Damage of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> biofilms by carolacton,a secondary metabolite from the myxobacterium <Emphasis Type="Italic">Sorangium cellulosum</Emphasis>

Background  

Streptococcus mutans is a major pathogen in human dental caries. One of its important virulence properties is the ability to form biofilms (dental plaque) on tooth surfaces. Eradication of such biofilms is extremely difficult. We therefore screened a library of secondary metabolites from myxobacteria for their ability to damage biofilms of S. mutans.     

A three-species biofilm model for the evaluation of enamel and dentin demineralization

Although Streptococcus mutans biofilms have been useful for evaluating the cariogenic potential of dietary carbohydrates and the effects of fluoride on dental demineralization, a more appropriate biofilm should be developed to demonstrate the influence of other oral bacteria on cariogenic biofilms. This study describes the development and validation of a three-species biofilm model comprising Streptococcus mutans, Actinomyces naeslundii, and Streptococcus gordonii for the evaluation of enamel and dentin demineralization after cariogenic challenges and fluoride exposure. Single- or three-species biofilms were developed on dental substrata for 96?h, and biofilms were exposed to feast and famine episodes. The three-species biofilm model produced a large biomass, mostly comprising S. mutans (41%) and S. gordonii (44%), and produced significant demineralization in the dental substrata, although enamel demineralization was decreased by fluoride treatment. The findings indicate that the three-species biofilm model may be useful for evaluating the cariogenic potential of dietary carbohydrates other than sucrose and determining the effects of fluoride on dental substrata.     

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.     

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.     

Enhancement of fluoride activity against Streptococcus mutans biofilms by a substance separated from Polygonum cuspidatum

Polygonum cuspidatum is a plant with spreading rhizomes and numerous reddish-brown stems that has been used in Korean folk medicine to improve oral hygiene. Nevertheless, there are no reports related to its possible effect on the virulence of dental biofilms. In this study, the ability of a fraction (F1) separated from P. cuspidatum, alone or in combination with fluoride, to disrupt virulence factors and the composition of Streptococcus mutans biofilms was examined. F1 was mainly composed of resveratrol, emodin and physcion (approximately 16.2%, 18.9% and 2.07% of the weight of F1, respectively). F1 showed inhibitory effects on acid production and F-ATPase activity of S. mutans in biofilms, and could enhance fluoride activity against acid production and acid tolerance of S. mutans in biofilms. When S. mutans biofilms were briefly treated with F1 (10 min, a total of five times), the biomass accumulation, water-insoluble polysaccharides and intracellular iodophilic polysaccharides were reduced. Furthermore, the fluoride activity against biomass accumulation was enhanced by F1. These results suggest that F1 may be useful in the control of dental biofilms and in improving the cariostatic properties of fluoride without increasing its exposure.     

Structural organization and dynamics of exopolysaccharide matrix and microcolonies formation by Streptococcus mutans in biofilms

Aims: To investigate the structural organization and dynamics of exopolysaccharides (EPS) matrix and microcolonies formation by Streptococcus mutans during the biofilm development process. Methods and Results: Biofilms of Strep. mutans were formed on saliva‐coated hydroxyapatite (sHA) discs in the presence of glucose or sucrose (alone or mixed with starch). At specific time points, biofilms were subjected to confocal fluorescence imaging and computational analysis. EPS matrix was steadily formed on sHA surface in the presence of sucrose during the first 8 h followed by a threefold biomass increase between 8 and 30 h of biofilm development. The initial formation and further development of three‐dimensional microcolony structure occurred concomitantly with EPS matrix synthesis. Tridimensional renderings showed EPS closely associated with microcolonies throughout the biofilm development process forming four distinct domains (i) between sHA surface and microcolonies, (ii) within, (iii) covering and (iv) filling the spaces between microcolonies. The combination of starch and sucrose resulted in rapid formation of elevated amounts of EPS matrix and faster assembly of microcolonies by Strep. mutans, which altered their structural organization and susceptibility of the biofilm to acid killing (vs sucrose‐grown biofilms; P < 0·05). Conclusions: Our data indicate that EPS modulate the development, sequence of assembly and spatial distribution of microcolonies by Strep. mutans. Significance and Impact of the Study: Simultaneous visualization and analysis of EPS matrix and microcolonies provide a more precise examination of the structural organization of biofilms than labelling bacteria alone, which could be a useful approach to elucidate the exact mechanisms by which Strep. mutans influences oral biofilm formation and possibly identify novel targets for effective antibiofilm therapies.     

Effect of brief periodic fluoride treatments on the virulence and composition of a cariogenic biofilm

The present study investigated the effect of periodic 1-min fluoride treatments on Streptococcus mutans biofilms and then determined the relationship between anti-biofilm activity, treatment frequency, and fluoride concentration using a linear-fitting procedure. S. mutans biofilms were periodically treated (1-min/treatment) with fluoride during biofilm formation and analyzed using microbiological methods, confocal microscopy, and real-time PCR. The results indicated that reductions in the dry weight and acidogenicity of biofilms due to periodic fluoride treatment occurred in a concentration dependent manner. The reduction in dry weight without affecting bacterial cell viability was observed mainly due to the inhibitory effect of fluoride on gtfB and gtfC gene expression, which suppresses EPS production and avoids reduction of the pH below the critical point on the tooth surface. This study suggests that brief periodic exposure to appropriate fluoride concentrations through mouthwashes and toothpastes may affect the virulence and composition of cariogenic biofilms and subsequently prevent dental caries.     

DNA-microarrays identification of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> genes associated with biofilm thickness

Background  

A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms.     

Inhibitory Activity by Barley Coffee Components Towards Streptococcus Mutans Biofilm

It was shown that barley coffee (BC) interferes with Streptococcus mutans adsorption to hydroxyapatite. After BC component fractionation by dialysis and gel filtration chromatography (GFC), it was found that the low molecular mass (<1,000 Da) fraction (LMM fraction) containing polyphenols, zinc and fluoride ions and, above all, a high molecular mass (HMM > 1,000 kDa) melanoidin fraction display strong anti-adhesive properties towards S. mutans. In this study, we have further examined the potential of BC, BC LMM fraction and BC HMM melanoidin fraction as caries controlling agents by evaluating their anti-biofilm activity.The effects of BC and BC fractions on biofilm formation by S. mutans ATCC 25175 and its detachment from pre-developed biofilms were evaluated by microtiter plate assay. It was found that BC and its fractions, at concentrations ranging from 60 to 15 mg ml⁻¹ that are devoid of antimicrobial activity, inhibited S. mutans biofilm formation. An increase of S. mutans ATCC 25175 detachment from 24 h developed biofilm was observed at the highest tested concentrations. Interestingly, BC and BC fractions also showed anti-biofilm activity towards a variety of S. mutans clinical strains isolated from saliva, plaque and caries lesions of adult donors. In general, the HMM melanoidin fraction was more active than the LMM fraction. These findings, classifying BC LMM fraction and BC HMM melanoidin fractions as natural anti-biofilm agents, represent the basis for studying their possible use as anti-caries agents.     

Genetic adaptation of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> during biofilm formation on different types of surfaces

Background  

Adhesion and successful colonization of bacteria onto solid surfaces play a key role in biofilm formation. The initial adhesion and the colonization of bacteria may differ between the various types of surfaces found in oral cavity. Therefore, it is conceivable that diverse biofilms are developed on those various surfaces. The aim of the study was to investigate the molecular modifications occurring during in vitro biofilm development of Streptococcus mutans UA159 on several different dental surfaces.     

Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

Background

Microbial cell-cell interactions in the oral flora are believed to play an integral role in the development of dental plaque and ultimately, its pathogenicity. The effects of other species of oral bacteria on biofilm formation and virulence gene expression by Streptococcus mutans, the primary etiologic agent of dental caries, were evaluated using a dual-species biofilm model and RealTime-PCR analysis.

Results

As compared to mono-species biofilms, biofilm formation by S. mutans was significantly decreased when grown with Streptococcus sanguinis, but was modestly increased when co-cultivated with Lactobacillus casei. Co-cultivation with S. mutans significantly enhanced biofilm formation by Streptococcus oralis and L. casei, as compared to the respective mono-species biofilms. RealTime-PCR analysis showed that expression of spaP (for multi-functional adhesin SpaP, a surface-associated protein that S. mutans uses to bind to the tooth surface in the absence of sucrose), gtfB (for glucosyltransferase B that synthesizes α1,6-linked glucan polymers from sucrose and starch carbohydrates) and gbpB (for surface-associated protein GbpB, which binds to the glucan polymers) was decreased significantly when S. mutans were co-cultivated with L. casei. Similar results were also found with expression of spaP and gbpB, but not gtfB, when S. mutans was grown in biofilms with S. oralis. Compared to mono-species biofilms, the expression of luxS in S. mutans co-cultivated with S. oralis or L. casei was also significantly decreased. No significant differences were observed in expression of the selected genes when S. mutans was co-cultivated with S. sanguinis.

Conclusions

These results suggest that the presence of specific oral bacteria differentially affects biofilm formation and virulence gene expression by S. mutans.     

Comparative Effect of Chlorhexidine and Some Mouthrinses on Bacterial Biofilm Formation on Titanium Surface

The aim of the present study was to evaluate the effectiveness of chlorhexidine digluconate (CHX) and commonly used mouthrinses to single- and poly-species biofilms by S. mutans, S. aureus and P. aeruginosa, on titanium discs of grade IV. The formation of single- and poly-species biofilms at 16.5, 40.5 and 64.5-h incubation on titanium surface was evaluated by plate count (CFU ml⁻¹) before and after exposure to CHX and four mouthrinses (Curasept, Listerine, Meridol and Buccagel) and expressed as percentage of Inhibitory Activity (IA%). The application of the different anti-plaque formulations on biofilm can reduce the adhesion of bacteria to titanium surface with different degrees. The higher efficacy was observed for Listerine that shows IA% = 100 on the biofilm formed by S. mutans at 16.5 h. Log count of CFU was dependent to culture time and four mouthrinses for S. mutans and S. aureus, whilst was not dependent to culture time but to mouthrinses for P. aeruginosa. In general, the efficacy was particularly lesser to poly-species biofilms; no statistical differences were evidenced between all the mouthrinses and CHX as control group. The tested mouthrinses, compared to reference CHX 0.2%, have demonstrated a significant lower antibacterial activity than Listerine towards the experimental biofilms. This “in vitro” biofilm model should prove extremely useful for pre-clinical testing of anti-plaque agents, which inhibit biofilm formation, can prevent subsequent implant failure.     

An in vitro synergetic evaluation of the use of nisin and sodium fluoride or chlorhexidine against Streptococcus mutans

The objective of this study is to investigate the synergetic action between nisin and sodium fluoride or chlorhexidine against Streptococcus mutans, a primary cariogenic pathogen. In the antibacterial assay, a synergetic effect on S. mutans was found between nisin and sodium fluoride, but there was no interaction between nisin and chlorhexidine by the checkerboard, the fractional inhibitory concentration (FIC) and the fractional bactericidal concentration (FBC) tests. S. mutans survival rates showed a significant decline after treatment with a combination of nisin and sodium fluoride in a time-kill study. Scanning electron microscopy showed that the damage to S. mutans with the combined nisin and sodium fluoride treatment was the most severe among all of the different single and combined antimicrobial treatments. Furthermore, in the antibiofilm test, nisin in combination with sodium fluoride produced a stronger bactericidal effect on a S. mutans biofilm for 4 h and 16 h compared with sodium fluoride alone by confocal laser scanning microscopy. Nisin in combination with sodium fluoride exerted a high bactericidal effect on S. mutans and thereby has the potential to be used as an effective drug combination to prevent dental caries.     

Interaction of Salivary alpha-Amylase and Amylase-Binding-Protein A (AbpA) of <Emphasis Type="Italic">Streptococcus gordonii</Emphasis> with Glucosyltransferase of <Emphasis Type="Italic">S. gordonii</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Background  

Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation byStreptococcus gordoniiandStreptococcus mutans. The alpha-amylase-binding protein A (AbpA) ofS. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs ofS. gordoniiandS. mutans.     

Mutanase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. MP-1 produced inductively by fungal α-1,3-glucan and its potential for the degradation of mutan and <Emphasis Type="Italic">Streptococcus mutans</Emphasis> biofilm

Laetiporus sulphureus is a source of α-1,3-glucan that can substitute for the commercially-unavailable streptococcal mutan used to induce microbial mutanases. The water-insoluble fraction of its fruiting bodies from 0.15 to 0.2% (w/v) induced mutanase activity in Paenibacillus sp. MP-1 at 0.35 μ ml⁻¹. The mutanase extensively hydrolyzed streptococcal mutan, giving 23% of saccharification, and 83% of solubilization of glucan after 6 h. It also degraded α-1,3-polymers of biofilms, formed in vitro by Streptococcus mutans, even after only 3 min of contact.     

Pleurotus ostreatus biofilm‐forming ability and ultrastructure are significantly influenced by growth medium and support type

Aims

To investigate the effect of support and growth medium (GM) on Pleurotus ostreatus biofilm production, specific metabolic activity (SMA) and ultrastructure.

Methods and Results

Biofilms were developed on membranes covering a broad range of surface properties and, due to the applicative implications of mixed biofilms, on standard bacterial GM in stationary and shaken culture. Hydrophilic (glass fibre, Duran glass and hydroxyapatite) and mild hydrophobic (polyurethane, stainless steel, polycarbonate, nylon) supports were more adequate for biofilm attachment than the hydrophobic Teflon. Among the GM, sucrose–asparagine (SA) was more conducive to biofilm production than Luria–Bertani and M9. GM was more influential than support type on biofilm ultrastructure, and a high compactness was evident in biofilms developed on SA. Biofilms on Duran glass were more efficient than planktonic cultures in olive‐mill wastewater treatment.

Conclusions

The main effects of support and GM variables and their binary interactions on both biofilm production and SMA were all highly significant (P < 0·001): thus, the magnitude of the effect of each variable strongly depended on the level of the other one.

Significance and Impact of the Study

There is a lack of basic information regarding physiology and ultrastructure of P. ostreatus biofilms. To our knowledge, this is the first attempt to fill this gap, thus representing a basis for future studies.     

Do oral biofilms influence the wear and corrosion behavior of titanium?

The main aim of this work was to study the simultaneous wear-corrosion of titanium (Ti) in the presence of biofilms composed of Streptococcus mutans and Candida albicans. Both organisms were separately grown in specific growth media, and then mixed in a medium supplemented with a high sucrose concentration. Corrosion and tribocorrosion tests were performed after 48 h and 216 h of biofilm growth. Electrochemical corrosion tests indicated a decrease in the corrosion resistance of Ti in the presence of the biofilms although the TiO₂ film presented the characteristics of a compact oxide film. While the open circuit potential of Ti indicated a tendency to corrosion in the presence of the biofilms, tribocorrosion tests revealed a low friction on biofilm covered Ti. The properties of the biofilms were similar to those of the lubricant agents used to decrease the wear rate of materials. However, the pH-lowering promoted by microbial species, can lead to corrosion of Ti-based oral rehabilitation systems.     

Production,purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

Background  

The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances.     

Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions

Homeostasis of oral microbiota can be maintained through microbial interactions. Previous studies showed that Streptococcus oligofermentans, a non-mutans streptococci frequently isolated from caries-free subjects, inhibited the cariogenic Streptococcus mutans by the production of hydrogen peroxide (HP). Since pH is a critical factor in caries formation, we aimed to study the influence of pH on the competition between S. oligofermentans and S. mutans in biofilms. To this end, S. mutans and S. oligofermentans were inoculated alone or mixed at 1:1 ratio in buffered biofilm medium in a 96-well active attachment model. The single- and dual-species biofilms were grown under either constantly neutral pH or pH-cycling conditions. The latter includes two cycles of 8 h neutral pH and 16 h pH 5.5, used to mimic cariogenic condition. The 48 h biofilms were analysed for the viable cell counts, lactate and HP production. The last two measurements were carried out after incubating the 48 h biofilms in buffers supplemented with 1% glucose (pH 7.0) for 4 h. The results showed that S. oligofermentans inhibited the growth of S. mutans in dual-species biofilms under both tested pH conditions. The lactic acid production of dual-species biofilms was significantly lower than that of single-species S. mutans biofilms. Moreover, dual-species and single-species S. oligofermentans biofilms grown under pH-cycling conditions (with a 16 h low pH period) produced a significantly higher amount of HP than those grown under constantly neutral pH. In conclusion, S. oligofermentans inhibited S. mutans in biofilms not only under neutral pH, but also under pH-cycling conditions, likely through HP production. S. oligofermentans may be a compelling probiotic candidate against caries.