Streptococcal Mutans Research Articles

Effect of immediate and delayed finishing and polishing procedure on Streptococcal mutans adhesion and micro-hardness of composite resin surface: An in-vitro study

Proper finishing and polishing have become the indispensable step in clinical restorative dentistry due to increased aesthetic demands. There are several polishing systems containing fine particle size abrasives. The adhesion of bacterial plaque to restoration depends on the surface properties and composition of materials. In literature, surface roughness after polishing is well documented, but the timing, i.e., immediate or delayed polishing affecting the bacterial adherence and microhardness on the composite needs research. As the Streptococcus mutans has a major role in primary and secondary caries, so in this light, this study is conducted to investigate the best timing of polishing affecting the bacterial adhesion and microhardness of composite resin.: To evaluate the Streptococcal mutans adhesion and microhardness of composite resin after polishing them, immediately and after 24 hours of curing.: Sixty specimens were made from composite resin Filtek Z 350 XT (3M ESPE) of 6mm diameter and 4mm height, was cured by placing a Mylar strip and divided into 3 groups- Group 1 the control group with only MYLAR-STRIP, Group 2- Polishing system SOF-LEX (3M ESPE Dental products St. Paul, MN,USA.) Group 3- Polishing with SUPER-SNAP (Shofu Inc., Kyoto, Japan). Group 2 and 3 were further divided into two subgroups according to the polishing time- immediate and delayed. The Streptococcal mutans adhesion for each group was measured by colony forming units and compared. The mean log of CFU/mL present in the biofilm was also calculated. The results of bacterial adherence was analysed by three-way ANOVA (analysis of variance) (p<0.01) and the ANOVA two-way of variance was used for checking microhardness (p<0.01). A p value <0.01 was considered to indicate a statistically significant for both bacterial adhesion and hardness. Polishing after 24 hours of curing showed less bacterial adherence on the composite surface, regardless of the polishing treatment performed(p<0.01). Polishing with SOF-LEX(Group-2) had the lower bacterial adherence than SUPER-SNAP (Group-3), Control group (Mylar matrix strip) promoted the lowest bacterial adhesion on the surface composites. Micro hardness of composite was lowest in Mylar matrix group. Immediate polishing procedure showed lower microhardness values as compared to polishing that was delayed for 24hours for both polishing groups. SOF-LEX system can be used for chairside polishing of composite restorations in clinic and delayed finishing and polishing of composite shows better surface microhardness values.

Nanoscale adhesion forces of glucosyltransferase B and C genes regulated Streptococcal mutans probed by AFM.

Glucosyltransferases (Gtfs), represented by GtfB and GtfC, are important virulence factors of Streptococcus mutans and the major etiologic pathogens of tooth decay. However, the individual roles of gtfB and gtfC in the initial attachment of S. mutans are not known. We used atomic force microscopy to explore the contribution of gtfB and gtfC, as well as enamel-surface roughness, on the initial attachment of S. mutans. Adhesion forces of four S. mutans strains (wild-type, ΔgtfB, ΔgtfC, and ΔgtfBC), onto etched enamel surfaces, were determined. Force curves showed that, with increasing etching time from 0 to 10s, the forces of all strains increased accordingly with acid-exposure time, the adhesion forces of wild-type strains were significantly greater than those of mutant strains (p<.05), and the forces of the three mutants were similar (p<.05). When the etching time was increased from 10 to 30s, difference in force between 20 and 30s was not observed, and adhesion forces among ΔgtfB, ΔgtfC, and wild-type strains were not significantly different when the etching time was >20s (p>.05). These data suggest that the roughness and morphology of enamel surfaces may have a significant influence upon the initial attachment of bacteria, and that gtfB and gtfC are essential for the adhesion activity of bacteria. Furthermore, gtfB seems to be more important than gtfC for bacterial-biofilm formation, and gtfB inactivation is an effective strategy to inhibit the virulence of cariogenic biofilms.

Antimicrobial efficacy of commercially available mouthrinses: An in vitro study

Introduction: Oral cavity ecosystem represents a dynamic pattern. An effective plaque control measure should target plaque formation before the mature plaque is formed. Various types of chemotherapeutic agents are coming up with different antimicrobial agents in them. Hence, this study has been undertaken to know whether these antimicrobial agents are effective on common microorganisms of oral cavity which directly and indirectly contributes to plaque formation Aim: The aim of this study was to determine antimicrobial efficacy of different mouthrinses against the oral pathogens in vitro. Materials and Methods: A total of seven mouthrinses were tested for their antimicrobial activity against three oral pathogens, namely, Streptococcus mutans (MTCC 890), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 10231) by well agar diffusion assay. Statistical analysis was performed using Kruskal–Wallis test. The level of significance used was P< 0.05. Results: Mouthrinse with chlorhexidine (CHX) gluconate, triclosan as main ingredients showed maximum zone of inhibition (P = 0.003) against streptococcal mutans and E. coli at 1:16 dilution and mouthrinse with CHX gluconate and zinc chloride showed maximum zone of inhibition at 1:16 dilution against Candida among seven mouthrinses used in the present study. It was also observed that zone of inhibition of all the mouthrinses decreased with the increase in dilution. Conclusion: Among mouthrinses formulations, CHX combined with other active ingredients was found to be more effective.

Purification and properties of an α-(1 → 3)-glucanase (EC 3.2.1.84) from Trichoderma harzianum and its use for reduction of artificial dental plaque accumulation.

Extracellular α-(1 → 3)-glucanase (mutanase, EC 3.2.1.84) produced by Trichoderma harzianum CCM F-340 was purified to homogeneity by ultrafiltration followed by ion exchange and hydrophobic interaction chromatography, and final chromatofocusing. The enzyme was recovered with an 18.4-fold increase in specific activity and a yield of 4.3%. Some properties of the α-(1 → 3)-glucanase were investigated. The molecular mass of the enzyme is 67 kDa, as estimated by SDS/PAGE, its isoelectric point 7.1, and the carbohydrate content 3%. The pH and temperature optima are 5.5 and 45°C, respectively. The enzyme is stable over a pH range of 4.5-6.0 and up to 45°C for 1 h. The Km and Vmax under standard assay conditions are 0.73 mg/ml and 11.39 x 10(-2) µmol/min/mg protein, respectively. The enzyme activity is stimulated by addition of Mg(2+) and Na(+), and significantly inhibited by Hg(2+). The α-(1 → 3)-glucanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-(1 → 3)-glucans. The 20-residue N-terminal sequence of the enzyme is identical with those of other α-(1 → 3)-glucanases from the genus Trichoderma, and highly similar to those from other fungi. The purified α-(1 → 3)-glucanase was effective in preventing artificial dental plaque formation. The easy purification from fermentation broth and high stability, and the effective inhibition of oral biofilm accumulation make this α-(1 → 3)-glucanase highly useful for industrial and medical application.

Structural Diversity of Streptococcal Mutans Synthesized under Different Culture and Environmental Conditions and Its Effect on Mutanase Synthesis

Streptococcal mutans synthesized under different conditions by growing cultures or by their glucosyltransferases were shown to exhibit a great structural and property diversity. Culturing and environmental factors causing structural differences in mutans were specified. All of the obtained biopolymers (76 samples) were water-insoluble and most of them (72) had a structure with a predominance of α-(1→3)-linked glucose (i.e., the content of α-(1→3)-linkages in the glucan was always higher than 50%, but did not exceed 76%). An exception were four glucans containing more than 50% of α-(1→6)-sequences. In these structurally unique mutans, the ratio of α-(1→3)- to α-(1→6)-bonds ranged from 0.75 to 0.97. Aside from one polymer, all others had a heavily branched structures and differed in the number of α-(1→3), α-(1→6), and α-(1→3,6) linkages and their mutual proportion. The induction of mutanase production in shaken flask cultures of Trichoderma harzianum by the structurally diverse mutans resulted in enzyme activities ranging from 0.144 to 1.051 U/mL. No statistical correlation was found between the total percentage content of α-(1→3)-linkages in the α-glucan and mutanase activity. Thus, despite biosynthetic differences causing structural variation in the mutans, it did not matter which mutan structures were used to induce mutanase production.

Gene cloning, expression, and characterization of mutanase from Paenibacillus curdlanolyticus MP-1

Mutanases hydrolyze d-glucosidic linkages of α-1,3-linked polysaccharides which are important components of dental plaque. Therefore, these enzymes can be useful in preventive oral hygiene. A gene encoding mutanase was cloned from soil-isolated Paenibacillus curdlanolyticus MP-1 and expressed in Escherichia coli, and the resulting recombinant enzyme was characterized. The nucleotide sequence of the mutanase gene consisted of 3786 nucleotides encoding a protein of 1261 amino acids with a theoretical molecular weight of 131.62kDa. The deduced amino acid sequence exhibited a high degree of similarity with mutanases of Paenibacillus sp. KSM-M126 and Paenibacillus humicus NA1123, with 84% and 80% identity, respectively. The recombinant enzyme was purified 17.5-fold to homogeneity with a recovery of 37%. The purified mutanase showed optimal activity at pH 6.0 and 45°C, and was completely stable at pH 4.0–9.5 and up to 45°C. The enzyme was specific for α-1,3-glucosidic linkages and effectively solubilized fungal α-1,3-glucans and streptococcal mutans, releasing nigerooligosaccharides. The mutanase did not hydrolyze a synthetic substrate readily hydrolyzed by exoglucanases and the enzyme activity was not suppressed in the presence of deoxynojirimycin, an inhibitor of exo-type enzymes. These results suggest an endohydrolytic mode of action.

PURIFICATION AND CHARACTERIZATION OF MUTANASE PRODUCED BY Paenibacillus curdlanolyticus MP-1

Mutanases are enzymes that catalyze hydrolysis of α-1,3-glucosidic bonds in various α-glucans. One of such glucans, mutan, which is synthesized by cariogenic streptococci, is a major virulence factor for induction of dental caries. This means that mutan-degrading enzymes have potential in caries prophylaxis. In this study, we report the purification, characterization, and partial amino acid sequence of extracellular mutanase produced by the MP-1 strain of Paenibacillus curdlanolyticus, bacterium isolated from soil. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular mass 134 kD, while native gel filtration chromatography confirmed that the enzyme was a monomer of 142 kD. Mutanase showed a pH optimum in the range from pH 5.5 to 6.5 and a temperature optimum around 40–45°C. It was thermostable up to 45°C, and retained 50% activity after 1 hr at 50°C. The enzyme was fully stable at a pH range of 4 to 10. The enzyme activity was stimulated by the addition of Tween 20, Tween 80, and Ca2+, but it was significantly inhibited by Hg2+, Ag+, and Fe2+, and also by p-chloromercuribenzoate, iodoacetamide, and ethylenediamine tetraacetic acid (EDTA). Mutanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-1,3-glucans. It also showed binding activity to insoluble α-1,3-glucans. The N-terminal amino acid sequence was NH2-Ala-Gly-Gly-Thr-Asn-Leu-Ala-Leu-Gly-Lys-Asn-Val-Thr-Ala-Ser-Gly-Gln. This sequence indicated an analogy of the enzyme to α-1,3-glucanases from other Paenibacillus and Bacillus species.

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.

Production and use of mutanase from Trichoderma harzianum for effective degradation of streptococcal mutans

Basic cultural parameters affecting mutanase production by Trichoderma harzianum F-340 in shaken flasks and aerated fermenter cultures have been standardized. The best medium for enzyme production was Mandels medium A with initial pH 5.3, supplemented with 0.3% mutan and 0.05% peptone and inoculated with 20% of the 72-h mycelium as inoculum. It was shown that mycelial mass, used in the culture medium as a sole carbon source, induced mutanase synthesis and could be utilized as an inexpensive and easily available substitute for bacterial mutan. Application of optimized medium and cultural conditions enabled us to obtain a high mutanase yield (0.6-0.7 U/mL, 2.0-2.5 U/mg protein) in a short period of time (3-5 days), which was much higher than the best reported in literature. The enzyme in crude state was stable in the pH range of 4.5-6.0, and at temperatures of up to 40oC; its maximum activity was recorded at 45oC and at pH 5.5. The mutanase preparation obtained from the T. harzianum fungus was relatively stable under storage conditions, and showed a high hydrolytic potential in reaction with a mixed-linkage (a-1,3, a-1,6) water-insoluble mutan of streptococcal origin (hydrolysis yield reached a value of 69% in 24 h). Steady-state measurement of the enzymic reaction products during the hydrolysis revealed that mutanase exhibited an exo type of action on mutan. Thin-layer chromatographic analysis showed that glucose was the primary final product of mutan hydrolysis with mutanase. The potential application of mutanase in dentistry is discussed.