Determination of Pseudomonas aeruginosa MexXY-OprM substrate profile in a major efflux knockout system reveals distinct antibiotic substrate classes.

Defining the substrates of resistance-nodulation-division systems is often complicated by the presence of multiple systems in a single bacterial species. Using a major efflux knockout strain, we developed a Pseudomonas aeruginosa system that enables controlled expression of MexXY-OprM in the absence of background efflux complexes with overlapping substrate profiles. This system identified three groups of potential substrates: substrates, partial substrates, and nonsubstrates, and defined trimethoprim as a new substrate for MexXY-OprM.IMPORTANCEIncreasing antibiotic resistance in pathogenic bacteria is a major public health concern. One way bacteria can sustain growth in the presence of antibiotics is through active removal of these molecules via efflux pumps. In Pseudomonas aeruginosa, expression of the MexXY-OprM efflux pump is induced by treatment with certain antibiotics, resulting in the efflux of a diverse set of antimicrobials. Here, we designed an inducible system that allows for the determination of preferred antibiotic substrates for efflux by MexXY-OprM in a major efflux knockout strain. We show there are three distinct substrate classes-substrates, partial substrates, and nonsubstrates-and, for the first time, that the clinically important antibiotic trimethoprim is a substrate for MexXY-OprM. Our system's flexible design will enable future studies of other P. aeruginosa efflux systems that can tease apart the overlapping substrate profiles of multidrug efflux pumps that can contribute to treatment failures.