Amino Acid Substitutions Account for Most MexS Alterations in Clinical nfxC Mutants of Pseudomonas aeruginosa

Multidrug-resistant mutants of Pseudomonas aeruginosa that overproduce the active efflux system MexEF-OprN (called nfxC mutants) have rarely been characterized in the hospital setting. Screening of 221 clinical strains exhibiting a reduced susceptibility to ciprofloxacin (a substrate of MexEF-OprN) and imipenem (a substrate of the negatively coregulated porin OprD) led to the identification of 43 (19.5%) nfxC mutants. Subsequent analysis of 22 nonredundant mutants showed that, in contrast to their in vitro-selected counterparts, only 3 of them (13.6%) harbored a disrupted mexS gene, which codes for the oxidoreductase MexS, whose inactivation is known to activate the mexEF-oprN operon through a LysR-type regulator, MexT. Nine (40.9%) of the clinical nfxC mutants contained single amino acid mutations in MexS, and these were associated with moderate effects on resistance and virulence factor production in 8/9 strains. Finally, the remaining 10 (45.5%) nfxC mutants did not display mutations in any of the regulators known to control mexEF-oprN expression (the mexS, mexT, mvaT, and ampR genes), confirming that other loci are responsible for pump upregulation in patients. Collectively, these data demonstrate that nfxC mutants are probably more frequent in the hospital than previously thought and have genetic and phenotypic features somewhat different from those of in vitro-selected mutants.