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 (RND) 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.