Determination of <i>Pseudomonas aeruginosa</i> 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 <i>Pseudomonas aeruginosa</i> 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 <i>Pseudomonas aeruginosa,</i> 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 <i>P. aeruginosa</i> efflux systems that can tease apart the overlapping substrate profiles of multidrug efflux pumps that can contribute to treatment failures.