Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in‐vitro oral biofilms

Abstract

Objective Chlorhexidine mouthrinses are marketed in different formulations. This study aimed at investigating qualitative and quantitative changes in in‐vitro multispecies oral biofilms, induced by different chlorhexidine‐containing mouthrinses. Background data Earlier studies comparing chlorhexidine mouthrinses are either clinical studies or in‐vitro studies assessing the antimicrobial efficacy of the mouthrinses. However, no clear investigations are available regarding ecological impact of different chlorhexidine formulations on in‐vitro multispecies oral biofilms after rinsing with different chlorhexidine formulations. Methods Nine commercially available chlorhexidine mouthrinses were selected. Multispecies oral communities (14 species) were grown for 48 h in a Biostat‐B Twin bioreactor. After that, they were used to develop biofilms on the surface of hydroxyapatite disks in 24‐well pates for 48 h. Biofilms were then rinsed once or multiple times with the corresponding mouthrinse. Biofilms were collected before starting the rinsing experiment and every 24 h for 3 days and vitality quantitative PCR was performed. The experiment was repeated 3 independent times on 3 different days and the results were analyzed using a linear mixed model. Results The mouthrinses provoked different effects in terms of change in total viable bacterial load (VBL), ecology, and community structure of the multispecies biofilms. There was no relation between chlorhexidine concentrations, presence, or absence of cetylpyridinium chloride and/or alcohol, and the observed effects. Some tested chlorhexidine mouthrinses (MC, HG, HH, and HI) strongly lowered the total VBL (≈1007 Geq/ml), but disrupted biofilm symbiosis (≥40% of the biofilms communities are pathobionts). On the other hand, other tested chlorhexidine mouthrinses (MD, ME, and HF) had limited impact on total VBL (≥1010 Geq/ml), but improved the biofilm ecology and community structure (≤10% of the biofilms communities are pathobionts). Conclusion Not all chlorhexidine mouthrinses have the same effect on oral biofilms. Their effect seems to be strongly product dependent and vary according to their compositions and formulations.

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

“…Although the oral microbiota is known to be highly resilient, particularly as compared to the intestinal microbiota (Wade, 2021), clinicians should be aware of potential detrimental effects of long-term use of antiseptic mouthwashes with regard to oral microbial ecology (Bescos et al, 2020b), which may potentially further perturb the commensal microbiota rather than shifting to a health-associated state (Chatzigiannidou et al, 2020). However, it needs to be considered that here the effects of "pure" antiseptics were investigated, whereas the effects of antiseptic mouthwashes seem to be strongly dependent on their respective compositions and formulations, as recently shown (Zayed et al, 2022). Also, a commercially available mouthwash comprising both CHX and CPC showed capability to even improve the microbial ecology of a 14-species biofilm in vitro reducing the level of pathobionts to less than 10% (Zayed et al, 2022), whereas in our study, CHX led to enrichment of rather caries-associated saccharolytic taxa and CPC led to enrichment of rather gingivitis-associated proteolytic taxa.…”

“…However, it needs to be considered that here the effects of "pure" antiseptics were investigated, whereas the effects of antiseptic mouthwashes seem to be strongly dependent on their respective compositions and formulations, as recently shown (Zayed et al, 2022). Also, a commercially available mouthwash comprising both CHX and CPC showed capability to even improve the microbial ecology of a 14-species biofilm in vitro reducing the level of pathobionts to less than 10% (Zayed et al, 2022), whereas in our study, CHX led to enrichment of rather caries-associated saccharolytic taxa and CPC led to enrichment of rather gingivitis-associated proteolytic taxa.…”

“…Antiseptics are in widespread use in dental practice and also included in numerous over-the-counter oral care products ( Haps et al, 2008 ; Sanz et al, 2013 ; van der Weijden et al, 2015 ), but the effects of routine antiseptic use on microbial composition of oral biofilms ( Chatzigiannidou et al, 2020 ; Zayed et al, 2022 ) and on the emergence of resistant phenotypes are still unclear ( Cieplik et al, 2019a ; Mao et al, 2020 ). Therefore, this study aimed to investigate the ecological effects of daily treatment with CHX or CPC on mature saliva-grown biofilms and whether a suchlike treatment selects for resistant phenotypes.…”

“…Despite those concerns about limited antibacterial efficacy and potential risks of resistance, it is also not entirely understood which ecological changes in microbial composition of oral biofilms are induced by the routine use of antiseptic mouthwashes ( Al-Kamel et al, 2019 ; Bescos et al, 2020a ; Chatzigiannidou et al, 2020 ; Brookes et al, 2021 ; Zayed et al, 2022 ). Two recent studies have shown that antiseptic treatment of in vitro biofilms affected their microbial composition and may potentially result in ecological shifts toward increased abundance of pathobionts ( Chatzigiannidou et al, 2020 ; Zayed et al, 2022 ).…”

Oral Biofilm Cryotherapy as a Novel Ecological Modulation Approach

“…The 14-species communities were grown in a Biostat-B Twin bioreactor using Brain Hearth Infusion 2 (BHI-2) broth (Herrero, Slomka, Boon, et al 2016). Biofilms were formed on hydroxyapatite discs (HADs) under microaerophilic conditions for 48 h (Zayed et al 2022). Symbiotic and dysbiotic biofilm models were used ( n = 5 each).…”