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1.  Targeted Antimicrobial Treatment to Re-establish a Healthy Microbial Flora for Long-term Protection 
Advances in Dental Research  2012;24(2):94-97.
Streptococcus mutans has been implicated as the major acid-producing (cariogenic) bacterium. Dietary sugars and other factors may cause an imbalance of oral microflora that enables S. mutans to become dominant in the multi-species biofilms on the tooth surface, which could lead to dental caries. The application of broad-spectrum antimicrobials often results in re-colonization and re-dominance of S. mutans within oral flora, while in contrast, therapies capable of selective elimination of S. mutans from oral microbial communities may help to re-establish the normal flora and provide long-term protection. C16G2, a novel synthetic antimicrobial peptide with specificity for S. mutans, was found to have robust killing efficacy and selectivity for S. mutans in vitro. A subsequent pilot human study found that a single application of C16G2 in the oral cavity (formulated in a mouthrinse vehicle) was associated with a reduction in plaque and salivary S. mutans, lactic acid production, and enamel demineralization during the entire 4-day testing period. C16G2 is now being developed as a new anticaries drug.
doi:10.1177/0022034512453725
PMCID: PMC3420366  PMID: 22899688
microbial ecology; microbiology; microbial genetics; caries; dental biofilm; microbiota
2.  Clinical Efficacy of a Specifically Targeted Antimicrobial Peptide Mouth Rinse: Targeted Elimination of Streptococcus mutans and Prevention of Demineralization 
Caries Research  2011;45(5):415-428.
Background/Aims
Streptococcus mutans, the major etiological agent of dental caries, has a measurable impact on domestic and global health care costs. Though persistent in the oral cavity despite conventional oral hygiene, S. mutans can be excluded from intact oral biofilms through competitive exclusion by other microorganisms. This suggests that therapies capable of selectively eliminating S. mutans while limiting the damage to the normal oral flora might be effective long-term interventions to fight cariogenesis. To meet this challenge, we designed C16G2, a novel synthetic specifically targeted antimicrobial peptide with specificity for S. mutans. C16G2 consists of a S. mutans-selective ‘targeting region’ comprised of a fragment from S. mutans competence stimulation peptide (CSP) conjoined to a ‘killing region’ consisting of a broad-spectrum antimicrobial peptide (G2). In vitro studies have indicated that C16G2 has robust efficacy and selectivity for S. mutans, and not other oral bacteria, and affects targeted bacteria within seconds of contact.
Methods
In the present study, we evaluated C16G2 for clinical utility in vitro, followed by a pilot efficacy study to examine the impact of a 0.04% (w/v) C16G2 rinse in an intra-oral remineralization/demineralization model.
Results and Conclusions
C16G2 rinse usage was associated with reductions in plaque and salivary S. mutans, lactic acid production, and enamel demineralization. The impact on total plaque bacteria was minimal. These results suggest that C16G2 is effective against S. mutans in vivo and should be evaluated further in the clinic.
doi:10.1159/000330510
PMCID: PMC3169368  PMID: 21860239
Antimicrobial; Antimicrobial peptide; Caries; Demineralization; Dental plaque; Lactic acid; Mouth rinse; Oral therapeutic; Selective antibiotic; Selective therapeutic; Specifically targeted antimicrobial peptide; Streptococcus mutans; Targeted antimicrobial
3.  Characterization of Recombinant, Ureolytic Streptococcus mutans Demonstrates an Inverse Relationship between Dental Plaque Ureolytic Capacity and Cariogenicity 
Infection and Immunity  2000;68(5):2621-2629.
Dental caries results from prolonged plaque acidification that leads to the establishment of a cariogenic microflora and demineralization of the tooth. Urease enzymes of oral bacteria hydrolyze urea to ammonia, which can neutralize plaque acids. To begin to examine the relationship between plaque ureolytic activity and the incidence of dental caries, recombinant, ureolytic strains of Streptococcus mutans were constructed. Specifically, the ureABCEFGD operon from Streptococcus salivarius 57.I was integrated into the S. mutans chromosome in such a way that the operon was transcribed from a weak, cognate promoter in S. mutans ACUS4 or a stronger promoter in S. mutans ACUS6. Both strains expressed NiCl2-dependent urease activity, but the maximal urease levels in ACUS6 were threefold higher than those in ACUS4. In vitro pH drop experiments demonstrated that the ability of the recombinant S. mutans strains to moderate a decrease in pH during the simultaneous metabolism of glucose and urea increased proportionately with the level of urease activity expressed. Specific-pathogen-free rats that were infected with ACUS6 and fed a cariogenic diet with drinking water containing 25 mM urea and 50 μM NiCl2 had relatively high levels of oral urease activity, as well as dramatic decreases in the prevalence of smooth-surface caries and the severity of sulcal caries, relative to controls. Urease activity appears to influence plaque biochemistry and metabolism in a manner that reduces cariogenicity, suggesting that recombinant, ureolytic bacteria may be useful to promote dental health.
PMCID: PMC97468  PMID: 10768953
4.  Inhibitory effect of zingiber officinale towards Streptococcus mutans virulence and caries development: in vitro and in vivo studies 
BMC Microbiology  2015;15(1):1.
Background
Streptococcus mutans is known as a key causative agent of dental caries. It metabolizes dietary carbohydrate to produce acids which reduce the environmental pH leading to tooth demineralization. The ability of this bacterium to tolerate acids coupled with acid production, allows its effective colonization in the oral cavity leading to the establishment of highly cariogenic plaque. For this reason, S. mutans is the only bacterium found in significantly higher numbers than other bacteria in the dental plaque. The aim of this study was to evaluate the effect of crude extract and methanolic fraction of Z. officinale against S. mutans virulence properties.
Results
We investigated in vitro and in vivo activity of crude extract and methanolic fraction at sub- MIC levels against cariogenic properties of S. mutans. We found that these extracts strongly inhibited a variety of virulence properties which are critical for its pathogenesis. The biofilm formation in S. mutans was found to be reduced during critical growth phases. Furthermore, the glucan synthesis and adherence was also found to be inhibited. Nevertheless, the insoluble glucan synthesis and sucrose dependent adherence were apparently more reduced as compared to soluble glucan synthesis and sucrose- independent adherence. Biofilm architecture inspected with the help of confocal and scanning electron microscopy, showed dispersion of cells in the treated group as compared to the control. The Quantitative Real Time PCR (qRT-PCR) data had shown the down regulation of the virulence genes, which is believed to be one of the major reasons responsible for the observed reduction in the virulence properties. The incredible reduction of caries development was found in treated group of rats as compared to the untreated group which further validate our in vitro data.
Conclusion
The whole study concludes a prospective role of crude extract and methanolic fraction of Z. officinale in targeting complete array of cariogenic properties of S. mutans, thus reducing its pathogenesis. Hence, it may be strongly proposed as a putative anti- cariogenic agent.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0320-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12866-014-0320-5
PMCID: PMC4316655  PMID: 25591663
Streptococcus mutans; qRT- PCR; Biofilm; Dental caries; Gas chromatography-mass spectrometry; Glucosyltransferases; Dental plaque
5.  The Exopolysaccharide Matrix Modulates the Interaction between 3D Architecture and Virulence of a Mixed-Species Oral Biofilm 
PLoS Pathogens  2012;8(4):e1002623.
Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.
Author Summary
Virulent biofilms formed on surfaces are associated with many human infections. The disease dental caries, expressed as cavities, is a prime example of the consequences arising from interactions between bacteria and sugars on tooth-surfaces. When Streptococcus mutans metabolize sugars, they produce a glue-like polymer termed glucan, helping them to adhere firmly to teeth. Glucan is also formed on bacterial surfaces in the mouth, and will accumulate and enmesh additional microorganisms creating the gelatinous formation known as dental plaque-biofilm. We found unique islets of bacteria within these biofilms, particularly close to the tooth-surface, providing safe havens in which bacteria thrive and produce acids that erode teeth. One intriguing mystery is why acids accumulate on the tooth-surface when there is an abundance of neutral-pH saliva surrounding the teeth. We found that bacterial-islets are particularly protected by glucan, which retards neutralization. We noticed that, within biofilms, the interiors of these islets are acidic, where only acid-tolerant bacteria can prosper, ensuring continued localized acid production. Our study demonstrates that construction of biofilms mediated by glucans forms complex 3D architectures, creating a variety of acidic-microenvironments that are essential for virulence expression. These results may aid in the development of enhanced methods to modulate biofilm formation.
doi:10.1371/journal.ppat.1002623
PMCID: PMC3320608  PMID: 22496649
6.  Clinical Implications of Power Toothbrushing on Fluoride Delivery: Effects on Biofilm Plaque Metabolism and Physiology 
Dental biofilms are implicated in the formation of caries and periodontal disease. A major constituent of the supragingival biofilm is Streptococcus mutans, which produces lactic acid from sucrose fermentation, enhancing enamel demineralization and eventual caries development. Caries prevention through F inhibits enamel demineralization and promotes remineralization. Fluoride also exerts effects on metabolic activities in the supragingival biofilm such as aerobic respiration, acid fermentation and dentrification. In experimental S. mutans biofilms, adding 1000 ppm F to an acidogenic biofilm resulting from 10% sucrose addition increased pH to pre-sucrose levels, suggesting inhibition of acid fermentation. F effects on metabolic activity and sucrose utilization in interproximal plaque biofilms were also recorded. Addition of 10% sucrose reduced pH from neutral to 4.2, but subsequent addition of 1000 ppm F increased pH by 1 unit, inhibiting acid fermentation. 10% Sucrose addition also stimulated denitrification, increasing production of nitrous oxide (N2O). Addition of 1000 ppm F suppressed denitrification, indicating an additional mechanism by which F exerts effects in the active interproximal biofilm. Finally, fluid dynamic activity by power tooth brushing enhanced F delivery and retention in an experimental S. mutans biofilm, suggesting a potential novel benefit for this intervention beyond mechanical plaque removal.
doi:10.1155/2010/651869
PMCID: PMC2855952  PMID: 20414341
7.  Effects of pH and salinity on the antimicrobial properties of clavanins. 
Infection and Immunity  1997;65(7):2898-2903.
Clavanins are histidine-rich, amidated alpha-helical antimicrobial peptides that were originally isolated from the leukocytes (hemocytes) of a tunicate, Styela clava. The activities of clavanin A amide and clavanin A acid against Escherichia coli, Listeria monocytogenes, and Candida albicans were substantially greater at pH 5.5 than at pH 7.4. In contrast, clavanin AK, a synthetic variant of clavanin A acid containing 4 histidine-->lysine substitutions exerted substantial activity at both pH 7.4 and pH 5.5. Each of these three clavanins permeabilized the outer and inner membranes of E. coli very effectively at pH 5.5, but only clavanin AK did so at pH 7.4. Unlike magainin 1 and cecropin P1, alpha-helical antimicrobial peptides from frog skin and porcine intestine, respectively, clavanins were broadly effective against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, as well as gram-negative organisms. Because clavanins exert substantial antimicrobial activity in 0.1 to 0.3 M NaCl, they provide templates for designing broad-spectrum peptide antibiotics intended to function in extracellular environments containing normal or elevated NaCl concentrations. The pH-dependent properties of histidine-rich antimicrobial peptides may allow the design of agents that would function selectively in acidic compartments, such as the gastric lumen, or within phagolysosomes.
PMCID: PMC175407  PMID: 9199465
8.  Lab-Test® 4: Dental caries and bacteriological analysis 
Dental Research Journal  2012;9(Suppl 2):S139-S141.
Dental caries is one of the most common infectious ultifactorial diseases worldwide, characterized by the progressive demineralization of the tooth, following the action of bacterial acid metabolism. The main factors predisposing the onset of the carious process are: 1) the presence of bacterial species able to lower the pH until critical values of 5.5, 2) the absence of adequate oral hygiene, 3) an inefficient immune response anti-caries, 4) the type of alimentary diet and 5) the structure of the teeth. Among the 200 bacterial species isolated from dental plaque the most pathogenic for dental caries are: Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Actinomices viscusus and Bifidobacterium dentium. Our laboratory (LAB® s.r.l., Codigoro, Ferrara, Italy) has developed a test for absolute and relative quantification of the most common oral cariogenic bacteria. The test uses specific primers and probes for the amplification of bacteria genome sequences in Polymerase Chain Reaction Real Time. The results provide a profile of patient infection, helpful for improving the diagnosis and planning of preventive treatment to reduce the bacterial load.
doi:10.4103/1735-3327.109723
PMCID: PMC3692161  PMID: 23814571
Bacteria; dental caries; real-time polymerase chain reaction
9.  Development and evaluation of a safe and effective sugar-free herbal lollipop that kills cavity-causing bacteria 
Dental caries (tooth decay) is caused by a specific group of cariogenic bacteria, like Streptococcus mutans, which convert dietary sugars into acids that dissolve the mineral in tooth structure. Killing cariogenic bacteria is an effective way to control or prevent tooth decay. In a previous study, we discovered a novel compound (Glycyrrhizol A), from the extraction of licorice roots, with strong antimicrobial activity against cariogenic bacteria. In the current study, we developed a method to produce these specific herbal extracts in large quantities, and then used these extracts to develop a sugar-free lollipop that effectively kills cariogenic bacteria like Streptococcus mutans. Further studies showed that these sugar-free lollipops are safe and their antimicrobial activity is stable. Two pilot human studies indicate that a brief application of these lollipops (twice a day for ten days) led to a marked reduction of cariogenic bacteria in oral cavity among most human subjects tested. This herbal lollipop could be a novel tool to promote oral health through functional foods.
doi:10.4248/IJOS11005
PMCID: PMC3469870  PMID: 21449211
antimicrobial therapy; licorice; Streptococcus mutans
10.  Hyperosmotic response of streptococcus mutans: from microscopic physiology to transcriptomic profile 
BMC Microbiology  2013;13:275.
Background
Oral streptococci metabolize carbohydrate to produce organic acids, which not only decrease the environmental pH, but also increase osmolality of dental plaque fluid due to tooth demineralization and consequent calcium and phosphate accumulation. Despite these unfavorable environmental changes, the bacteria continue to thrive. The aim of this study was to obtain a global view on strategies taken by Streptococcus mutans to deal with physiologically relevant elevated osmolality, and perseveres within a cariogenic dental plaque.
Results
We investigated phenotypic change of S. mutans biofilm upon hyperosmotic challenge. We found that the hyperosmotic condition was able to initiate S. mutans biofilm dispersal by reducing both microbial content and extracellular polysaccharides matrix. We then used whole-genome microarray with quantitative RT-PCR validation to systemically investigate the underlying molecular machineries of this bacterium in response to the hyperosmotic stimuli. Among those identified 40 deferentially regulated genes, down-regulation of gtfB and comC were believed to be responsible for the observed biofilm dispersal. Further analysis of microarray data showed significant up-regulation of genes and pathways involved in carbohydrate metabolism. Specific genes involved in heat shock response and acid tolerance were also upregulated, indicating potential cross-talk between hyperosmotic and other environmental stress.
Conclusions
Hyperosmotic condition induces significant stress response on S. mutans at both phenotypic and transcriptomic levels. In the meantime, it may take full advantage of these environmental stimuli to better fit the fluctuating environments within oral cavity, and thus emerges as numeric-predominant bacterium under cariogenic conditions.
doi:10.1186/1471-2180-13-275
PMCID: PMC4219374  PMID: 24289739
Streptococcus mutans; Hyperosmotic condition; Transcriptional profile; Biofilm dispersal; Environmental fitness; Dental plaque
11.  Streptococcus mutans strains recovered from caries-active or caries-free individuals differ in sensitivity to host anti-microbial peptides 
Molecular oral microbiology  2011;26(3):187-199.
SUMMARY
Antimicrobial peptides (AMPs) are among the repertoire of host innate immune defenses. In the oral cavity, several AMPs are present in saliva and have antimicrobial activities against oral bacteria, including Streptococcus mutans, a primary etiologic agent of dental caries. In this study, we hypothesized that unique S. mutans strains as determined by DNA fingerprinting from sixty 13 year-old subjects with or without caries experience would have different susceptibilities to α-defensins-1-3 (HNP-1-3), β-defensins-2-3 (HBD-2-3) and LL-37. The salivary levels of these peptides in subjects also were measured by enzyme-linked immunosorbent assays (ELISA). We found that S. mutans strains from caries-active subjects showed greater resistance to salivary HNP-1-2, HBD-2-3 and LL-37 at varying concentrations than those from caries-free subjects. In addition, combinations of these peptides increased their antimicrobial activity against S. mutans either additively or synergistically. The salivary levels of these peptides were highly variable among subjects with no correlation to host caries experience. However, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. Our findings suggest that the relative ability of S. mutans to resist host salivary AMPs may be considered a potential virulence factor for this species such that S. mutans strains that are more resistant to these peptides may have an ecological advantage to preferentially colonize within dental plaque and increase the risk of dental caries.
doi:10.1111/j.2041-1014.2011.00607.x
PMCID: PMC3092152  PMID: 21545696
Dental caries; defensins; S. mutans; innate immunity; saliva
12.  Metatranscriptomics reveals overall active bacterial composition in caries lesions 
Journal of Oral Microbiology  2014;6:10.3402/jom.v6.25443.
Background
Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms.
Objective
To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay.
Design
Non-cavitated enamel caries lesions (n=15) and dentin caries lesions samples (n=12) were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition.
Results
A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.02–0.73% of the total bacterial community.
Conclusions
The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci detected confirms that they are a minority and questions its importance as the main etiological agent of tooth decay. Future experimental work should be performed to confirm the cariogenicity of the identified bacteria.
doi:10.3402/jom.v6.25443
PMCID: PMC4247497  PMID: 25626770
16S rRNA; pyrosequencing; Streptococcus mutans; polymicrobial disease; tissue-dependent hypothesis; caries etiology
13.  Antibacterial and physical properties of calcium-phosphate and calcium-fluoride nanocomposites with chlorhexidine 
Dental Materials  2012;28(5):573-583.
Objectives
Previous studies have developed calcium phosphate and fluoride releasing composites. Other studies have incorporated chlorhexidine (CHX) particles into dental composites. However, CHX has not been incorporated in calcium phosphate and fluoride composites. The objectives of this study were to develop nanocomposites containing amorphous calcium phosphate (ACP) or calcium fluoride (CaF2) nanoparticles and CHX particles, and investigate S. mutans biofilm formation and lactic acid production for the first time.
Methods
Chlorhexidine was frozen via liquid nitrogen and ground to obtain a particle size of 0.62 µm. Four nanocomposites were fabricated with fillers of: Nano ACP; nano ACP+10% CHX; nano CaF2; nano CaF2+10% CHX. Three commercial materials were tested as controls: A resin-modified glass ionomer, and two composites. S. mutans live/dead assay, colony-forming unit (CFU) counts, biofilm metabolic activity, and lactic acid were measured.
Results
Adding CHX fillers to ACP and CaF2 nanocomposites greatly increased their antimicrobial capability. ACP and CaF2 nanocomposites with CHX that were inoculated with S. mutans had a growth medium pH > 6.5 after 3 d, while the control commercial composites had a cariogenic pH of 4.2. Nanocomposites with CHX reduced the biofilm metabolic activity by 10–20 folds and reduced the acid production, compared to the controls. CFU on nanocomposites with CHX were three orders of magnitude less than that on commercial composite. Mechanical properties of nanocomposites with CHX matched a commercial composite without fluoride.
Significance
The novel calcium phosphate and fluoride nanocomposites could be rendered antibacterial with CHX to greatly reduce biofilm formation, acid production, CFU and metabolic activity. The antimicrobial and remineralizing nanocomposites with good mechanical properties may be promising for a wide range of tooth restorations with anti-caries capabilities.
doi:10.1016/j.dental.2012.01.006
PMCID: PMC3322264  PMID: 22317794
dental nanocomposite; calcium phosphate; calcium fluoride; chlorhexidine; stress-bearing; S. mutans biofilm; caries inhibition
14.  Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿  
Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents.
doi:10.1128/AAC.01270-06
PMCID: PMC1855471  PMID: 17296741
15.  Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis 
PLoS ONE  2012;7(10):e47722.
Dental caries in very young children may be severe, result in serious infection, and require general anesthesia for treatment. Dental caries results from a shift within the biofilm community specific to the tooth surface, and acidogenic species are responsible for caries. Streptococcus mutans, the most common acid producer in caries, is not always present and occurs as part of a complex microbial community. Understanding the degree to which multiple acidogenic species provide functional redundancy and resilience to caries-associated communities will be important for developing biologic interventions. In addition, microbial community interactions in health and caries pathogenesis are not well understood. The purpose of this study was to investigate bacterial community profiles associated with the onset of caries in the primary dentition. In a combination cross-sectional and longitudinal design, bacterial community profiles at progressive stages of caries and over time were examined and compared to those of health. 16S rRNA gene sequencing was used for bacterial community analysis. Streptococcus mutans was the dominant species in many, but not all, subjects with caries. Elevated levels of S. salivarius, S. sobrinus, and S. parasanguinis were also associated with caries, especially in subjects with no or low levels of S. mutans, suggesting these species are alternative pathogens, and that multiple species may need to be targeted for interventions. Veillonella, which metabolizes lactate, was associated with caries and was highly correlated with total acid producing species. Among children without previous history of caries, Veillonella, but not S. mutans or other acid-producing species, predicted future caries. Bacterial community diversity was reduced in caries as compared to health, as many species appeared to occur at lower levels or be lost as caries advanced, including the Streptococcus mitis group, Neisseria, and Streptococcus sanguinis. This may have implications for bacterial community resilience and the restoration of oral health.
doi:10.1371/journal.pone.0047722
PMCID: PMC3472979  PMID: 23091642
16.  A Lactotransferrin Single Nucleotide Polymorphism Demonstrates Biological Activity That Can Reduce Susceptibility to Caries 
Infection and Immunity  2013;81(5):1596-1605.
Streptococcus mutans is prominently linked to dental caries. Saliva's influence on caries is incompletely understood. Our goal was to identify a salivary protein with anti-S. mutans activity, characterize its genotype, and determine genotypic variants associated with S. mutans activity and reduced caries. An S. mutans affinity column was used to isolate active moieties from saliva obtained from a subject with minimal caries. The bound and eluted protein was identified as lactotransferrin (LTF) by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis and confirmed by Western blotting with LTF antibody. A single nucleotide polymorphism (SNP) that produced a shift from arginine (R) to lysine (K) at amino acid position 47 in the LTF antimicrobial region (rs: 1126478) killed S. mutans in vitro. Saliva from a subject with moderate caries and with the LTF “wild-type” R form at position 47 had no such activity. A pilot genetic study (n = 30) showed that KK subjects were more likely to have anti-S. mutans activity than RR subjects (P = 0.001; relative risk = 3.6; 95% confidence interval [95% CI] = 1.5 to 11.13). Pretreatment of KK saliva with antibody to LTF reduced S. mutans killing in a dose-dependent manner (P = 0.02). KK subjects were less likely to have caries (P = 0.02). A synthetic 11-mer LTF/K peptide killed S. mutans and other caries-related bacteria, while the LTF/R peptide had no effect (P = 0.01). Our results provide functional evidence that the LTF/K variant results in both anti-S. mutans activity and reduced decay. We suggest that the LTF/K variant can influence oral microbial ecology in general and caries-provoking microbes specifically.
doi:10.1128/IAI.01063-12
PMCID: PMC3647982  PMID: 23460521
17.  Protective potential of casein phosphopeptide amorphous calcium phosphate containing paste on enamel surfaces 
Background:
Dental caries remains the most common dental disease facing mankind. Prevention of initiation and interruption in progression of early lesions are the desirable modes of caries management. There is a scope for agents, which may be used to enhance anti - caries activity. This need has redirected research to develop novel preventive agents that can act as an adjunct to fluoride or independent of it. Casein Phosphopeptide – Amorphous Calcium Phosphate (CPP-ACP) is one such agent that has been proposed to have anti cariogenic properties.
Aim:
The purpose of this in vitro study was to evaluate the effect of paste containing CPP-ACP, MI Paste, on enamel remineralization.
Materials and Methods:
This study consisted of 30 samples embedded in orthodontic resin with either the buccal or lingual surface exposed. The samples were assigned to either a CPP-ACP containing paste; Fluoridated toothpaste; or a control group. The groups were then subjected to cycling in a demineralizing solution and a remineralizing solution. Groups II and III received prior application of MI paste and Fluoridated toothpaste respectively followed by cycling in a demineralizing solution and a remineralizing solution. Following 14 days of cycling, the samples were sectioned and examined using confocal microscopy. The lesion depth, were evaluated.
Statistical Analysis:
Image Proplus software was used to analyze the images. The values were statistically evaluated using one – way ANOVA and Scheffe's Test.
Results and Conclusion:
Within the limitations of the study it was concluded that enamel surfaces treated with the CPP-ACP paste exhibited the least lesion depths followed by the enamel surfaces treated with the fluoridated tooth paste and control group respectively.
doi:10.4103/0972-0707.108199
PMCID: PMC3659862  PMID: 23716969
Caries; casein phosphopeptide-amorphous calcium phosphate; fluoride
18.  Effects of mushroom and chicory extracts on the shape, physiology and proteome of the cariogenic bacterium Streptococcus mutans 
Background
Dental caries is an infectious disease which results from the acidic demineralisation of the tooth enamel and dentine as a consequence of the dental plaque (a microbial biofilm) accumulation. Research showed that several foods contain some components with antibacterial and antiplaque activity. Previous studies indicated antimicrobial and antiplaque activities in a low-molecular-mass (LMM) fraction of extracts from either an edible mushroom (Lentinus edodes) or from Italian red chicory (Cichorium intybus).
Methods
We have evaluated the antimicrobial mode of action of these fractions on Streptococcus mutans, the etiological agent of human dental caries. The effects on shape, macromolecular syntheses and cell proteome were analysed.
Results
The best antimicrobial activity has been displayed by the LMM mushroom extract with a bacteriostatic effect. At the MIC of both extracts DNA synthesis was the main macromolecular synthesis inhibited, RNA synthesis was less inhibited than that of DNA and protein synthesis was inhibited only by roughly 50%. The partial inhibition of protein synthesis is compatible with the observed significant increase in cell mass. The increase in these parameters is linked to the morphological alteration with transition from cocci of the untreated control to elongated cells. Interestingly, these modifications were also observed at sub-MIC concentrations. Finally, membrane and cytosol proteome analysis was conducted under LMM mushroom extract treatment in comparison with untreated S. mutans cells. Significant changes were observed for 31 membrane proteins and 20 of the cytosol fractions. The possible role of the changed proteins is discussed.
Conclusions
This report has shown an antibiotic-like mode of action of mushroom and chicory extracts as demonstrated by induced morphogenetic effects and inhibition of specific macromolecular synthesis. This feature as well as the safe use of this extract as result of its natural origin render the LMM both mushroom and chicory extracts suitable for the formulation into products for daily oral hygiene such as mouthwashes or toothpastes.
doi:10.1186/1472-6882-13-117
PMCID: PMC3672068  PMID: 23714053
Streptococcus mutans; Dental caries; Anticaries compounds; Food components
19.  Elevated Incidence of Dental Caries in a Mouse Model of Cystic Fibrosis 
PLoS ONE  2011;6(1):e16549.
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.
doi:10.1371/journal.pone.0016549
PMCID: PMC3031584  PMID: 21304986
20.  Remineralization of Demineralized Enamel via Calcium Phosphate Nanocomposite 
Journal of Dental Research  2012;91(10):979-984.
Secondary caries remains the main problem limiting the longevity of composite restorations. The objective of this study was to investigate the remineralization of demineralized human enamel in vitro via a nanocomposite containing nanoparticles of amorphous calcium phosphate (NACP). NACP were synthesized by a spray-drying technique and incorporated into a dental resin. First, caries-like subsurface enamel lesions were created via an acidic solution. Then, NACP nanocomposite or a commercial fluoride-releasing control composite was placed on the demineralized enamel, along with control enamel without a composite. These specimens were then treated with a cyclic demineralization/remineralization regimen for 30 days. Quantitative microradiography showed typical enamel subsurface demineralization before cyclic demineralization/remineralization treatment, and significant remineralization in enamel under the NACP nanocomposite after the demineralization/remineralization treatment. The NACP nanocomposite had the highest enamel remineralization (mean ± SD; n = 6) of 21.8 ± 3.7%, significantly higher than the 5.7 ± 6.9% for fluoride-releasing composite (p < 0.05). The enamel group without composite had further demineralization of −26.1 ± 16.2%. In conclusion, a novel NACP nanocomposite was effective in remineralizing enamel lesions in vitro. Its enamel remineralization was 4-fold that of a fluoride-releasing composite control. Combined with the good mechanical and acid-neutralization properties reported earlier, the new NACP nanocomposite is promising for remineralization of demineralized tooth structures.
doi:10.1177/0022034512458288
PMCID: PMC3446834  PMID: 22933607
dental nanocomposite; calcium phosphate nanoparticles; human enamel; lesion remineralization; contact microradiography; caries inhibition
21.  Dental plaque microcosm biofilm behavior on calcium phosphate nanocomposite with quaternary ammonium 
Dental Materials  2012;28(8):853-862.
Objectives
Half of dental restorations fail in 10 years, with secondary caries as the main reason. Calcium phosphate composites could remineralize tooth lesions. The objectives of this study were to: (1) Impart antibacterial activity to a composite with nanoparticles of amorphous calcium phosphate (NACP); and (2) investigate the effect of quaternary ammonium dimethacrylate (QADM) on mechanical and dental plaque microcosm biofilm properties for the first time.
Methods
The NACP and glass particles were filled into a dental resin that contained bis(2-methacryloyloxy-ethyl) dimethyl-ammonium bromide, the QADM. NACP nanocomposites containing 0%, 7%, 14%, and 17.5% of QADM by mass, respectively, were photo-cured. A commercial composite with no antibacterial activity was used as control. Mechanical properties were measured in three-point flexure. A human saliva microcosm model was used to grow biofilms on composites. Live/dead assay, metabolic activity, colony-forming unit (CFU) counts, and lactic acid production of biofilms on the composites were measured.
Results
Increasing QADM mass fraction monotonically reduced the biofilm viability, CFU and lactic acid. Biofilms on NACP nanocomposite with 17.5% QADM had metabolic activity that was 30% that on a commercial composite control (p<0.05). Total microorganisms, total streptococci, and mutans streptococci CFU counts (mean±sd; n=6) on composite control was 6-fold those on NACP+17.5% QADM nanocomposite. Composite control had long strings of cells with normal short-rod shapes, while some cells on NACP-QADM nanocomposites disintegrated into pieces. Adding QADM to NACP did not decrease the strength and elastic modulus, which matched (p>0.1) those of a commercial composite without Ca-PO4 or antibacterial activity.
Significance
A dental plaque microcosm model was used to evaluate the novel NACP-QADM nanocomposite. The nanocomposite greatly reduced the biofilm viability, metabolic activity and lactic acid, while its mechanical properties matched those of a commercial composite. NACP-QADM nanocomposite with calcium phosphate fillers, good mechanical properties and a strong antibacterial activity may have potential for anti-biofilm and anti-caries restorations.
doi:10.1016/j.dental.2012.04.024
PMCID: PMC3393817  PMID: 22578992
Antibacterial nanocomposite; amorphous calcium phosphate nanoparticles; quaternary ammonium; dental plaque microcosm biofilm; stress-bearing; dental caries
22.  The oral metagenome in health and disease 
The ISME Journal  2011;6(1):46-56.
The oral cavity of humans is inhabited by hundreds of bacterial species and some of them have a key role in the development of oral diseases, mainly dental caries and periodontitis. We describe for the first time the metagenome of the human oral cavity under health and diseased conditions, with a focus on supragingival dental plaque and cavities. Direct pyrosequencing of eight samples with different oral-health status produced 1 Gbp of sequence without the biases imposed by PCR or cloning. These data show that cavities are not dominated by Streptococcus mutans (the species originally identified as the ethiological agent of dental caries) but are in fact a complex community formed by tens of bacterial species, in agreement with the view that caries is a polymicrobial disease. The analysis of the reads indicated that the oral cavity is functionally a different environment from the gut, with many functional categories enriched in one of the two environments and depleted in the other. Individuals who had never suffered from dental caries showed an over-representation of several functional categories, like genes for antimicrobial peptides and quorum sensing. In addition, they did not have mutans streptococci but displayed high recruitment of other species. Several isolates belonging to these dominant bacteria in healthy individuals were cultured and shown to inhibit the growth of cariogenic bacteria, suggesting the use of these commensal bacterial strains as probiotics to promote oral health and prevent dental caries.
doi:10.1038/ismej.2011.85
PMCID: PMC3246241  PMID: 21716308
metagenomics; human microbiome; dental caries; Streptococcus mutans; pyrosequencing; probiotics
23.  Activity of an Antimicrobial Peptide Mimetic against Planktonic and Biofilm Cultures of Oral Pathogens▿ †  
Antimicrobial Agents and Chemotherapy  2007;51(11):4125-4132.
Antimicrobial peptides (AMPs) are naturally occurring, broad-spectrum antimicrobial agents that have recently been examined for their utility as therapeutic antibiotics. Unfortunately, they are expensive to produce and are often sensitive to protease digestion. To address this problem, we have examined the activity of a peptide mimetic whose design was based on the structure of magainin, exhibiting its amphiphilic structure. We demonstrate that this compound, meta-phenylene ethynylene (mPE), exhibits antimicrobial activity at nanomolar concentrations against a variety of bacterial and Candida species found in oral infections. Since Streptococcus mutans, an etiological agent of dental caries, colonizes the tooth surface and forms a biofilm, we quantified the activity of this compound against S. mutans growing under conditions that favor biofilm formation. Our results indicate that mPE can prevent the formation of a biofilm at nanomolar concentrations. Incubation with 5 nM mPE prevents further growth of the biofilm, and 100 nM mPE reduces viable bacteria in the biofilm by 3 logs. Structure-function analyses suggest that mPE inhibits the bioactivity of lipopolysaccharide and binds DNA at equimolar ratios, suggesting that it may act both as a membrane-active molecule, similar to magainin, and as an intracellular antibiotic, similar to other AMPs. We conclude that mPE and similar molecules display great potential for development as therapeutic antimicrobials.
doi:10.1128/AAC.00208-07
PMCID: PMC2151458  PMID: 17785509
24.  Recovery of Crystallographic Texture in Remineralized Dental Enamel 
PLoS ONE  2014;9(10):e108879.
Dental caries is the most prevalent disease encountered by people of all ages around the world. Chemical changes occurring in the oral environment during the caries process alter the crystallography and microstructure of dental enamel resulting in loss of mechanical function. Little is known about the crystallographic effects of demineralization and remineralization. The motivation for this study was to develop understanding of the caries process at the crystallographic level in order to contribute towards a long term solution. In this study synchrotron X-ray diffraction combined with scanning electron microscopy and scanning microradiography have been used to correlate enamel crystallography, microstructure and mineral concentration respectively in enamel affected by natural caries and following artificial demineralization and remineralization regimes. In particular, the extent of destruction and re-formation of this complex structure has been measured. 2D diffraction patterns collected at the European Synchrotron Radiation Facility were used to quantify changes in the preferred orientation (crystallographic texture) and position of the (002) Bragg reflection within selected regions of interest in each tooth slice, and then correlated with the microstructure and local mineral mass. The results revealed that caries and artificial demineralization cause a large reduction in crystallographic texture which is coupled with the loss of mineral mass. Remineralization restores the texture to the original level seen in healthy enamel and restores mineral density. The results also showed that remineralization promotes ordered formation of new crystallites and growth of pre-existing crystallites which match the preferred orientation of healthy enamel. Combining microstructural and crystallographic characterization aids the understanding of caries and erosion processes and assists in the progress towards developing therapeutic treatments to allow affected enamel to regain structural integrity.
doi:10.1371/journal.pone.0108879
PMCID: PMC4215832  PMID: 25360532
25.  Dental plaque as a biofilm and a microbial community – implications for health and disease 
BMC Oral Health  2006;6(Suppl 1):S14.
Dental plaque is a structurally- and functionally-organized biofilm. Plaque forms in an ordered way and has a diverse microbial composition that, in health, remains relatively stable over time (microbial homeostasis). The predominant species from diseased sites are different from those found in healthy sites, although the putative pathogens can often be detected in low numbers at normal sites. In dental caries, there is a shift toward community dominance by acidogenic and acid-tolerating species such as mutans streptococci and lactobacilli, although other species with relevant traits may be involved. Strategies to control caries could include inhibition of biofilm development (e.g. prevention of attachment of cariogenic bacteria, manipulation of cell signaling mechanisms, delivery of effective antimicrobials, etc.), or enhancement of the host defenses. Additionally, these more conventional approaches could be augmented by interference with the factors that enable the cariogenic bacteria to escape from the normal homeostatic mechanisms that restrict their growth in plaque and out compete the organisms associated with health. Evidence suggests that regular conditions of low pH in plaque select for mutans streptococci and lactobacilli. Therefore, the suppression of sugar catabolism and acid production by the use of metabolic inhibitors and non-fermentable artificial sweeteners in snacks, or the stimulation of saliva flow, could assist in the maintenance of homeostasis in plaque. Arguments will be presented that an appreciation of ecological principles will enable a more holistic approach to be taken in caries control.
doi:10.1186/1472-6831-6-S1-S14
PMCID: PMC2147593  PMID: 16934115

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