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

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 s repressor (leading to overexpression of the 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 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 . clinical isolates, we have confirmed that deleterious mutations in commonly arise in and are significantly associated with reduced susceptibility to CHD and other cationic biocides. Mutations in were also associated with CHD tolerance. Conversely, mutations in and the response regulator (which governs lipid A modifications that alter LPS surface charge) were associated with increased susceptibility to several biocides. Several isolates harbouring mutations displayed incongruous phenotypes, exhibiting relatively modest CHD tolerance, which could not be accounted for by co-occurring mutations in and or defects in LPS (as assessed by polymyxin B susceptibility). Further analysis of these isolates revealed mutations in the LPS core biosynthesis gene , leading to LPS truncation from the inner core region. Directed evolution experiments further reinforced the importance of inactivation in biocide adaptation in 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.