In vitro Phenotypic Adaptation Towards Common Oral Antiseptics

Objectives: Bacterial resistances against antibiotics are known as a serious threat for public health, but much less attention has been given to the directly related problem of resistance towards antiseptics that are routinely used in dental practice. The aim of this study was to investigate potential phenotypic adaptation of selected oral and dermal bacterial strains upon repeated exposure to sublethal concentrations of the antiseptics chlorhexidine digluconate (CHX), benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC).

Methods: Actinomyces naeslundii (DSM-43013), Enterococcus faecalis (ATCC-29212), Fusobacterium nucelatum (DSM-20482), Streptococcus mutans (DSM-20523), Escherichia coli (ATCC-25922) and Staphylococcus aureus (ATCC-29213) were cultured as planktonic cultures and minimum inhibitory concentrations (MICs) were recorded for CHX, BAC and CPC. Bacteria from the sub-MIC population (highest antiseptic concentration with visible bacterial growth) were re-grown in antiseptic-free medium before starting a new passage of MIC-testing. This procedure was repeated 10 times (passage 1-10; n=6). Strains showing higher MICs after 10 passages as compared to passage 1, were then re-grown in antiseptic-free medium after storage and re-evaluated in order to assess the stability of the phenotypic adaptation. Protein expression profiles of adapted strains were compared to wildtype strains by means of SDS-PAGE.

Results: Phenotypic adaptation as shown by stable MIC-increases were found for E. coli towards BAC (22.5 µg/mL; 2-fold increase), CHX (3.9 µg/mL; 2-fold) and CPC (42.38 µg/mL; 4-fold), E. faecalis towards BAC (5.62 µg/mL; 2-fold) and CHX (15.6 µg/mL; 4-fold) and S. aureus towards CPC (2.64 µg/mL; 2-fold). The protein expression profile of adapted E. coli was different to the one of wildtype E. coli.

Conclusions: Stable phenotypic adaptation was found in three out of six of the tested bacterial strains towards at least one of the tested antiseptics encouraging further studies on the risk of resistance development towards antiseptics and its underlying molecular mechanisms.