De-repression of the smvA efflux system arises in clinical isolates of Proteus mirabilis and reduces susceptibility to chlorhexidine and other biocides

Proteus mirabilis is a common pathogen of the catheterized urinary tract and often described as intrinsically resistant to the biocide chlorhexidine (CHD). Here, we demonstrate that derepression of the smvA efflux system has occurred in clinical isolates of P. mirabilis and reduces susceptibility to CHD and other cationic biocides. Compared with other isolates examined, P. mirabilis RS47 exhibited a significantly higher CHD MIC (≥512 μg/ml) and significantly greater expression of smvA. Comparison of the RS47 smvA and cognate smvR repressor with sequences from other isolates indicated that RS47 carries an inactivated smvR. Complementation of RS47 with a functional smvR from isolate RS50a (which exhibited the lowest smvA expression and lowest CHD MIC) reduced smvA expression by ~59-fold and markedly lowered the MIC of CHD and other cationic biocides. Although complementation of RS47 did not reduce MICs to concentrations observed in isolate RS50a, the significantly lower polymyxin B MIC of RS50a indicated that differences in lipopolysaccharide (LPS) structure are also a factor in P. mirabilis CHD susceptibility. To determine if exposure to CHD can select for mutations in smvR, clinical isolates with the lowest CHD MICs were adapted to grow at increasing concentrations of CHD up to 512 μg/ ml. Analysis of the smvR in adapted populations indicated that mutations predicted to inactivate smvR occurred following CHD exposure in some isolates. Collectively, our data show that smvA derepression contributes to reduced biocide susceptibility in P. mirabilis, but differences in LPS structure between strains are also likely to be an important factor.