Characterization of Spontaneously Arising Chlorhexidine-Tolerant Variants inStreptococcus mutans

Abstract

ABSTRACTBroad spectrum antimicrobials, both in dental products and within the clinic, have been used in the suppression of cariogenic bacteria such asStreptococcus mutansfor over 40 years. One such antimicrobial is chlorhexidine (CHX), and serves as a standard in dental research against which other antimicrobial therapies are compared against for their efficacy. However, very little is known about the mode of action for CHX against Streptococci and whether tolerance can be developed from repeated exposures. Here, we begin to answer such questions by passagingS. mutanswith increasing concentrations of CHX and isolating spontaneously-arising tolerant variants (CTVs) from separate lineages. We find that these CTVs display a higher minimal inhibitory concentration (MIC) against CHX than the wild-type strain and have altered virulence properties such as acid tolerance and biofilm formation. We record higher MICs for the variants against both daptomycin and bacitracin, but find increased sensitivity to triclosan and sodium fluoride. Measurements of antagonistic capabilities against other health-associated oral streptococci show decreased bacteriocin production compared to wild-type and increased sensitivity to hydrogen peroxide. Finally whole genome sequencing of the CTVs show common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a glycolipid synthesis enzyme, altering LTA accumulation and potentially lipid profile of the cell wall. Together, these findings confirm that streptococci may develop tolerance to antimicrobial agents such as CHX but in the case ofS. mutans,increased tolerance may come at a fitness cost for survival within oral biofilms that keeps variants suppressed within the population.