Human Caries Research Articles

Antibacterial Activity of the Essential Oil from Rosmarinus offi cinalis and its Major Components against Oral Pathogens

The essential oil of Rosmarinus officinalis L. (rosemary) was obtained by hydro-distillation and analysed by gas chromatography-mass spectrometry. Sixty-two constituents were identified, representing 98.06% of the total oil content. Oxygenated monoterpenes were the predominant components. The rosemary oil was characterized as having prominent (> 5%) contents of camphor (18.9%), verbenone (11.3%), a-pinene (9.6%), beta-myrcene (8.6%), 1,8-cineole (8.0%), and beta-caryophyllene (5.1%). The antimicrobial activity of the oil as well as of its major constituents was tested against the following microorganisms: Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus sobrinus, and Enterococcus faecalis, which are potentially responsible for the formation of dental caries in humans. The microdilution method was used for determination of the minimum inhibitory concentration (MIC) during evaluation of the antibacterial activity. The essential oil displayed low activity against the selected microorganisms. In the present study, the pure major compounds were more active than the essential oil. Among all the microorganisms tested, the pathogen S. mitis was the most susceptible and E. faecalis was the most resistant to the evaluated samples. This is the first report on antimicrobial activity of the major components of rosemary oil against oral pathogens.

Characterization of the Streptococcus sobrinus acid-stress response by interspecies microarrays and proteomics

Streptococcus mutans and Streptococcus sobrinus are considered the primary organisms responsible for human dental caries. The ability to generate acids and to adapt to low pH conditions is directly associated with the cariogenic potential of these bacteria. To survive acidic conditions, both species have been shown to mount an acid-tolerance response (ATR). However, previous characterization of the S.sobrinus ATR identified critical differences in the mechanisms of acid adaptation between S.mutans and S.sobrinus. Here, interspecies microarray and proteomic approaches were used to identify novel, previously unrecognized genes and pathways that participate in the S.sobrinus acid-stress response. The results revealed that, among other things, metabolic alterations that enhance energy generation and upregulation of the malolactic fermentation enzyme activity constitute important acid-resistance properties in S.sobrinus. Some of these acid adaptive traits are shared by S.mutans and might be considered optimal targets for therapeutic treatments designed to control dental caries.

Users’ perceptions and efficacy of indigenous adjunct teeth-cleansing agents on the bacterial flora of human dental caries

Background and Aim: The aim of this study was to conduct in vitro and in vivo studies for the evaluation of antimicrobial potential of four Nigerian indigenous chewing sticks and to compare the oral effects of the popular Nigerian chewing sticks with toothpastes and other teeth-cleansing agents on a total of 21 and 17 oral bacterial strains isolated from children and adult dental caries respectively. Materials and Methods: Teeth cleansing agents were assayed in vitro against the isolated bacterial carries flora on de Man Rogosa and Sharpe (MRS) agar using modified agar well-diffusion methods. The simulated in vivo studies were carried out using twenty four human subjects. Results: The results obtained indicated that the bacterial flora exhibited different degrees of in vitro inhibitions of between low and moderate susceptibility against the teeth-cleansing agents. Toothpastes recorded the highest rates of inhibition against the dental caries isolates, while H2O2 was the most inhibitory among the chemical teethcleansing agents against the dental caries isolates from children (47.6%) and adults (52.9%). No species differential susceptibility to the teeth-cleansing agents was observed among the dental caries isolates. Conclusions: This study showed that combined teeth cleansing hygiene with toothpastes, chewing sticks and chemical teeth cleansing agent (H2O2) gave the best mouth feel and were found to be the most preferred.Keywords: Chewing sticks, dental caries, indigenous, oral health and hygiene, teeth-cleansing agents.

Crystal Structure of the Apo Form of D-Alanine:D-Alanine Ligase (DDl) from Streptococcus mutans

D-Alanine:D-Alanine ligase (DDl) catalyzes the formation of D-Alanine:D-Alanine dipeptide and is an essential enzyme in bacterial cell wall biosynthesis.. This enzyme does not have a human ortholog, making it an attractive target for developing new antibiotic drugs. We determined the crystal structure at 2.23 A resolution of DDl from Streptococcus mutans (SmDDl), the principal aetiological agent of human dental caries. This structure reveals that SmDDl is a dimer and has a disordered omega-loop region.

The Delta Subunit of RNA Polymerase, RpoE, Is a Global Modulator ofStreptococcus mutansEnvironmental Adaptation

The delta subunit of RNA polymerase, RpoE, is widespread in low-G+C Gram-positive bacteria and is thought to play a role in enhancing transcriptional specificity by blocking RNA polymerase binding at weak promoter sites and stimulating RNA synthesis by accelerating core enzyme recycling. Despite the well-studied biochemical properties of RpoE, a role for this protein in vivo has not been defined in depth. In this study, we show that inactivation of rpoE in the human dental caries pathogen Streptococcus mutans causes impaired growth and loss of important virulence traits, including biofilm formation, resistance to antibiotics, and tolerance to environmental stresses. Complementation of the mutant with rpoE expressed in trans restored its phenotype to wild type. The luciferase fusion reporter showed that rpoE was highly transcribed throughout growth and that acid and hydrogen peroxide stresses repressed rpoE expression. Transcriptome profiling of wild-type and ΔrpoE cells in the exponential and early stationary phase of growth, under acid and hydrogen peroxide stress and under both stresses combined, revealed that genes involved in histidine synthesis, malolactic fermentation, biofilm formation, and antibiotic resistance were downregulated in the ΔrpoE mutant under all conditions. Moreover, the loss of RpoE resulted in dramatic changes in transport and metabolism of carbohydrates and amino acids. Interestingly, differential expression, mostly upregulation, of 330 noncoding regions was found. In conclusion, this study demonstrates that RpoE is an important global modulator of gene expression in S. mutans which is required for optimal growth and environmental adaptation.

Damage of Streptococcus mutans biofilms by carolacton, a secondary metabolite from the myxobacterium Sorangium cellulosum

BackgroundStreptococcus 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.ResultsHere we show that carolacton, a secondary metabolite isolated from Sorangium cellulosum, has high antibacterial activity against biofilms of S. mutans. Planktonic growth of bacteria was only slightly impaired and no acute cytotoxicity against mouse fibroblasts could be observed. Carolacton caused death of S. mutans biofilm cells, elongation of cell chains, and changes in cell morphology. At a concentration of 10 nM carolacton, biofilm damage was already at 35% under anaerobic conditions. A knock-out mutant for comD, encoding a histidine kinase specific for the competence stimulating peptide (CSP), was slightly less sensitive to carolacton than the wildtype. Expression of the competence related alternate sigma factor ComX was strongly reduced by carolacton, as determined by a pcomX luciferase reporter strain.ConclusionsCarolacton possibly interferes with the density dependent signalling systems in S. mutans and may represent a novel approach for the prevention of dental caries.

Alterations in immunodominance of the Streptococcus mutans P1 adhesin: lessons learned from immunomodulatory monoclonal antibodies (38.4)

Abstract S. mutans is the bacterial agent of human dental caries. P1 (aka AgI/II, PAc) is a surface localized multifunctional protein adhesin that has been studied as an anti-caries vaccine antigen. Our laboratory has identified several anti-P1 MAbs that redirect the host immune response via an Fc-independent mechanism toward one of improved inhibition of bacterial adherence when administered as part of an immune complex (IC) with bacterial cells in BALB/c mice. A common feature of our immunomodulatory MAbs is a requirement for certain intramolecular interactions between discontinuous segments of P1 for their epitope formation. Binding of P1 by these MAbs appears to cause a structural alteration of the cell surface adhesin exposing normally cryptic and protective epitopes. We now utilize information gained by evaluating changes in serum Abs after immunization with IC to design and evaluate structural variants of the P1 protein itself as more effective immunogens. Also, an anti-AgI/II MAb previously reported as a passive immunotherapy in clinical trials was evaluated and shown to be immunomodulatory. Results varied with MAb concentration, thus providing an explanation of long-term protection observed in human subjects and the difficulty in reproducing these results in all studies. Our results indicate a delicate balance exists between P1 structure and the availability of putative T helper cell epitopes, but suggests development of a protein-based vaccine is achievable. (NIH DE13882)

Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans

Previously we reported a novel strategy of "targeted killing" through the design of narrow-spectrum molecules known as specifically targeted antimicrobial peptides (STAMPs) (R. Eckert et al., Antimicrob. Agents Chemother. 50:3651-3657, 2006; R. Eckert et al., Antimicrob. Agents Chemother. 50:1480-1488, 2006). Construction of these molecules requires the identification and the subsequent utilization of two conjoined yet functionally independent peptide components: the targeting and killing regions. In this study, we sought to design and synthesize a large number of STAMPs targeting Streptococcus mutans, the primary etiologic agent of human dental caries, in order to identify candidate peptides with increased killing speed and selectivity compared with their unmodified precursor antimicrobial peptides (AMPs). We hypothesized that a combinatorial approach, utilizing a set number of AMP, targeting, and linker regions, would be an effective method for the identification of STAMPs with the desired level of activity. STAMPs composed of the Sm6 S. mutans binding peptide and the PL-135 AMP displayed selectivity at MICs after incubation for 18 to 24 h. A STAMP where PL-135 was replaced by the B-33 killing domain exhibited both selectivity and rapid killing within 1 min of exposure and displayed activity against multispecies biofilms grown in the presence of saliva. These results suggest that potent and selective STAMP molecules can be designed and improved via a tunable "building-block" approach.

Production of novel bioactive compounds by enzymes, and their application to food

Recently, people have been paying greater attention to their health and, as a result, the need to use physiologically functional foods was found on the market. For these reasons, the market size of “foods for specified health use” (FOSHU) in Japan has grown and was approximately ¥700 billion in 2008. Many enzymes such as amylases and proteases have been used in food manufacturing because of their diversity, specificity, and mild condition in reaction. The aim of this investigation was the production of novel bioactive compounds by three kinds of transglycosylation reaction of the amylolytic enzymes, and the research and development of physiologically functional foods using these compounds. Phosphoryl oligosaccharides of calcium (POs-Ca) are a complex with Ca and phosphoryl oligosaccharides prepared from potato starch by a hydrolysis (transglycosylation to H2O) of amylolytic enzymes. The chewing gum included POs-Ca prevented dental caries in humans. Highly branched cyclic dextrin (HBCD) was produced from amylopectin by branching enzyme (intramolecular transglycosylation), which had a relatively narrow molecular-weight distribution compared with commercially available dextrins. The sports drink containing HBCD enhanced swimming endurance in mice and humans. α-Glycosylhesperidin (G-Hsp) was produced from starch and hesperidin, a flavonoid found abundantly in citrus fruits, by the intermolecular transglycosylation using cyclodextrin glucanotransferase. Oral administration of G-Hsp improved rheumatoid arthritis in mice and humans, and poor blood circulation in women. In this study, we looked to prove that this enzymatic modification technique was useful in creating unique and effective physiologically functional foods. These functional foods are expected to improve the health and quality of life of many people.

Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

BackgroundOne of the key virulence determinants of Streptococcus mutans, the primary etiological agent of human dental caries, is its strong acid tolerance. The acid tolerance response (ATR) of S. mutans comprises several mechanisms that are induced at low pH and allow the cells to quickly adapt to a lethal pH environment. Malolactic fermentation (MLF) converts L-malate to L-lactate and carbon dioxide and furthermore regenerates ATP, which is used to translocate protons across the membrane. Thus, MLF may contribute to the aciduricity of S. mutans but has not been associated with the ATR so far.ResultsHere we show that the malolactic fermentation (mle) genes are under the control of acid inducible promoters which are induced within the first 30 minutes upon acid shock in the absence of malate. Thus, MLF is part of the early acid tolerance response of S. mutans. However, acidic conditions, the presence of the regulator MleR and L-malate were required to achieve maximal expression of all genes, including mleR itself. Deletion of mleR resulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation.ConclusionThis study shows that the MLF gene cluster is part of the early acid tolerance response in S. mutans and is induced by both low pH and L-malate.

ClpP of Streptococcus mutans Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance

Streptococcus mutans is the primary etiological agent of human dental caries and, at times, of infective endocarditis. Within the oral cavity, the pathogen is subjected to conditions of stress. A well-conserved protein complex named ClpP (caseinolytic protease) plays a vital role in adaptation under stress conditions. To gain a better understanding of the global role of the ClpP protease in cellular homeostasis, a transcriptome analysis was performed using a DeltaclpP mutant strain. The expression levels of more than 100 genes were up- or downregulated in the DeltaclpP mutant compared to the wild type. Notably, the expression of genes in several genomic islands, such as TnSmu1 and TnSmu2, was differentially modulated in the DeltaclpP mutant strain. ClpP deficiency also increased the expression of genes associated with a putative CRISPR locus. Furthermore, several stress-related genes and genes encoding bacteriocin-related peptides and many transcription factors were also found to be altered in the DeltaclpP mutant strain. A comparative analysis of the two-dimensional protein profile of the wild type and the DeltaclpP mutant strains showed altered protein profiles. Comparison of the transcriptome data with the proteomic data identified four common gene products, suggesting that the observed altered protein expression of these genes could be due to altered transcription. The results presented here indicate that ClpP-mediated proteolysis plays an important global role in the regulation of several important traits in this pathogen.

Genome-Wide Characterization of the SloR Metalloregulome in Streptococcus mutans

Streptococcus mutans is the primary causative agent of human dental caries, a ubiquitous infectious disease for which effective treatment strategies remain elusive. We investigated a 25-kDa SloR metalloregulatory protein in this oral pathogen, along with its target genes that contribute to cariogenesis. Previous studies have demonstrated manganese- and SloR-dependent repression of the sloABCR metal ion transport operon in S. mutans. In the present study, we demonstrate that S. mutans coordinates this repression with that of certain virulence attributes. Specifically, we noted virulence gene repression in a manganese-containing medium when SloR binds to promoter-proximal sequence palindromes on the S. mutans chromosome. We applied a genome-wide approach to elucidate the sequences to which SloR binds and to reveal additional "class I" genes that are subject to SloR- and manganese-dependent repression. These analyses identified 204 S. mutans genes that are preceded by one or more conserved palindromic SloR recognition elements (SREs). We cross-referenced these genes with those that we had identified previously as SloR and/or manganese modulated in microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) experiments. From this analysis, we identified a number of S. mutans virulence genes that are subject to transcriptional upregulation by SloR and noted that such "class II"-type regulation is dependent on direct SloR binding to promoter-distal SREs. These observations are consistent with a bifunctional role for the SloR metalloregulator and implicate it as a target for the development of therapies aimed at alleviating S. mutans-induced caries formation.

Bioinformatics and Structural Characterization of a Hypothetical Protein from Streptococcus mutans: Implication of Antibiotic Resistance

As an oral bacterial pathogen, Streptococcus mutans has been known as the aetiologic agent of human dental caries. Among a total of 1960 identified proteins within the genome of this organism, there are about 500 without any known functions. One of these proteins, SMU.440, has very few homologs in the current protein databases and it does not fall into any protein functional families. Phylogenetic studies showed that SMU.440 is related to a particular ecological niche and conserved specifically in some oral pathogens, due to lateral gene transfer. The co-occurrence of a MarR protein within the same operon among these oral pathogens suggests that SMU.440 may be associated with antibiotic resistance. The structure determination of SMU.440 revealed that it shares the same fold and a similar pocket as polyketide cyclases, which indicated that it is very likely to bind some polyketide-like molecules. From the interlinking structural and bioinformatics studies, we have concluded that SMU.440 could be involved in polyketide-like antibiotic resistance, providing a better understanding of this hypothetical protein. Besides, the combination of multiple methods in this study can be used as a general approach for functional studies of a protein with unknown function.

Changes in Biochemical and Phenotypic Properties of Streptococcus mutans during Growth with Aeration

Oxygen has a potent influence on the expression of genes and the activity of physiological and biochemical pathways in bacteria. We have found that oxygen significantly altered virulence-related phenotypic properties of Streptococcus mutans, the primary etiological agent of human dental caries. Transport of glucose, fructose, or mannose by the sugar:phosphotransferase system was significantly enhanced by growth under aerobic conditions, whereas aeration caused an extended lag phase and slower growth of S. mutans in medium containing glucose, fructose, or mannose as the carbohydrate source. Aeration resulted in a decrease in the glycolytic rate and enhanced the production of intracellular storage polysaccharides. Although aeration decreased the acid tolerance of S. mutans, aerobically grown cells had higher F-ATPase activity. Aeration altered biofilm architecture but did not change the ability of S. mutans to interact with salivary agglutinin. Growth in air resulted in enhanced cell-associated glucosyltransferase (Gtf) activity at the expense of cell-free Gtf activity. These results demonstrate that S. mutans can dramatically alter its pathogenic potential in response to exposure to oxygen, suggesting that the phenotype of the organism may be highly variable in the human oral cavity depending on the maturity of the dental plaque biofilm.

Genes involved in the repression of mutacin I production in Streptococcus mutans

Streptococcus mutans is considered a primary pathogen for human dental caries. Its ability to produce a variety of peptide antibiotics called mutacins may play an important role in its invasion and establishment in the dental biofilm. S. mutans strain UA140 produces two types of mutacins, the lantibiotic mutacin I and the non-lantibiotic mutacin IV. In a previous study, we constructed a random insertional-mutation library to screen for genes involved in regulating mutacin I production, and found 25 genes/operons that have a positive effect on mutacin I production. In this study, we continued our previous work to identify genes that are negatively involved in mutacin I production. By using a high-phosphate brain heart infusion agar medium that inhibited mutacin I production of the wild-type, we isolated 77 clones that consistently produced mutacin I under repressive conditions. From the 34 clones for which we were able to obtain a sequence, 17 unique genes were identified. These genes encompass a variety of functional groups, including central metabolism, surface binding and sugar transport, and unknown functions. Some of the 17 mutations were further characterized and shown to increase mutacin gene expression during growth when the gene is usually not expressed in the wild-type. These results further demonstrate an intimate and intricate connection between mutacin production and the overall cellular homeostasis.

Fabrication and Antibacterial Performance of Bio-Graded Nano-Composite Materials by Using Inkjet Printing Method

The graded composite materials were fabricated by direct inkjet printing of nano-electropolar mineral and nano-hydroxyapatite powders for the study of inhibiting function on streptococcus mutans, which is a primary pathogen for human dental caries. The electropolar mineral can be utilized in many fields due to its spontaneous permanent polarity and far infrared emission properties. In this study, the graded distribution of the streptococcus mutans on the surface of graded composite materials was investigated by energy dispersion spectrometry, and the mechanism of which is that the streptococcus mutans was remained in the culture of pH at about 8.5 self-adjustment induced by the electropolar mineral. This culture inhibits growth and acid production of streptococcus mutans. Therefore, this composite materials have inhibitory effects on streptococcus mutans, and can be used as a promising biomaterial for preventing human teeth from caries.

Oral Therapeutic Vaccination withStreptococcus sobrinusRecombinant Enolase Confers Protection against Dental Caries in Rats

Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.

A phenotypic microarray analysis of a Streptococcus mutans liaS mutant

Streptococcus mutans, a biofilm-forming Gram-positive bacterium that resides in the human oral cavity, is considered to be the primary aetiological agent of human dental caries. A cell-envelope stress-sensing histidine kinase, LiaS, is considered to be important for expression of virulence factors such as glucan-binding protein C and mutacin production. In this study, a liaS mutant was subjected to phenotypic microarray (PM) analysis of about 2000 phenotypes, including utilization of various carbon, nitrogen, phosphate and sulfur sources; osmolytes; metabolic inhibitors; and susceptibility to toxic compounds, including several types of antibiotics. Compared to the parental strain UA159, the liaS mutant strain (IBS148) was more tolerant to various inhibitors that target protein synthesis, DNA synthesis and cell-wall biosynthesis. Some of the key findings of the PM analysis were confirmed in independent growth studies and by using antibiotic discs and E-test strips for susceptibility testing.

Dental Caries in the Qafzeh 3 Middle Paleolithic Modern Human

La denture incomplète de Qafzeh 3, un homme moderne ancien du Paléolithique moyen, présente quatre défauts antemortem de l’émail : sur les faces mésiale et distale de la I1 supérieure, sur la face occlusale de la I2 supérieure et sur la face distale de la M 1 inférieure. L’observation directe des dents complétée par une analyse au microscope électronique à balayage montrent que l’une des lésions (celle de la face distale de la I1) est une fracture antemortem de la dent et que les trois autres correspondent à des caries. Ces lésions viennent augmenter le petit nombre de caries dentaires connues chez les fossiles archaïques récents et modernes anciens du Paléolithique moyen du pourtour méditerranéen. La fréquence de caries chez ces hommes fossiles reste cependant très faible.

Can Coffee Prevent Caries? - An In-Vitro Study

Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. The micro-organisms produce 3 types of glucosyltransferase, and synthesize an adherent and water-insoluble glucan from sucrose by their cooperative action, which causes the organisms to adhere firmly to the tooth surface. The adherent glucan also contributes to the formation of dental plaque, in which the accumulation of acids leads to localized decalcification of the enamel surface. Roasted coffee extract possesses antibacterial activity against a wide range of microorganisms, including Staphylococcus aureus and Streptococcus mutans. The in-vitro study conducted at Oral Maxillofacial Pathology and Microbiology laboratory at KLE Society’s Institute of Dental Sciences showed that coffee has antibacterial action to streptococcus mutans and hence has an anticariogenic action but no antiadhesive action. This was studied by counting the colony forming units of the Strep. Mutans and the zone of inhibition around the different combinations of coffee.