Difference in formation of a dental multi-species biofilm according to substratum direction.

OBJECTIVES: The aim of this study was to investigate the difference in dental biofilm formation according to substratum direction, using an artificial biofilm model. METHODS: A three-species biofilm, consisting of Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, was formed on saliva-coated hydroxyapatite (sHA) discs oriented in three directions: downward (the discs placed in the direction of gravity), vertical (the discs placed parallel to the direction of gravity), and upward (the discs placed in opposite direction of gravity). The biofilms at 22 h and 46 h of age were analyzed using microbiological and biochemical methods, fluorescence-based assays, and scanning electron microscopy to investigate difference in bacterial adhesion, early and mature biofilm formation. RESULTS: The biofilms formed in the upward direction displayed the most complex structure, with the highest number and biovolume of bacteria, as well as the lowest pH conditions at both time points. The vertical and downward directions, however, had only scattered and small bacterial colonies. In the 22-h-old biofilms, the proportion of S. oralis was similar to, or slightly higher than, that of S. mutans in all directions of substratum surfaces. However, in the 46-h-old biofilms, S. mutans became the dominant bacteria in all directions, especially in the vertical and upward directions. CONCLUSIONS: The direction of the substratum surface could impact the proportion of bacteria and cariogenic properties of the multi-species biofilm. Biofilms in an upward direction may exhibit a higher cariogenic potential, followed by those in the vertical and downward directions, which could be related to gravity.

Biofilm ecology associated with dental caries: understanding of microbial interactions in oral communities leads to development of therapeutic strategies targeting cariogenic biofilms.

A biofilm is a sessile community characterized by cells attached to the surface and organized into a complex structural arrangement. Dental caries is a biofilm-dependent oral disease caused by infection with cariogenic pathogens, such as Streptococcus mutans, and associated with frequent exposure to a sugar-rich diet and poor oral hygiene. The virulence of cariogenic biofilms is often associated with the spatial organization of S. mutans enmeshed with exopolysaccharides on tooth surfaces. However, in the oral cavity, S. mutans does not act alone, and several other microbes contribute to cariogenic biofilm formation. Microbial communities in cariogenic biofilms are spatially organized into complex structural arrangements of various microbes and extracellular matrices. The balance of microbiota diversity with reduced diversity and a high proportion of acidogenic-aciduric microbiota within the biofilm is closely related to the disease state. Understanding the characteristics of polymicrobial biofilms and the association of microbial interactions within the biofilm (e.g., symbiosis, cooperation, and competition) in terms of their potential role in the pathogenesis of oral disease would help develop new strategies for interventions in virulent biofilm formation.

The reciprocal interaction between fluoride release of glass ionomers and acid production of Streptococcus mutans biofilm.

Objectives: The aim of this study was to demonstrate the mode of action of glass ionomers (G-Is) against cariogenic biofilms in the slow fluoride release phase by analyzing the reciprocal interaction between fluoride release from G-Is and acid production of Streptococcus mutans biofilm. Methods: G-Is discs in the slow fluoride release phase were prepared and 51 h-old S. mutans biofilms were formed on these discs. The interrelationship between the acid production of the biofilm and the fluoride release of the G-Is discs was investigated by analyzing both factors simultaneously during the biofilm formation period. The composition of the 51 h-old biofilms was then examined using microbiological, biochemical, and confocal laser scanning microscopic methods. Results: Acid production by the cariogenic biofilm, particularly at < pH 5, promotes G-Is fluoride release. Conversely, G-Is fluoride release inhibits the acid production of the cariogenic biofilm. This reciprocal interaction results in the reduction of virulence such as extracellular polysaccharides formation and cariogenic biofilm bio-mass, which may reduce the potential of secondary caries development around G-Is. Conclusions: These results suggest that G-Is may play a role in preventing the development of secondary caries during the slow fluoride release phase.

Collagen Peptide in a Combinatorial Treatment with Lactobacillus rhamnosus Inhibits the Cariogenic Properties of Streptococcus mutans: An In Vitro Study.

Dental caries is caused by the formation of cariogenic biofilm, leading to localized areas of enamel demineralization. Streptococcus mutans, a cariogenic pathogen, has long been considered as a microbial etiology of dental caries. We hypothesized that an antagonistic approach using a prebiotic collagen peptide in combination with probiotic Lactobacillus rhamnosus would modulate the virulence of this cariogenic biofilm. In vitro S. mutans biofilms were formed on saliva-coated hydroxyapatite discs, and the inhibitory effect of a combination of L. rhamnosus and collagen peptide on S. mutans biofilms were evaluated using microbiological, biochemical, confocal imaging, and transcriptomic analyses. The combination of L. rhamnosus with collagen peptide altered acid production by S. mutans, significantly increasing culture pH at an early stage of biofilm formation. Moreover, the 3D architecture of the S. mutans biofilm was greatly compromised when it was in the presence of L. rhamnosus with collagen peptide, resulting in a significant reduction in exopolysaccharide with unstructured and mixed bacterial organization. The presence of L. rhamnosus with collagen peptide modulated the virulence potential of S. mutans via down-regulation of eno, ldh, and atpD corresponding to acid production and proton transportation, whereas aguD associated with alkali production was up-regulated. Gly-Pro-Hyp, a common tripeptide unit of collagen, consistently modulated the cariogenic potential of S. mutans by inhibiting acid production, similar to the bioactivity of a collagen peptide. It also enhanced the relative abundance of commensal streptococci (S. oralis) in a mixed-species biofilm by inhibiting S. mutans colonization and dome-like microcolony formation. This work demonstrates that food-derived synbiotics may offer a useful means of disrupting cariogenic communities and maintaining microbial homeostasis.

Relationship between sucrose concentration and bacteria proportion in a multispecies biofilm: Short title: Sucrose challenges to a multispecies biofilm.

Objective: The aim of this study was to evaluate the relationship between sucrose concentration and bacteria proportion in a multispecies biofilm model. Methods: Streptococcus mutans (S. mutans), Streptococcus oralis (S. oralis), and Actinomyces naeslundii (A. naeslundii) were chose to form a multispecies biofilm. Different concentration (0-40%) of sucrose was introduced to the multispecies biofilm 3 times per day (30 min per time). And then the bacteria proportion and acid production of the biofilms were analyzed. Results: Increasing sucrose level increased CFU count of S. mutans up to a certain concentration (5% sucrose), after which the number of S. mutans slightly decreased, but the CFU counts of S. oralis and A. naeslundii continually decreased with sucrose concentration increase, especially, from 5% sucrose, the reduction was significant, and S. mutans became the dominant species in the biofilms. Furthermore, the acid production ability of the multispecies biofilm gradually increased and slightly decreased with sucrose concentration increased, and the turning concentration was 5%. Conclusion: Our findings suggest that increasing sucrose level could increase the competitiveness of S. mutans in the multispecies biofilm, which may shift the biofilm to a more cariogenic one, and 5% sucrose formed a most cariogenic biofilm in this study.

Sucrose challenges to Streptococcus mutans biofilms and the curve fitting for the biofilm changes.

The relationship between sugar level and development of dental caries has long been a main topic in dentistry. However, as a ubiquitous component of the modern diet, sucrose is mainly derived from three meals a day, rather than a long time exposure. In this study, various concentrations of sucrose were provided to Streptococcus mutans biofilms for 1 h per exposure (three times per day) to imitate a human meal pattern. And then the relationship between sucrose concentration and changes in the treated biofilms was determined. The results indicated that the components and acid production of the treated biofilms changed in a second-order polynomial curve pattern with sucrose concentration increase, which were confirmed by CLSM and SEM analyses. However, gene expression related to extracellular polysaccharides (EPS) formation, acid production and tolerance was up-regulated with sucrose concentration increase, which might have been due to compensation for the decrease in EPS formation and acid production by the biofilms at higher concentrations of sucrose. These findings suggest that sucrose in the range of 1%-5% can support the highest acid production and accumulation of S. mutans biofilms, which may further increase its cariogenic potential. However, additional studies are required to confirm the relationships in human cariogenic biofilms.

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis.

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.

Functional Relationship between Sucrose and a Cariogenic Biofilm Formation.

Sucrose is an important dietary factor in cariogenic biofilm formation and subsequent initiation of dental caries. This study investigated the functional relationships between sucrose concentration and Streptococcus mutans adherence and biofilm formation. Changes in morphological characteristics of the biofilms with increasing sucrose concentration were also evaluated. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs in culture medium containing 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, or 40% (w/v) sucrose. The adherence (in 4-hour biofilms) and biofilm composition (in 46-hour biofilms) of the biofilms were analyzed using microbiological, biochemical, laser scanning confocal fluorescence microscopic, and scanning electron microscopic methods. To determine the relationships, 2nd order polynomial curve fitting was performed. In this study, the influence of sucrose on bacterial adhesion, biofilm composition (dry weight, bacterial counts, and water-insoluble extracellular polysaccharide (EPS) content), and acidogenicity followed a 2nd order polynomial curve with concentration dependence, and the maximum effective concentrations (MECs) of sucrose ranged from 0.45 to 2.4%. The bacterial and EPS bio-volume and thickness in the biofilms also gradually increased and then decreased as sucrose concentration increased. Furthermore, the size and shape of the micro-colonies of the biofilms depended on the sucrose concentration. Around the MECs, the micro-colonies were bigger and more homogeneous than those at 0 and 40%, and were surrounded by enough EPSs to support their structure. These results suggest that the relationship between sucrose concentration and cariogenic biofilm formation in the oral cavity could be described by a functional relationship.

Long-term anti-cariogenic biofilm activity of glass ionomers related to fluoride release.

OBJECTIVES: The aim of this study was to evaluate the difference between anti-cariogenic biofilm activities of glass ionomers (G-Is) during the initial and second fluoride release phases and to define relationships between the anti-biofilm activities and fluoride release. METHODS: Fluoride release of three commercially available G-Is in a buffer was evaluated for 770 h, and then 70-h-old Streptococcus mutans UA159 biofilms were formed on the G-Is that had been immersed in the buffer for 0, 100, 200, or 700 h. The dry weight, bacterial cell number, water-insoluble extracellular polysaccharides (EPSs), and accumulated fluoride concentration of the 70-h-old biofilms and fluoride release and acid production rates during biofilm formation were determined. Relationships between the experimental variables and fluoride release rate were also evaluated using linear regression analysis. RESULTS: In this study, fluoride release of the tested G-Is did not exhibit a biphasic pattern during biofilm formation. The release was sustained or did not rapidly decrease even over long immersion periods and was strongly correlated with an increase in accumulated fluoride concentration of the biofilms (R=0.99, R(2)=0.98) and reductions in dry weight, water-insoluble EPSs, and acid production rate of the biofilms (R=-0.99 to -0.96, R(2)=0.92-0.98). CONCLUSIONS: This study suggests that G-Is can effectively affect acid production, EPS formation, and accumulation of cariogenic biofilms even during the second fluoride release phase, and that the anti-cariogenic biofilm activity is strongly correlated with fluoride release, which may be enhanced by acid production of cariogenic biofilms. CLINICAL SIGNIFICANCE: G-Is can affect cariogenic biofilm formation even during the second fluoride release phase.

Effects of combined oleic acid and fluoride at sub-MIC levels on EPS formation and viability of Streptococcus mutans UA159 biofilms.

Despite the widespread use of fluoride, dental caries, a biofilm-related disease, remains an important health problem. This study investigated whether oleic acid, a monounsaturated fatty acid, can enhance the effect of fluoride on extracellular polysaccharide (EPS) formation by Streptococcus mutans UA159 biofilms at sub-minimum inhibitory concentration levels, via microbiological and biochemical methods, confocal fluorescence microscopy, and real-time PCR. The combination of oleic acid with fluoride inhibited EPS formation more strongly than did fluoride or oleic acid alone. The superior inhibition of EPS formation was due to the combination of the inhibitory effects of oleic acid and fluoride against glucosyltransferases (GTFs) and GTF-related gene (gtfB, gtfC, and gtfD) expression, respectively. In addition, the combination of oleic acid with fluoride altered the bacterial biovolume of the biofilms without bactericidal activity. These results suggest that oleic acid may be useful for enhancing fluoride inhibition of EPS formation by S. mutans biofilms, without killing the bacterium.

Effect of 1-Minute Fluoride Treatment on Potential Virulence and Viability of a Cariogenic Biofilm.

Fluoride is a well-studied and widely used agent for the prevention of dental caries. Although dental caries is strongly related to cariogenic biofilms, the effect of brief fluoride treatment on the virulence properties of biofilms has not been well studied. This study evaluated the effect of a 1-min fluoride treatment on the virulence properties and viability of cariogenic biofilms, using a Streptococcus mutans biofilm model. For this study, 46-hour-old S. mutans biofilms were formed on saliva-coated hydroxyapatite discs and were treated with fluoride (0, 30, 300, 1,000, and 2,000 ppm F(-)) for 1 min. Viability and changes in acidogenicity, aciduricity and extracellular polysaccharide (EPS) formation of the biofilms were analyzed using biochemical and microbiological methods (pH drop, H(+) permeability, acid killing, and bacterial colony-forming unit assays). Laser scanning confocal fluorescence microscopy study was also performed. After the 1-min fluoride treatment, acidogenicity, aciduricity, and EPS formation of 46-hour-old S. mutans biofilms were significantly reduced when treated with concentrations ≥300 ppm F(-). The antivirulence activities of the 1-min fluoride treatment increased in a concentration-dependent pattern. However, the 1-min fluoride treatments did not affect viability, biovolume, and microcolony appearance of biofilm bacteria, even at high concentrations. These results suggest that the brief treatment with fluoride at concentrations ≥300 ppm F(-) is an effective measure for controlling cariogenic biofilms.

Relationship between fluoride release rate and anti-cariogenic biofilm activity of glass ionomer cements.

OBJECTIVES: The aim of this study was to evaluate acidogenicity and composition of Streptococcus mutans biofilms on glass ionomer cements (GICs) and then to determine the relationship between the anti-S. mutans biofilm activity and fluoride release rate of the GICs. METHODS: S. mutans biofilms were formed on discs prepared using five commercial GICs. Acid production and fluoride release rates of the biofilms on the GIC discs during biofilm formation (0-94 h) were determined. Next, 94-h-old S. mutans biofilms on GIC discs were analyzed to evaluate the biofilm composition (dry weight, bacterial cell number, and extra-cellular polysaccharide (EPS) amount) using microbiological, biochemical, and confocal laser scanning microscopy (CLSM) methods. Lastly, relationships between the fluoride release rate and changes in acidogenicity and composition of the biofilms were determined using a linear-fitting procedure. RESULTS: All of the tested GICs released fluoride ions. Of the GICs, the two that showed the highest fluoride release rates strongly affected acidogenicity, dry weight, and EPS formation of the biofilms. Furthermore, they reduced the bacterial and EPS bio-volumes and EPS thickness. However, the number of colony forming units (CFUs) of the biofilms was higher than that of the control. Generally, changes in the acidogenicity and composition (except for CFU count) of the biofilms on the GICs followed a negative linear-pattern of fluoride release rate-dependence (R=-0.850 to -0.995, R(2)=0.723-0.990). SIGNIFICANCE: These results suggest that the anti-cariogenic biofilm activity of GICs is closely correlated with their fluoride release rate during biofilm formation.