Regulatory Effect of Irresistin-16 on Competitive Dual-Species Biofilms Composed of Streptococcus mutans and Streptococcus sanguinis

Abstract

Based on the ecological plaque hypothesis, suppressing opportunistic pathogens within biofilms, rather than killing microbes indiscriminately, could be a biofilm control strategy for managing dental caries. The present study aimed to evaluate the effects of irresistin-16 (IRS-16) on competitive dual-species biofilms, which consisted of the conditional cariogenic agent Streptococcus mutans (S. mutans) and oral commensal bacteria Streptococcus sanguinis (S. sanguinis). Bacterial growth and biofilm formation were monitored using growth curve and crystal violet staining, respectively. The microbial proportion was determined using fluorescence in situ hybridization. A 2, 5-diphenyltetrazolium bromide assay was used to measure the metabolic activity of biofilms. Bacterial/extracellular polysaccharide (EPS) dyeing, together with water-insoluble EPS measurements, were used to estimate EPS synthesis. A lactic acid assay was performed to detect lactic acid generation in biofilms. The cytotoxicity of IRS-16 was evaluated in mouse fibroblast L929 cells using a live/dead cell viability assay and cell counting kit-8 assay. Our results showed that IRS-16 exhibited selective anti-biofilm activity, leading to a remarkable survival disadvantage of S. mutans within competitive dual-species biofilms. In addition, the metabolic activity, EPS synthesis, and acid generation of dual-species biofilms were significantly reduced by IRS-16. Moreover, IRS-16 showed minimal cytotoxicity against mouse fibroblast L929 cells. In conclusion, IRS-16 exhibited remarkable regulatory effects on dual-species biofilms composed of S. mutans and S. sanguinis with low cytotoxicity, suggesting that it may have potential for use in caries management through ecological biofilm control.

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“…Lactate dehydrogenase enzyme was utilized to quantify the lactate levels in the BPW by measuring the optical density at 340 nm using a microplate reader (Spectra-Max M5), as previously explained. The experiment to produce lactic acid was conducted three times [ 31 ].…”

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“…Then, 150 μL of 33% acetic acid solution was added to elute the crystal violet stain from the biofilms. The eluent was transferred to another 96-well plates, and the OD at 590 nm was recorded by a microplate reader (SpectraMaxM5, Molecular Devices, USA) [ 19 ].…”