Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis .

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. , an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in , we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn insert in the gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with inactivation. Disruption of was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in crystalline biofilm formation.