Phenotypic Adaptation to Antiseptics and Effects on Biofilm Formation Capacity and Antibiotic Resistance in Clinical Isolates of Early Colonizers in Dental Plaque

Abstract

Despite the wide-spread use of antiseptics in dental practice and oral care products, there is little public awareness of potential risks associated with antiseptic resistance and potentially concomitant cross-resistance. Therefore, the aim of this study was to investigate potential phenotypic adaptation in 177 clinical isolates of early colonizers of dental plaque (Streptococcus, Actinomyces, Rothia and Veillonella spp.) upon repeated exposure to subinhibitory concentrations of chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) over 10 passages using a modified microdilution method. Stability of phenotypic adaptation was re-evaluated after culture in antiseptic-free nutrient broth for 24 or 72 h. Strains showing 8-fold minimal inhibitory concentration (MIC)-increase were further examined regarding their biofilm formation capacity, phenotypic antibiotic resistance and presence of antibiotic resistance genes (ARGs). Eight-fold MIC-increases to CHX were detected in four Streptococcus isolates. These strains mostly exhibited significantly increased biofilm formation capacity compared to their respective wild-type strains. Phenotypic antibiotic resistance was detected to tetracycline and erythromycin, consistent with the detected ARGs. In conclusion, this study shows that clinical isolates of early colonizers of dental plaque can phenotypically adapt toward antiseptics such as CHX upon repeated exposure. The underlying mechanisms at genomic and transcriptomic levels need to be investigated in future studies.

Keywords: adaptation; antibiotic; antiseptic; biocide; cetylpyridinium chloride; chlorhexidine; oral biofilm; resistance.

Figure 1

Biofilm formation capacity of P10 replicates showing 8-fold MIC increase toward CHX and corresponding WT strains. Asterisks indicate statistically significant differences from pairwise comparisons between P10 replicates and their respective WT strains. E. faecalis strain T9 was used as positive control for biofilm formation capacity [38].