Prevalence of mutations associated with tolerance to chlorhexidine and other cationic biocides among Proteus mirabilis clinical isolates

Proteus mirabilis is a frequent cause of catheter-associated urinary tract infection and often exhibits high tolerance to chlorhexidine (CHD), a biocide used widely in healthcare settings. We previously demonstrated that inactivation of the smvR repressor (leading to overexpression of the smvA efflux system), truncation of the MltA-interacting protein MipA and aspects of lipopolysaccharide (LPS) structure modulate CHD susceptibility in this organism. However, the prevalence of these mechanisms among P. mirabilis clinical isolates, the conditions under which they can be acquired and their impact on susceptibility to other cationic biocides require further study. Through phenotypic and genomic analysis of a panel of 78 P. mirabilis clinical isolates, we have confirmed that deleterious mutations in smvR commonly arise in P. mirabilis and are significantly associated with reduced susceptibility to CHD and other cationic biocides. Mutations in mipA were also associated with CHD tolerance. Conversely, mutations in smvA and the rppA response regulator (which governs lipid A modifications that alter LPS surface charge) were associated with increased susceptibility to several biocides. Several isolates harbouring smvR mutations displayed incongruous phenotypes, exhibiting relatively modest CHD tolerance, which could not be accounted for by co-occurring mutations in smvA and rppA or defects in LPS (as assessed by polymyxin B susceptibility). Further analysis of these isolates revealed mutations in the LPS core biosynthesis gene waaG, leading to LPS truncation from the inner core region. Directed evolution experiments further reinforced the importance of smvR inactivation in biocide adaptation in P. mirabilis and demonstrated that relevant mutations can be selected for by exposure to CHD concentrations up to four times lower than the minimum inhibitory concentration. Taken together, these results expand our understanding of mechanisms underlying tolerance to cationic biocides in this species and provide evidence for common mechanisms of cationic biocide tolerance.