Impact of OqxR loss of function on the envelope proteome of Klebsiella pneumoniae and susceptibility to antimicrobials

Background: In Klebsiella pneumoniae, loss-of-function mutations in the transcriptional repressors RamR and OqxR both have an impact on the production of efflux pumps and porins relevant to antimicrobial efflux/entry. Objectives: To define, in an otherwise isogenic background, the relative effects of OqxR and RamR loss-offunction mutations on envelope protein production, envelope permeability and antimicrobial susceptibility. We also investigated the clinical relevance of an OqxR loss-of-function mutation, particularly in the context of beta-lactam susceptibility. Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. Antimicrobial susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and quantitative RT-PCR was used tomeasure transcript levels. Results: Loss of RamR or OqxR reduced envelope permeability in K. pneumoniae by 45%-55% relative to the WT. RamR loss activated AcrAB efflux pump production similar to 5-fold and this reduced b-lactam susceptibility, conferring ertapenem non-susceptibility even in the absence of a carbapenemase. In contrast, OqxR loss specifically activated OqxAB efflux pump production >10 000-fold. This reduced fluoroquinolone susceptibility but had little impact on beta-lactamsusceptibility even in the presence of alpha beta-lactamase. Conclusions: Whilst OqxR loss and RamR loss are both seen in K. pneumoniae clinical isolates, only RamR loss significantly stimulates AcrAB efflux pump production. This means that only RamR mutants have significantly reduced beta-lactamase-mediated beta-lactamsusceptibility and therefore represent a greater clinical threat.