Characterization of Spontaneously Arising Chlorhexidine-Tolerant Variants inStreptococcus mutans

Abstract

22Broad spectrum antimicrobials, both in dental products and within the clinic, have been 23 used in the suppression of cariogenic bacteria such as Streptococcus mutans for over 40 years. 24One such antimicrobial is chlorhexidine (CHX), and serves as a standard in dental research 25 against which other antimicrobial therapies are compared against for their efficacy. However, 26 very little is known about the mode of action for CHX against Streptococci and whether 27 tolerance can be developed from repeated exposures. Here, we begin to answer such questions 28 by passaging S. mutans with increasing concentrations of CHX and isolating spontaneously-29 arising tolerant variants (CTVs) from separate lineages. We find that these CTVs display a 30 higher minimal inhibitory concentration (MIC) against CHX than the wild-type strain and have 31 altered virulence properties such as acid tolerance and biofilm formation. We record higher 32 MICs for the variants against both daptomycin and bacitracin, but find increased sensitivity to 33 triclosan and sodium fluoride. Measurements of antagonistic capabilities against other health-34 associated oral streptococci show decreased bacteriocin production compared to wild-type and 35 increased sensitivity to hydrogen peroxide. Finally whole genome sequencing of the CTVs show 36 common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a 37 glycolipid synthesis enzyme, altering LTA accumulation and potentially lipid profile of the cell 38 wall. Together, these findings confirm that streptococci may develop tolerance to antimicrobial 39 agents such as CHX but in the case of S. mutans, increased tolerance may come at a fitness 40 cost for survival within oral biofilms that keeps variants suppressed within the population. 42 INTRODUCTION 43Controlling microbial biofilm infections in humans is challenging in part due to the 44 presence of extracellular polymeric substances (EPS) in which the organisms are embedded, 45 the presence of persister cells and sub-populations of cells with low metabolic activity, and 46 exchange of antimicrobial resistance genes through horizontal gene transfer. In the human oral 47 cavity, accumulation of biofilms on the teeth can lead to destruction of tooth mineral when there 48 is frequent and significant localized acidification by organic acids produced as metabolic end 49 products from fermentable carbohydrates (1). The initiation and progression of dental caries can 50 be controlled to a degree through non-specific mechanical removal of the biofilms or by 51 treatment with various chemical agents that include fluoride, xylitol or chlorhexidine (CHX) (2, 52 3). While the effectiveness of CHX in caries prevention is not definitively established and there 53 can be undesirable side effects (e.g. staining, irritation of host tissues) (4), CHX remains a 54 standard against which antimicrobial therapies are compared for their efficacy in the 55 suppression of caries-causing bacteria, such as Streptococcus mutans (5-7). Currently...