The Envelope Proteome Changes Driven by RamA Overproduction inKlebsiella pneumoniaethat Enhance Acquired β-Lactam Resistance

OBJECTIVES In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA over-production can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes seen upon RamA overproduction during growth in low and high osmolarity media. METHODS Envelope permeability was estimated using a fluorescent dye accumulation assay. Antibiotic susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels quantified by Real Time RT-PCR. RESULTS RamA overproduction enhanced β-lactamase mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF over-expression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 out of 4 carbapenemase producers, 1/21 CTX-M producers and 2/19 strains not carrying CTX-M or carbapenemases. CONCLUSIONS Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.