AUTHOR=Dulyayangkul Punyawee , Satapoomin Naphat , Avison Matthew B. , Charoenlap Nisanart , Vattanaviboon Paiboon , Mongkolsuk Skorn 

TITLE=Over-Expression of Hypochlorite Inducible Major Facilitator Superfamily (MFS) Pumps Reduces Antimicrobial Drug Susceptibility by Increasing the Production of MexXY Mediated by ArmZ in Pseudomonas aeruginosa

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.592153

DOI=10.3389/fmicb.2020.592153

ISSN=1664-302X

ABSTRACT=<p><italic>Pseudomonas aeruginosa</italic>, a well<bold>-</bold>known cause of nosocomial infection, is frequently antibiotic resistant and this complicates treatment. Links between oxidative stress responses inducing antibiotic resistance through over<bold>-</bold>production of RND<bold>-</bold>type efflux pumps have been reported in <italic>P. aeruginosa</italic>, but this has not previously been associated with MFS<bold>-</bold>type efflux pumps. Two MFS efflux pumps encoded by <italic>mfs1</italic> and <italic>mfs2</italic> were selected for study because they were found to be sodium hypochlorite (NaOCl) inducible. Antibiotic susceptibility testing was used to define the importance of these MFS pumps in antibiotic resistance and proteomics was used to characterize the resistance mechanisms involved. The results revealed that <italic>mfs1</italic> is NaOCl inducible whereas <italic>mfs2</italic> is NaOCl, N<bold>-</bold>Ethylmaleimide and t<bold>-</bold>butyl hydroperoxide inducible. Deletion of <italic>mfs1</italic> or <italic>mfs2</italic> did not affect antibiotic or paraquat susceptibility. However, over<bold>-</bold>production of Mfs1 and Mfs2 reduced susceptibility to aminoglycosides, quinolones, and paraquat. Proteomics, gene expression analysis and targeted mutagenesis showed that over<bold>-</bold>production of the MexXY RND<bold>-</bold>type efflux pump in a manner dependent upon <italic>armZ</italic>, but not <italic>amgRS</italic>, is the cause of reduced antibiotic susceptibility upon over<bold>-</bold>production of Mfs1 and Mfs2. <italic>mexXY operon</italic> expression analysis in strains carrying various lengths of <italic>mfs1</italic> and <italic>mfs2</italic> revealed that at least three transmembrane domains are necessary for <italic>mexXY</italic> over<bold>-</bold>expression and decreased antibiotic susceptibility. Over-expression of the MFS-type efflux pump gene <italic>tetA</italic>(C) did not give the same effect. Changes in paraquat susceptibility were independent of <italic>mexXY</italic> and <italic>armZ</italic> suggesting that it is a substrate of Mfs1 and Mfs2. Altogether, this is the first evidence of cascade effects where the over<bold>-</bold>production of an MFS pump causes over<bold>-</bold>production of an RND pump, in this case MexXY via increased <italic>armZ</italic> expression.</p>