Influence of Fluoride-Resistant Streptococcus mutans Within Antagonistic Dual-Species Biofilms Under Fluoride In Vitro

Abstract

The widespread application of fluoride, an extremely effective caries prevention agent, induces the generation of fluoride-resistant strains of opportunistic cariogenic bacteria such as fluoride-resistant Streptococcus mutans (S. mutans). However, the influence of this fluoride-resistant strain on oral microecological homeostasis under fluoride remains unknown. In this study, an antagonistic dual-species biofilm model composed of S. mutans and Streptococcus sanguinis (S. sanguinis) was used to investigate the influence of fluoride-resistant S. mutans on dual-species biofilm formation and pre-formed biofilms under fluoride to further elucidate whether fluoride-resistant strains would influence the anti-caries effect of fluoride from the point of biofilm control. The ratio of bacteria within dual-species biofilms was investigated using quantitative real-time PCR and fluorescence in situ hybridization. Cristal violet staining, scanning electron microscopy imaging, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay were used to evaluate biofilm biomass, biofilm structure, and metabolic activity, respectively. Biofilm acidogenicity was determined using lactic acid and pH measurements. The anthrone method and exopolysaccharide (EPS) staining were used to study the EPS production of biofilms. We found that, in biofilm formation, fluoride-resistant S. mutans occupied an overwhelming advantage in dual-species biofilms under fluoride, thus showing more biofilm biomass, more robust biofilm structure, and stronger metabolic activity (except for 0.275 g/L sodium fluoride [NaF]), EPS production, and acidogenicity within dual-species biofilms. However, in pre-formed biofilms, the advantage of fluoride-resistant S. mutans could not be fully highlighted for biofilm formation. Therefore, fluoride-resistant S. mutans could influence the anti-caries effect of fluoride on antagonistic dual-species biofilm formation while being heavily discounted in pre-formed biofilms from the perspective of biofilm control.

Ecological influence by colonization of fluoride-resistant Streptococcus mutans in oral biofilm

“…S. mutans was identified to play an important role in caries not only for its EPS-matrix generation ability and aciduricity but also for its cariogenicity. FR biofilms had lower pH at 275 ppm NaF at three time points, which were consistent with a previous study in which FR-related dual-species biofilms had lower supernatant pH at 275 ppm NaF whereas less difference was found at 1,250 ppm NaF (Zhang K. et al, 2022). Under low fluoride, FR may be responsible for enhanced cariogenicity depending on its better survivability and acid production under NaF, whereas the inhibitory effect of NaF on acid generation may play a dominant role at high NaF concentrations, hence less difference between FR and WT groups in addition to higher pH when compared with 275 ppm NaF.…”

“…Then, the biofilms were dehydrated using an ethanol gradient (50%, 60%, 70%, 80%, 90%, 95%, and absolute ethyl alcohol; 30 min incubation per concentration). Biofilms were dried, coated with gold-palladium, and imaged using a scanning electron microscope (SEM; Hitachi, Tokyo, Japan) at ×1,000 magnification ( Zhang K. et al., 2022 ).…”

“…Recently, it was reported that fluoride resistance influenced the development of competitive dual-species biofilms comprising S. mutans and Streptococcus sanguinis ( S. sanguinis ) under in vitro fluoride treatment ( Zhang K. et al., 2022 ). Fluoride resistance acquired an edge in competitive dual-species biofilm formation, resulting in a more robust biofilm formation, and increased cariogenic virulence.…”

Inhibitory effect of ficin on Candida albicans biofilm formation and pre-formed biofilms

“…Combined with the findings of this study, we conclude that ficin controls not only bacterial biofilms but also fungal biofilms. Pre-formed biofilms show stronger resistance to stress than biofilm formation [24]. Therefore, studies have reported that antibiofilm agents, including the antifungal fluconazole, inhibit biofilm formation but do not suppress pre-formed biofilms [24][25][26].…”

“…Pre-formed biofilms show stronger resistance to stress than biofilm formation [24]. Therefore, studies have reported that antibiofilm agents, including the antifungal fluconazole, inhibit biofilm formation but do not suppress pre-formed biofilms [24][25][26]. The effectiveness of ficin on both biofilm formation and pre-formed biofilm reveals its advantage over fluconazole to some extent, except for the preponderance of enzymatic degradation to control biofilms, rare resistance [13].…”