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bioRxiv · 10.1101/133918

The Envelope Proteome Changes Driven By RamA Overproduction in Klebsiella pneumoniae That Enhance Acquired β-Lactam Resistance

Jimenez-Castellanos, J.-C.·Wan Nur Ismah, W. A. K.·Takebayashi, Y.·Findlay, J.·Schneiders, T.·Heesom, K. J.·Avison, M. B.
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Abstract

OBJECTIVESIn 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 {beta}-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.\n\nMETHODSEnvelope 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.\n\nRESULTSRamA overproduction enhanced {beta}-lactamase mediated {beta}-lactam resistance, in some cases dramatically, without altering {beta}-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.\n\nCONCLUSIONSWhilst 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 {beta}-lactamase mediated {beta}-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.

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Jimenez-Castellanos, J.-C., Wan Nur Ismah, W. A. K., Takebayashi, Y., Findlay, J., Schneiders, T., Heesom, K. J., Avison, M. B.. 2017-05-03. The Envelope Proteome Changes Driven By RamA Overproduction in Klebsiella pneumoniae That Enhance Acquired β-Lactam Resistance. https://doi.org/10.1101/133918

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