Coordinate overexpression of twoRNDefflux systems,ParXYandTtgABC, is responsible for multidrug resistance inPseudomonas putida

Resistance Nodulation cell Division (RND) efflux pumps are known to contribute to the tolerance ofPseudomonas putidato aromatic hydrocarbons, but their role in antibiotic resistance has not been fully elucidated. In this study, two types of single-step multidrug-resistant (MDR) mutants were selectedin vitrofrom reference strain KT2440. Mutants of the first type were more resistant to fluoroquinolones and beta-lactams except imipenem, and overproduced the efflux system TtgABC as a result of mutations occurring in regulator TtgR. In addition to TtgABC, mutants of the second type such as HPG-5 were found to upregulate a novel RND pump, dubbed ParXY/TtgC, which accommodates cefepim, fluoroquinolones and aminoglycosides. As demonstrated by gene deletion experiments, TtgABC and ParXY/TtgC are both under the positive control of a two-component system, PpeRS. Whole-genome sequence analyses revealed that mutant HPG-5 harbours a mutation inactivating the gene (sucD) of succinyl-CoA synthetase, an enzyme of the tricarboxylic cycle. Disruption ofsucDin strain KT2440 reproduced the resistance phenotype of HPG-5, and activated the glyoxylate shunt. Finally, identification of two MDR clinical strains ofP.putidathat jointly overexpress TtgABC and ParXY/TtgC, of which one is asucDmutant, highlights the role of these efflux systems as determinants of antibiotic resistance.