Spontaneously Arising Streptococcus mutans Variants with Reduced Susceptibility to Chlorhexidine Display Genetic Defects and Diminished Fitness.

Chlorhexidine (CHX) has been used to control dental caries caused by acid-tolerant bacteria such as since the 1970s. Repeat CHX exposure for other bacterial species results in the development of variants with reduced susceptibility that also become more resistant to other antimicrobials. It has not been tested if such variants arise when streptococci are exposed to CHX. Here, we passaged in increasing concentrations of CHX and isolated spontaneously arising educed usceptibility ariants (RSVs) from separate lineages that have MICs that are up to 3-fold greater than the parental strain. The RSVs have increased growth rates at neutral pH and under acidic conditions in the presence of CHX but accumulate less biomass in biofilms. RSVs display higher MICs for daptomycin and clindamycin but increased sensitivity to dental-relevant antimicrobials triclosan and sodium fluoride. Plate-based assays for competition with health-associated oral streptococci revealed decreased bacteriocin production by the RSVs, increased sensitivity to hydrogen peroxide, and diminished competitive fitness in a human-derived biofilm consortium. Whole-genome sequencing identified common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a glycolipid synthesis enzyme, which could alter the accumulation of lipoteichoic acids and other envelope constituents, as well as a variety of mutations in other genes. Collectively, these findings confirm that and likely other streptococci can develop tolerance to CHX but that increased tolerance comes at a fitness cost, such that CHX-induced variants that spontaneously arise in the human oral cavity may not persist.