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Background: Acidogenicity and production of an extracellular matrix (ECM) are important virulence factors for the dental caries-associated bacteria, such as Streptococcus mutans, that live in biofilms on tooth surface. The ECM protects the bacteria from the flushing and buffering effects of saliva resulting in highly acidic microenvironments inside the biofilm. Materials and methods: In this in vitro study, we applied real-time assays to follow biofilm formation and pH decrease in a growth medium and saliva by three S. mutans strains, as well as acid neutralization inside the mature biofilm. Results were compared with the biofilm composition. Effects of a non-fermentable polyol, xylitol, on acid production and acid neutralization in mature biofilms were evaluated by real-time pH measurements and confocal microscopy. Results: Combination of real-time pH measurements with biofilm accumulation assays revealed growth media dependent differences in the pH decrease and biofilm accumulation, as well as strain differences in acid production and biofilm formation but not in the buffer diffusion through ECM. The presence of xylitol reduced the pH drop during biofilm formation of all strains. In addition, with strain Ingbritt xylitol reduced the amount of ECM in biofilm, which increased the rate of acid neutralization inside the biofilm after buffer exposure. Conclusion: Our results stress the importance of biofilm matrix in creating the acidic environment inside a S. mutans biofilm, especially in the presence of saliva. In addition, our results suggest a novel mechanism of xylitol action. The observed increase in the permeability of the S. mutans ECM after xylitol exposure may allow acid-neutralizing saliva to reach deeper layer of the biofilms and thus, in part, explain previous clinical observations of reduced plaque acidogenicity after frequent xylitol use.

Postbiotics offer better properties than probiotics. This study investigated the antimicrobial activity of Lactiplantibacillus plantarum EIR/IF-1 postbiotics against pH-adaptive bacteria, namely Prevotella denticola, Fusobacterium nucleatum, and Streptococcus sanguinis. Cell-free culture media of L. plantarum EIR/IF-1 were used as postbiotics in either crude (acidic) or neutralized form to also understand non-pH-dependent antimicrobial potential. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and viable cell counts were determined for crude and neutralized postbiotics. Culture media adjusted to different pH values were also compared to adjusted media with postbiotics to understand the strength of organic acids in postbiotics. Antibiofilm activity of postbiotics was determined against polymicrobial biofilm formation. Finally, the toxicity of crude postbiotics was tested on human periodontal ligament fibroblast cells (hPDLFCs). MIC values of crude postbiotics were 12.5 mg/mL for all strains. F. nucleatum and P. denticola strains were sensitive to neutralized postbiotics after 48 h of incubation. Moreover, 12.5 and 25 mg/mL postbiotics inhibited biofilm formation and 2.5 mg/mL and lower concentrations of crude postbiotics showed no cytotoxicity in hPDLFCs. This study showed that postbiotics have antimicrobial activity against pH-adaptive oral bacteria and no cytotoxic effect on hPDLFCs depending on the dose. The non-acidic antimicrobial components of postbiotics could also enable their safe use in the oral cavity.

Previous studies have indicated that the exopolysaccharides of lactic acid bacteria exhibit antibiofilm activity against non-oral bacteria by preventing their initial adhesion to surfaces and by downregulating the expression of genes responsible for their biofilm formation. The aims of this study were to (1) characterize the exopolysaccharides (EPSs) of Lactobacillus plantarum EIR/IF-1 postbiotics, (2) test their antibiofilm effect on dual biofilms, and (3) evaluate their bacterial auto-aggregation, co-aggregation, and hydrocarbon-binding inhibitory activity. The EPSs were characterized by FTIR, HPLC, and thermogravimetric analysis. Bacterial auto- and co-aggregation were tested by Kolenbrander’s method and hydrocarbon binding was tested by Rosenberg’s method. Dual biofilms were formed by culturing Fusobacterium nucleatum ATCC 25586 with one of the following bacteria: Prevotella denticola ATCC 33185, P. denticola AHN 33266, Porphyromonas gingivalis ATCC 33277, P. gingivalis AHN 24155, and Filifactor alocis ATCC 35896. The EPSs contained fractions with different molecular weights (51 and 841 kDa) and monosaccharides of glucose, galactose, and fructose. The EPSs showed antibiofilm activity in all the biofilm models tested. The EPSs may have inhibited bacterial aggregation and binding to hydrocarbons by reducing bacterial hydrophobicity. In conclusion, the EPSs of L. plantarum EIR/IF-1, which consists of two major fractions, exhibited antibiofilm activity against oral bacteria, which can be explained by the inhibitory effect of EPSs on the auto-aggregation and co-aggregation of bacteria and their binding to hydrocarbons.