted no direct effect on the dehydrogenase activity of ArnA. Treatment with the combination of DSF and PB but not with PB alone decreased both the transcription level of genes in the arn operon and the modification level of lipid A. DSF also strengthened the disruption of the cell membrane integrity of PB. Moreover, the enhanced PB antibacterial activity was effective against clinical PB-resistant strains. Conclusion We identified a new drug combination that can be used to reduce the necessary dosage of PB and overcome PB resistance, and this drug combination has good prospects for clinical application.

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 aim of this study was to determine whether there is an association between serum resistance, O serotypes, and the production of extended-spectrum beta-lactamases (ESBLs) in Klebsiella pneumoniae. Ninety ESBL-producing and 178 non-ESBL-producing K. pneumoniae isolates gathered in five European countries were O serotyped and tested for sensitivity to the serum's bactericidal effect. The frequency of serum-resistant isolates was higher among ESBL-producing strains (30%; 27/90 isolates) than among non-ESBL-producing strains (17.9%; 32/178 isolates) (P = 0.037; odds ratio [OR] = 1.96; 95% confidence interval [95% CI] = 1.08 to 3.53). Although O1 was the most common O serotype in both Klebsiella groups, its frequency among ESBL-producing strains was significantly higher (59%; 53/90 isolates) than among non-ESBL producers (36%; 64/178 isolates) (P = 0.0006; OR = 2.5; 95% CI = 1.52 to 4.29). Furthermore, the prevalence of the O1 serotype was higher among serum-resistant strains of both ESBL-producing (74%; 20/27isolates) and non-ESBL producers (75%; 24/32 isolates) than among serum-sensitive ESBL producers (52.4%; 33/63 isolates) and non-ESBL producers (27.4%; 40/146 isolates). Serum resistance among ESBL-producing strains (36%; 17/47 isolates) versus non-ESBL-producing strains (16%; 27/166 isolates) was also significantly higher after the exclusion of clonal strains (P = 0.0056; OR = 2.9; 95% CI = 1.41 to 6.01). Sixteen ESBL types were detected, among which the frequency of serum resistance was significantly lower among the SHV-producing strains (9/48 isolates) than among the TEM producers (16/35 isolates) (P = 0.016; OR = 3.65; CI = 1.3 to 9.7). Curing ESBL-coding plasmids did not influence the serum resistance of the bacteria; all six plasmid-cured derivatives maintained serum resistance. The present findings suggest that ESBL-producing strains have a greater pathogenic potential than non-ESBL-producing strains, but the linkage between O serotypes, serum resistance, and ESBL production remains unclear at this stage.

Project description:Polymyxins are last-line antibiotics employed against multidrug-resistant (MDR) Klebsiella pneumoniae. Worryingly, polymyxin resistance is rapidly on the rise globally. Polymyxins initially target lipid A of lipopolysaccharides (LPSs) in the cell outer membrane (OM), causing disorganization and cell lysis. While most studies focus on how genetic variations confer polymyxin resistance, the mechanisms of membrane remodeling and metabolic changes in polymyxin-resistant strains remain unclear, thus hampering the development of effective therapies to treat severe K. pneumoniae infections. In the present study, lipid A profiling, OM lipidomics, genomics, and metabolomics were integrated to elucidate the global mechanisms of polymyxin resistance and metabolic adaptation in a polymyxin-resistant strain (strain S01R; MIC of >128 mg/L) obtained from K. pneumoniae strain S01, a polymyxin-susceptible (MIC of 2 mg/L), New Delhi metallo-β-lactamase (NDM)-producing MDR clinical isolate. Genomic analysis revealed a novel in-frame deletion at position V258 of PhoQ in S01R, potentially leading to lipid A modification with 4-amino-4-deoxy-l-arabinose (L-Ara4N) despite the absence of polymyxin B. Comparative metabolomic analysis revealed slightly elevated levels of energy production and amino acid metabolism in S01R compared to their levels in S01. Exposure to polymyxin B (4 mg/L for S01 and 512 mg/L for S01R) substantially altered energy, nucleotide, and amino acid metabolism and resulted in greater accumulation of lipids in both strains. Furthermore, the change induced by polymyxin B treatment was dramatic at both 1 and 4 h in S01 but only significant at 4 h in S01R. Overall, profound metabolic adaptation was observed in S01R following polymyxin B treatment. These findings contribute to our understanding of polymyxin resistance mechanisms in problematic NDM-producing K. pneumoniae strains and may facilitate the discovery of novel therapeutic targets. IMPORTANCE Antimicrobial resistance (AMR) is a major threat to global health. The emergence of resistance to the polymyxins that are the last line of defense in so-called Gram-negative "superbugs" has further increased the urgency to develop novel therapies. There are frequent outbreaks of K. pneumoniae infections in hospitals being reported, and polymyxin usage is increasing remarkably. Importantly, the polymyxin-resistant K. pneumoniae strains are imposing more severe consequences to health systems. Using metabolomics, lipid A profiling, and outer membrane lipidomics, our findings reveal (i) changes in the pentose phosphate pathway and amino acid and nucleotide metabolism in a susceptible strain following polymyxin treatment and (ii) how cellular metabolism, lipid A modification, and outer membrane remodeling were altered in K. pneumoniae following the acquisition of polymyxin resistance. Our study provides, for the first time, mechanistic insights into metabolic responses to polymyxin treatment in a multidrug-resistant, NDM-producing K. pneumoniae clinical isolate with acquired polymyxin resistance. Overall, these results will assist in identifying new therapeutic targets to combat and prevent polymyxin resistance.

Dataset Information

Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A.

Publications

Molecular basis for the increased polymyxin susceptibility of Klebsiella pneumoniae strains with under-acylated lipid A.

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