Oral Biofilms: Pathogens, Matrix, and Polymicrobial Interactions in Microenvironments.

Biofilms are microbial communities embedded within an extracellular matrix, forming a highly organized structure that causes many human infections. Dental caries (tooth decay) is a polymicrobial biofilm disease driven by the diet and microbiota-matrix interactions that occur on a solid surface. Sugars fuel the emergence of pathogens, the assembly of the matrix, and the acidification of the biofilm microenvironment, promoting ecological changes and concerted multispecies efforts that are conducive to acid damage of the mineralized tooth tissue. Here, we discuss recent advances in the role of the biofilm matrix and interactions between opportunistic pathogens and commensals in the pathogenesis of dental caries. In addition, we highlight the importance of matrix-producing organisms in fostering a pathogenic habitat where interspecies competition and synergies occur to drive the disease process, which could have implications to other infections associated with polymicrobial biofilms.

Enzymology and Ultrastructure of the in situ Pellicle in Caries-Active and Caries-Inactive Patients.

AIM: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. METHODS: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4',6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. RESULTS: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. CONCLUSION: The enzyme activities as well as the pellicle's ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

Enzymes in the in-situ pellicle of children with different caries activity.

The present study investigated, for the first time, enzymes in the in-situ pellicle of children. Furthermore, it was purposed to detect glucosyltransferase (GTF) isoforms in each child's pellicle. Twenty-four children (5-9 yr of age) participated in the study. Twelve were caries free with no decayed, missing, and filled teeth (dmft), whereas 12 had active caries (dmft ≥ 2, indicating at least two untreated carious lesions). Bovine enamel slabs, fixed on individual upper jaw splints, were utilized for pellicle formation in situ. After 3 and 30 min, samples were tested for amylase, lysozyme, and peroxidase activities; total GTF activity was examined only in the 30-min pellicle. Gold-immunolabelling was used to quantify the GTF B, C, and D isoforms in the pellicle by transmission electron microscopy (TEM). All enzymes tested were detected in the children's in-situ pellicle in an active conformation, and there were no significant differences in their levels of activity between caries-free and caries-active children. All GTF isoforms were found to be randomly distributed within all pellicle layers, althoug GTF B was only detected very sporadically. A significantly higher amount of GTF D was detected in the pellicle of caries-active children. Pellicle formation in children is characterized by uniformity and selectivity. Glucosyltransferase D might represent a possible biomarker for high caries risk in children.

The collagen binding protein Cnm contributes to oral colonization and cariogenicity of Streptococcus mutans OMZ175.

Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm(+) native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm(+) isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity.

Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo.

Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived ß1,3-glucans contribute to the EPS matrix structure, while fungal mannan and ß-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

The Stephan Curve revisited.

The Stephan Curve has played a dominant role in caries research over the past several decades. What is so remarkable about the Stephan Curve is the plethora of interactions it illustrates and yet acid production remains the dominant focus. Using sophisticated technology, it is possible to measure pH changes in plaque; however, these observations may carry a false sense of accuracy. Recent observations have shown that there may be multiple pH values within the plaque matrix, thus emphasizing the importance of the milieu within which acid is formed. Although acid production is indeed the immediate proximate cause of tooth dissolution, the influence of alkali production within plaque has received relative scant attention. Excessive reliance on Stephan Curve leads to describing foods as "safe" if they do not lower the pH below the so-called "critical pH" at which point it is postulated enamel dissolves. Acid production is just one of many biological processes that occur within plaque when exposed to sugar. Exploration of methods to enhance alkali production could produce rich research dividends.

Rodent model in caries research.

The rodent model of dental caries has made fundamental contributions to the understanding of the etiology, pathogenesis, and prevention of this disease. In conducting animal-based investigations, important details considered essential for successful outcome are occasionally overlooked. Adequate number of animals of appropriate age should always be included to ensure sufficient statistical power to draw valid conclusions. Attention should be paid to litter source as it is clear that litter origin may have a profound effect on the outcome. Inclusion of additive in diet may also affect eating pattern. A range of special techniques are available which can expand the role of animals in exploring the pathogenesis of dental caries. The arrival of genetically altered microorganisms along with availability of knock-out rodents offers exciting challenges to further explore the pathogenesis of this still too common disease.

Elevated incidence of dental caries in a mouse model of cystic fibrosis.

BACKGROUND: Dental caries is the single most prevalent and costly infectious disease worldwide, affecting more than 90% of the population in the U.S. The development of dental cavities requires the colonization of the tooth surface by acid-producing bacteria, such as Streptococcus mutans. Saliva bicarbonate constitutes the main buffering system which neutralizes the pH fall generated by the plaque bacteria during sugar metabolism. We found that the saliva pH is severely decreased in a mouse model of cystic fibrosis disease (CF). Given the close relationship between pH and caries development, we hypothesized that caries incidence might be elevated in the mouse CF model. METHODOLOGY/PRINCIPAL FINDINGS: We induced carious lesions in CF and wildtype mice by infecting their oral cavity with S. mutans, a well-studied cariogenic bacterium. After infection, the mice were fed a high-sucrose diet for 5 weeks (diet 2000). The mice were then euthanized and their jaws removed for caries scoring and bacterial counting. A dramatic increase in caries and severity of lesions scores were apparent in CF mice compared to their wildtype littermates. The elevated incidence of carious lesions correlated with a striking increase in the S. mutans viable population in dental plaque (20-fold increase in CF vs. wildtype mice; p value < 0.003; t test). We also found that the pilocarpine-stimulated saliva bicarbonate concentration was significantly reduced in CF mice (16 ± 2 mM vs. 31 ± 2 mM, CF and wildtype mice, respectively; p value < 0.01; t test). CONCLUSIONS/SIGNIFICANCE: Considering that bicarbonate is the most important pH buffering system in saliva, and the adherence and survival of aciduric bacteria such as S. mutans are enhanced at low pH values, we speculate that the decrease in the bicarbonate content and pH buffering of the saliva is at least partially responsible for the increased severity of lesions observed in the CF mouse.

Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans biofilms.

Cranberry fruit is a rich source of polyphenols, and has shown biological activities against Streptococcus mutans. In the present study, we examined the influence of extracts of flavonols (FLAV), anthocyanins (A) and proanthocyanidins (PAC) from cranberry on virulence factors involved in Streptococcus mutans biofilm development and acidogenicity. PAC and FLAV, alone or in combination, inhibited the surface-adsorbed glucosyltransferases and F-ATPases activities, and the acid production by S. mutans cells. Furthermore, biofilm development and acidogenicity were significantly affected by topical applications of PAC and FLAV (P<0.05). Anthocyanins were devoid of any significant biological effects. The flavonols are comprised of mostly quercetin glycosides, and the PAC are largely A-type oligomers of epicatechin. Our data show that proanthocyanidins and flavonols are the active constituents of cranberry against S. mutans.

Influence of apigenin on gtf gene expression in Streptococcus mutans UA159.

Apigenin, a potent inhibitor of glucosyltransferase activity, affects the accumulation of Streptococcus mutans biofilms in vitro by reducing the formation of insoluble glucans and enhancing the soluble glucan content of the polysaccharide matrix. In the present study, we investigated the influence of apigenin on gtfB, gtfC, and gtfD expression in S. mutans UA159. Apigenin (0.1 mM) significantly decreased the expression of gtfB and gtfC mRNA (P < 0.05); in contrast, it increased the expression of gtfD in S. mutans growing in the planktonic state. The protein levels of GTF B, GTF C, and GTF D in culture supernatants were also affected; less GTF B and C were detected, whereas the level of GTF D was significantly elevated (P < 0.05). A similar profile of gtf expression was obtained with biofilms, although an elevated concentration (1 mM) of apigenin was required to elicit the effects. The influence of apigenin on gtf gene expression was independent of any effect on GTF activity, did not involve inhibition of growth or effects on pH, and was not affected by addition of sucrose. The data show that apigenin modulates the genetic expression of virulence factors in S. mutans.

The influence of a novel propolis on mutans streptococci biofilms and caries development in rats.

A flavonoids-free Brazilian propolis (type 6) showed biological effects against mutans streptococci and inhibited the activity of glucosyltransferases. This study evaluated the influence of the ethanolic extract of a novel type of propolis (EEP) and its purified hexane fraction (EEH) on mutans streptococci biofilms and the development of dental caries in rats. The chemical composition of the propolis extracts were examined by gas chromatography/mass spectrometry. The effects of EEP and EEH on Streptococcus mutans UA159 and Streptococcus sobrinus 6715 biofilms were analysed by time-kill and glycolytic pH drop assays. Their influence on proton-translocating F-ATPase activity was also tested. In the animal study, the rats were infected with S. sobrinus 6715 and fed with cariogenic diet 2000. The rats were treated topically twice a day with each of the extracts (or control) for 5 weeks. After the experimental period, the microbial composition of their dental plaque and their caries scores were determined. The results showed that fatty acids (oleic, palmitic, linoleic and stearic) were the main compounds identified in EEP and EEH. These extracts did not show major effects on the viability of mutans streptococci biofilms. However, EEP and EEH significantly reduced acid production by the biofilms and also inhibited the activity of F-ATPase (60-65%). Furthermore, both extracts significantly reduced the incidence of smooth surface caries in vivo without displaying a reduction of the percentage of S. sobriuns in the animals' plaque (P < 0.05). However, only EEH was able to reduce the incidence and severity of sulcal surface caries (P < 0.05). The data suggest that the cariostatic properties of propolis type 6 are related to its effect on acid production and acid tolerance of cariogenic streptococci; the biological activities may be attributed to its high content of fatty acids.

Comparison of the cariogenicity of cola, honey, cow milk, human milk, and sucrose.

OBJECTIVE: The purpose of this study was to determine and compare the cariogenicity of various fluids that are frequently fed to infants and toddlers. We chose to examine sucrose, cola drink, honey, human milk, cow milk, and water because some of these have been associated with development of early childhood caries, although direct experimental evidence is lacking. METHODS: We used our desalivated rat model because the approach mimics the situation found in infants, whereby the flow of saliva is interrupted through mechanical effects of a nipple. The animals received basic nutrition by gavage, and the fluids being tested were available ad libitum. Thus, the only substances that came in contact with teeth were the test fluids. The investigation continued for 14 days. RESULTS: Cola, sucrose, and honey were by far the most cariogenic. In addition, cola and honey induced considerable erosion. Human milk was significantly more cariogenic than cow milk probably because of its lower mineral content and higher level of lactose. CONCLUSIONS: Our data show that the use of honey, cola, and sucrose water in nursing bottles should be discouraged. Although human milk is more cariogenic than cow milk, it is no more cariogenic than are common infant formulas. Protracted exposure to human milk or formula through allowing an infant to sleep on the nipple should be discouraged, and the need for oral hygiene after tooth eruption should be emphasized.

In vitro and in vivo effects of isolated fractions of Brazilian propolis on caries development.

Recently, two chemically different types of Brazilian propolis (type-3 and -12) were shown to have cariostatic properties. This study aimed to evaluate the influence of their isolated fractions on mutans streptococci viability, glucosyltransferases (GTFs) activity and caries development in rats. The ethanolic extracts of propolis (EEPs) were serially fractionated into hexane (H-fr), chloroform, ethyl acetate, and ethanol. The ability of the four fractions and EEP to inhibit Streptococcus mutans and Streptococcus sobrinus growth and adherence to a glass surface was examined. The effect on GTFs B and C activity was also determined. For the caries study, 60 Wistar rats infected with Streptococcus sobrinus were treated topically twice daily as follows: (1) EEP type-3, (2) H-fr type-3, (3) EEP type-12, (4) H-fr type-12, and (5) control. In general, the H-fr from both types of propolis showed the highest antibacterial activity and GTFs inhibition. Furthermore, the EEP and H-fr type-3 and -12 were equally effective in reducing dental caries in rats. The data suggest that the putative cariostatic compounds of propolis type-3 and -12 are mostly non-polar; and H-fr should be the fraction of choice for identifying further potentially novel anti-caries agents.

The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases.

Glucanohydrolases, especially mutanase [alpha-(1-->3) glucanase; EC 3.2.1.59] and dextranase [alpha-(1-->6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of alpha-(1-->3)-linked glucans) or C [synthesis of alpha-(1-->6)- and alpha-(1-->3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1-->6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.

Fluorosis: is it really a problem?

BACKGROUND: Scientists have noted an association between mottled enamel and fluoride exposure since the early 1900s. By the mid-1900s, they also recognized that fluoride intake was related to lower caries incidence. To harness the protective effect of fluoride while limiting the occurrence of fluorosis, dental researchers have recommended that the fluoride level in chinking water be 1 part per million or less. OVERVIEW: Despite the recognition that fluoride levels in water can be controlled to offer caries protection with minimal risk of fluorosis, the cosmetic defect continues to appear. However, although the word "fluorosis" conjures up images of brown stained and pitted enamel, such severe cases rarely are seen in the United States. Children in this country are exposed to fluoride from numerous sources and the appearance of mild fluorosis is not unusual. CONCLUSIONS AND PRACTICE IMPLICATIONS: In most cases, fluorosis is a minor cosmetic defect that should not be cause for alarm. Dentists should educate their patients about the optimal range of fluoride intake for caries protection, sources of fluoride and the possibility of fluorosis.