Adaptation to the host environment reduces Pseudomonas aeruginosa susceptibility to antibiotics
File(s)
Author(s)
Stoneham, Simon
Type
Thesis
Abstract
Pseudomonas aeruginosa chronic lung infection is a major cause of morbidity and mortality in people with Cystic Fibrosis (pwCF). Although frequently treated with the polymyxin antibiotic colistin, chronic infection is often impossible to eradicate despite low rates of colistin resistance. In this work it was hypothesised that bacterial adaptation to the lung environment reduces susceptibility to colistin.
The established synthetic sputum model SCFM-2 was modified to investigate the impact of sputum composition on P. aeruginosa susceptibility to antimicrobials, using the well characterised strain PA14 and relevant CF clinical isolates. Synthetic sputum significantly reduced susceptibility to colistin in both PA14 and clinical isolates.
Extracellular DNA (eDNA) was found to be the key component responsible and was sufficient to reduce susceptibility to colistin in a dose-dependent manner. MALDI-ToF lipidomic analysis of lipid A revealed that eDNA induced LPS modification with phosphoethanolamine (pEtN). Mutants lacking either eptA or arnC confirmed modification of LPS with pEtN and 4-amino-4-deoxy L arabinose (L-ara4N) were important for eDNA-induced reductions in colistin susceptibility. Reduced susceptibility was shown to occur via multiple two-component systems: ColRS, PmrAB and CprRS. Next, it was found that eDNA also induced resistance to the novel antimicrobial murepavadin. However, eDNA-induced resistance was circumvented by exposing bacteria to colistin and murepavadin in combination. Bacteria could also be sensitised to colistin or murepavadin by targeted normalisation of pH in the presence of eDNA by NaHCO3 confirming environmental pH as an important trigger of reduced susceptibility.
This work demonstrates a previously unrecognised role for endogenous pEtN modification of LPS in host-triggered colistin resistance in P. aeruginosa. Both NaHCO3 and the novel antimicrobial murepavadin enhanced colistin-mediated killing in synthetic sputum indicating their potential value as therapeutics in pwCF.
The established synthetic sputum model SCFM-2 was modified to investigate the impact of sputum composition on P. aeruginosa susceptibility to antimicrobials, using the well characterised strain PA14 and relevant CF clinical isolates. Synthetic sputum significantly reduced susceptibility to colistin in both PA14 and clinical isolates.
Extracellular DNA (eDNA) was found to be the key component responsible and was sufficient to reduce susceptibility to colistin in a dose-dependent manner. MALDI-ToF lipidomic analysis of lipid A revealed that eDNA induced LPS modification with phosphoethanolamine (pEtN). Mutants lacking either eptA or arnC confirmed modification of LPS with pEtN and 4-amino-4-deoxy L arabinose (L-ara4N) were important for eDNA-induced reductions in colistin susceptibility. Reduced susceptibility was shown to occur via multiple two-component systems: ColRS, PmrAB and CprRS. Next, it was found that eDNA also induced resistance to the novel antimicrobial murepavadin. However, eDNA-induced resistance was circumvented by exposing bacteria to colistin and murepavadin in combination. Bacteria could also be sensitised to colistin or murepavadin by targeted normalisation of pH in the presence of eDNA by NaHCO3 confirming environmental pH as an important trigger of reduced susceptibility.
This work demonstrates a previously unrecognised role for endogenous pEtN modification of LPS in host-triggered colistin resistance in P. aeruginosa. Both NaHCO3 and the novel antimicrobial murepavadin enhanced colistin-mediated killing in synthetic sputum indicating their potential value as therapeutics in pwCF.
Version
Open Access
Date Issued
2025-02-03
Date Awarded
01/09/2025
License URL
Advisor
Edwards, Andrew
Davies, Jane
Sponsor
Medical Research Council (Great Britain)
Publisher Department
Department of Infectious Disease
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)