Proteomic analyses of chlorhexidine tolerance mechanisms in Delftia acidovorans biofilms

Protein expression and fatty acid profiles of biofilm cells of chlorhexidinetolerant Delftia acidovorans (MIC = 15 μg/ml) and its chlorhexidine-susceptible mutant (MIC = 1 μg/ml) were investigated. The chlorhexidine-susceptible mutant (MT51) was derived from the parental strain (WT15) using Tn5 transposon mutagenesis. The disrupted gene was identified as tolQ, a component of the tolQRAB gene cluster known to be involved in outer membrane stability. Proteomic responses of biofilm cells were compared by differential in-gel electrophoresis following exposure to chlorhexidine at sub-MIC (10 μg/ml) and above-MIC (30 μg/ml) concentrations. Numerous changes in protein abundance were observed in biofilm cells following chlorhexidine exposure, suggesting that molecular changes occurred during adaptation to chlorhexidine. Forty proteins showing significant differences (≥1.5- fold; P < 0.05) were identified by mass spectrometry and were associated with various functions, including amino acid and lipid biosynthesis, protein translation, energy metabolism, and stress-related functions (e.g., GroEL, aspartyl/ glutamyl-tRNA amidotransferase, elongation factor Tu, Clp protease, and hydroxymyristoyl-ACP dehydratase). Several proteins involved in fatty acid synthesis were affected by chlorhexidine, in agreement with fatty acid analysis, wherein chlorhexidine-induced shifts in the fatty acid profile were observed in the chlorhexidine-tolerant cells, primarily the cyclic fatty acids. Transmission electron microscopy revealed more prominent changes in the cell envelope of chlorhexidine-susceptible MT51 cells. This study suggests that multiple mechanisms involving both the cell envelope (and likely TolQ) and panmetabolic regulation play roles in chlorhexidine tolerance in D. acidovorans.