Ecological Effects of Daily Antiseptic Treatment on Microbial Composition of Saliva-Grown Microcosm Biofilms and Selection of Resistant Phenotypes

Abstract

Antiseptics are widely used in dental practice and included in numerous over-the-counter oral care products. However, the effects of routine antiseptic use on microbial composition of oral biofilms and on the emergence of resistant phenotypes remain unclear. Microcosm biofilms were inoculated from saliva samples of four donors and cultured in the Amsterdam Active Attachment biofilm model for 3 days. Then, they were treated two times daily with chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) for a period of 7 days. Ecological changes upon these multiple antiseptic treatments were evaluated by semiconductor-based sequencing of bacterial 16S rRNA genes and identification of amplicon sequence variants (ASVs). Furthermore, culture-based approaches were used for colony-forming units (CFU) assay, identification of antiseptic-resistant phenotypes using an agar dilution method, and evaluation of their antibiotic susceptibilities. Both CHX and CPC showed only slight effects on CFU and could not inhibit biofilm growth despite the two times daily treatment for 7 days. Both antiseptics showed significant ecological effects on the microbial compositions of the surviving microbiota, whereby CHX led to enrichment of rather caries-associated saccharolytic taxa and CPC led to enrichment of rather gingivitis-associated proteolytic taxa. Antiseptic-resistant phenotypes were isolated on antiseptic-containing agar plates, which also exhibited phenotypic resistance to various antibiotics. Our results highlight the need for further research into potential detrimental effects of antiseptics on the microbial composition of oral biofilms and on the spread of antimicrobial resistance in the context of their frequent use in oral healthcare.

Efficacy of a Mouthwash Containing CHX and CPC in SARS-CoV-2–Positive Patients: A Randomized Controlled Clinical Trial

“…It must further be considered that it is still unclear whether the observed reductions of SARS-CoV-2 infectivity after gargling with mouthwashes containing CPC or other active ingredients can lead to clinically relevant reductions in the risk of transmission of SARS-CoV-2 ( Cieplik and Jakubovics 2022 ). Furthermore, it must be kept in mind that the frequent use of antiseptics may also exert some negative effects such as inducing potentially detrimental ecological shifts in the oral microbiota ( Mao et al 2022 ) or development of antiseptic resistance in oral bacteria ( Verspecht et al 2019 ; Auer et al 2022 ; Muehler et al 2022 ).…”

Antibiotic Resistance of Selected Bacteria after Treatment of the Supragingival Biofilm with Subinhibitory Chlorhexidine Concentrations

“…Furthermore, we recently showed that twice-daily treatment of microcosm biofilms with CHX led to an ecological shift toward Gram-positive cocci in these biofilms. [ 19 ]. S. oralis was reported to have lower environmental requirements for a high growth rate in combination with a high adherence ability [ 40 , 41 ].…”

“…Furthermore, Mao et al recently showed that multi-resistant phenotypes with additional resistance to CHX could be isolated from microcosm biofilms that were cultured in vitro and subjected to antiseptic treatment twice daily [ 19 ].…”

Preprocedural Mouthwashes for Reduction of SARS-CoV-2 Viral Load and Infectivity

“…In addition, further work is needed to establish the link between the observed reduction in viral infectivity from CPC-containing mouthwashes and a clinically useful reduction in the risk of transmission of SARS-CoV-2. Ultimately, large-scale clinical studies will be needed to address this issue and identify the optimal approaches for delivering CPC or other antiseptics, while considering potentially detrimental ecologic shifts in the oral microbiota that may result from regular antiseptic use (Bescos et al 2020; Mao et al 2022).…”

“…Ultimately, large-scale clinical studies will be needed to address this issue and identify the optimal approaches for delivering CPC or other antiseptics, while considering potentially detrimental ecologic shifts in the oral microbiota that may result from regular antiseptic use ( Bescos et al. 2020 ; Mao et al 2022 ).…”