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Antibacterial effect of water-soluble chitosan on representative dental pathogens Streptococcus mutans and Lactobacilli brevis
Chen, Chih-Yu;Chung, Ying-Chien;
Journal of Applied Oral Science , 2012, DOI: 10.1590/S1678-77572012000600006
Abstract: dental caries is still a major oral health problem in most industrialized countries. the development of dental caries primarily involves lactobacilli spp. and streptococcus mutans. although antibacterial ingredients are used against oral bacteria to reduce dental caries, some reports that show partial antibacterial ingredients could result in side effects. objectives: the main objective is to test the antibacterial effect of water-soluble chitosan while the evaluation of the mouthwash appears as a secondary aim. material and methods: the chitosan was obtained from the application chemistry company (taiwan). the authors investigated the antibacterial effects of water-soluble chitosan against oral bacteria at different temperatures (25-37oc) and ph values (ph 5-8), and evaluated the antibacterial activities of a self-made water-soluble chitosan-containing mouthwash by in vitro and in vivo experiments, and analyzed the acute toxicity of the mouthwashes. the acute toxicity was analyzed with the pollen tube growth (ptg) test. the growth inhibition values against the logarithmic scale of the test concentrations produced a concentrationresponse curve. the ic50 value was calculated by interpolation from the data. results: the effect of the ph variation (5-8) on the antibacterial activity of water-soluble chitosan against tested oral bacteria was not significant. the maximal antibacterial activity of water-soluble chitosan occurred at 37oc. the minimum bactericidal concentration (mbc) of water-soluble chitosan on streptococcus mutans and lactobacilli brevis were 400 μg/ml and 500 μg/ml, respectively. only 5 s of contact between water-soluble chitosan and oral bacteria attained at least 99.60% antibacterial activity at a concentration of 500 μg/ml. the water-soluble chitosan-containing mouthwash significantly demonstrated antibacterial activity that was similar to that of commercial mouthwashes (>99.91%) in both in vitro and in vivo experiments. in addition, the alcohol-free
Antibacterial effect of water-soluble chitosan on representative dental pathogens Streptococcus mutans and Lactobacilli brevis  [cached]
Chih-Yu Chen,Ying-Chien Chung
Journal of Applied Oral Science , 2012,
Abstract: Dental caries is still a major oral health problem in most industrialized countries. The development of dental caries primarily involves Lactobacilli spp. and Streptococcus mutans. Although antibacterial ingredients are used against oral bacteria to reduce dental caries, some reports that show partial antibacterial ingredients could result in side effects. OBJECTIVES: The main objective is to test the antibacterial effect of water-soluble chitosan while the evaluation of the mouthwash appears as a secondary aim. MATERIAL AND METHODS: The chitosan was obtained from the Application Chemistry Company (Taiwan). The authors investigated the antibacterial effects of water-soluble chitosan against oral bacteria at different temperatures (25-37oC) and pH values (pH 5-8), and evaluated the antibacterial activities of a self-made water-soluble chitosan-containing mouthwash by in vitro and in vivo experiments, and analyzed the acute toxicity of the mouthwashes. The acute toxicity was analyzed with the pollen tube growth (PTG) test. The growth inhibition values against the logarithmic scale of the test concentrations produced a concentrationresponse curve. The IC50 value was calculated by interpolation from the data. RESULTS: The effect of the pH variation (5-8) on the antibacterial activity of water-soluble chitosan against tested oral bacteria was not significant. The maximal antibacterial activity of water-soluble chitosan occurred at 37oC. The minimum bactericidal concentration (MBC) of water-soluble chitosan on Streptococcus mutans and Lactobacilli brevis were 400 μg/mL and 500 μg/mL, respectively. Only 5 s of contact between water-soluble chitosan and oral bacteria attained at least 99.60% antibacterial activity at a concentration of 500 μg/mL. The water-soluble chitosan-containing mouthwash significantly demonstrated antibacterial activity that was similar to that of commercial mouthwashes (>99.91%) in both in vitro and in vivo experiments. In addition, the alcohol-free mouthwash exhibited no cytotoxicity and no oral stinging. To the best of our knowledge, this was the first study to combine in vitro and in vivo investigations to analyze the antibacterial properties of water-soluble chitosan-containing mouthwash. CONCLUSIONS: This study illustrated that water-soluble chitosan may be a viable alternative to commercial mouthwashes in the future.
Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms
Jae-Gyu Jeon, Marlise I Klein, Jin Xiao, Stacy Gregoire, Pedro L Rosalen, Hyun Koo
BMC Microbiology , 2009, DOI: 10.1186/1471-2180-9-228
Abstract: Twice-daily treatment (one-minute exposure) during biofilm formation affected the gene expression by S. mutans both at early (49-h) and later (97-h) stages of biofilm development. Biofilms treated with combination of agents displayed lower mRNA levels for gtfB and gtfD (associated with exopolysaccharides synthesis) and aguD (associated with S. mutans acid tolerance) than those treated with vehicle-control (p < 0.05). Furthermore, treatment with combination of agents markedly affected the structure-architecture of S. mutans biofilms by reducing the biovolume (biomass) and proportions of both EPS and bacterial cells across the biofilm depth, especially in the middle and outer layers (vs. vehicle-control, p < 0.05). The biofilms treated with combination of agents were also less acidogenic, and had reduced amounts of extracellular insoluble glucans and intracellular polysaccharides than vehicle-treated biofilms (p < 0.05).The data show that the combination of naturally-occurring agents with fluoride effectively disrupted the expression of specific virulence genes, structural organization and accumulation of S. mutans biofilms, which may explain the enhanced cariostatic effect of our chemotherapeutic approach.Oral diseases related to dental biofilms, such as dental caries, continue to afflict the majority of the World's population [1]. This ubiquitous disease results from the interaction of specific bacteria with constituents of the diet within a biofilm known as plaque. Streptococcus mutans effectively colonizes tooth surfaces, and is a key contributor to the formation of cariogenic biofilms because this bacterium (i) utilizes dietary sucrose to synthesize large amounts of extracellular polysaccharides (EPS), (ii) adheres tenaciously to glucan-coated surfaces, and (iii) is also highly acidogenic and acid-tolerant [2,3].The majority of biofilm matrices are rich in polysaccharides, and dental biofilms are no exception. Polysaccharides of dental biofilms are mostly glucans
The effect of iron on Streptococcus mutans biofilm and on enamel demineralization
Ribeiro, Cecília Cláudia Costa;Ccahuana-Vásquez, Renzo Alberto;Carmo, Cadidja Dayane Sousa do;Alves, Cláudia Maria Coêlho;Leit?o, Tarcísio Jorge;Vidotti, Lisandra Rocha;Cury, Jaime Aparecido;
Brazilian Oral Research , 2012, DOI: 10.1590/S1806-83242012000400003
Abstract: iron (fe) may have an anticaries effect by specific inhibition of glycosyltransferase (gtf) enzymes of streptococcus mutans, but this hypothesis has not yet been clarified. in this study, s. mutans biofilms were formed on blocks of bovine dental enamel of a predetermined surface hardness (sh). these biofilms were exposed eight times/day to 10% sucrose, and two times/day they were subjected to one of the following treatments: g1, 0.9% nacl as a negative control; g2, 0.12% chlorhexidine digluconate (chx) as a positive antibacterial control; g3, 0.05% naf (225 ppm f) as a positive anticaries control; g4, g5, and g6, ferrous sulfate (fe2+) at concentrations of 1.0, 10.0, and 100.0 μg fe/ml, respectively. the experiment was performed in triplicate and was repeated three times (n = 9). the ph of the culture medium was determined every 24 h as an indicator of the biofilm's acidogenicity. the biofilm formed on each block was collected for determination of the viable bacteria and concentration of extracellular polysaccharides (eps). enamel sh was again determined and the percentage of sh loss (%shl) was calculated as an indicator of demineralization. iron treatment reduced the number of viable bacteria formed in the s. mutans biofilm (p = 0.04), in a dose-dependent manner, and also reduced the enamel's %shl (p = 0.005). at 100 μg/ml, fe reduced enamel demineralization as effectively as chx and naf (p < 0.05), but it did not inhibit eps production. in conclusion, the data suggest that the anticaries mechanism of action of fe may not involve the oxidative inhibition of gtfs.
Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms
Cury, Jaime Aparecido;Seils, Jennifer;Koo, Hyun;
Brazilian Oral Research , 2008, DOI: 10.1590/S1806-83242008000300005
Abstract: the presence of extracellular polysaccharides matrix makes extraction and purification of rna from streptococcus mutans within biofilms challenging. in this study, several approaches to purify rna extracted from s. mutans in suspension cultures and biofilms were examined. the combination of sonication (3 pulses of 30 s at 7 w), suspension in naes buffer (50 mm sodium acetate buffer, 10 mm edta and 1% sds; ph 5.0) and homogenization-mechanical cells disruption in naes- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg) of crude preparation of rna per 100 mg of total cell (or biofilm) dry-weight. the crude rna preparations were subjected to various dnase i treatments. the combination of dnase i in silica-gel based column followed by recombinant dnase i in solution provided the best genomic dna removal, resulting in 4.35 mg (or 0.06 mg) of purified rna per 100 mg of total cell (or biofilm) dry-weight. the cdnas generated from the purified rna sample were efficiently amplified using gtfb s. mutans-specific primers. the results showed a method that yields high-quality rna from both planktonic cells and biofilms of s. mutans in sufficient quantity and quality for real-time rt-pcr analyses.
CovR and VicRK Regulate Cell Surface Biogenesis Genes Required for Biofilm Formation in Streptococcus mutans  [PDF]
Rafael N. Stipp, Heike Boisvert, Daniel J. Smith, José F. H?fling, Margaret J. Duncan, Renata O. Mattos-Graner
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0058271
Abstract: The two-component system VicRK and the orphan regulator CovR of Streptococcus mutans co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of vicK and covR display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with vicK (UAvic) or covR (UAcov) deletion mutants together with electrophoretic motility shift assays (EMSA), we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including gbpB, wapE, smaA, SMU.2146c, and lysM. Deletion mutants of genes regulated by VicR and CovR (wapE, lysM, smaA), or regulated only by VicR (SMU.2146c) or CovR (epsC) promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of smaA, wapE, lysM, SMU.2146c and epsC, implying roles in cell wall biogenesis. WapE and lysM mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, vicR, covR and VicRK/CovR-targets (gbpB, wapE, smaA, SMU.2146c, lysM, epsC) are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose.
Mutacins of Streptococcus mutans
Kamiya, Regianne Umeko;Taiete, Tiago;Gon?alves, Reginaldo Bruno;
Brazilian Journal of Microbiology , 2011, DOI: 10.1590/S1517-83822011000400001
Abstract: the colonization and accumulation of streptococcus mutans are influenced by various factors in the oral cavity, such as nutrition and hygiene conditions of the host, salivary components, cleaning power and salivary flow and characteristics related with microbial virulence factors. among these virulence factors, the ability to synthesize glucan of adhesion, glucan-binding proteins, lactic acid and bacteriocins could modify the infection process and pathogenesis of this species in the dental biofilm. this review will describe the role of mutacins in transmission, colonization, and/or establishment of s. mutans, the major etiological agent of human dental caries. in addition, we will describe the method for detecting the production of these inhibitory substances in vitro (mutacin typing), classification and diversity of mutacins and the regulatory mechanisms related to its synthesis.
Generation of Diversity in Streptococcus mutans Genes Demonstrated by MLST  [PDF]
Thuy Do,Steven C. Gilbert,Douglas Clark,Farida Ali,Clarissa C. Fatturi Parolo,Marisa Maltz,Roy R. Russell,Peter Holbrook,William G. Wade,David Beighton
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009073
Abstract: Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However π, nucleotide diversity per site, was low for all loci being in the range 0.019–0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3–1.15] compared to 8.3 [95% confidence interval 5.0–14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.
Streptococcus mutans, Caries and Simulation Models  [PDF]
Sofia D. Forssten,Marika Bj?rklund,Arthur C. Ouwehand
Nutrients , 2010, DOI: 10.3390/nu2030290
Abstract: Dental caries and dental plaque are among the most common diseases worldwide, and are caused by a mixture of microorganisms and food debris. Specific types of acid-producing bacteria, especially Streptococcus mutans, colonize the dental surface and cause damage to the hard tooth structure in the presence of fermentable carbohydrates e.g., sucrose and fructose. This paper reviews the link between S. mutans and caries, as well as different simulation models that are available for studying caries. These models offer a valuable approach to study cariogenicity of different substrates as well as colonization of S. mutans.
Interaction of Salivary alpha-Amylase and Amylase-Binding-Protein A (AbpA) of Streptococcus gordonii with Glucosyltransferase of S. gordonii and Streptococcus mutans
Biswendu Chaudhuri, Jennifer Rojek, M Vickerman, Jason M Tanzer, Frank A Scannapieco
BMC Microbiology , 2007, DOI: 10.1186/1471-2180-7-60
Abstract: The addition of salivary alpha-amylase to culture supernatants of S. gordonii precipitated a protein complex containing amylase, AbpA, amylase-binding protein B (AbpB), and the glucosyltransferase produced by S. gordonii (Gtf-G). rAbpA was expressed from an inducible plasmid, purified from Escherichia coli and characterized. Purified rAbpA, along with purified amylase, interacted with and precipitated Gtfs from culture supernatants of both S. gordonii and S. mutans. The presence of amylase and/or rAbpA increased both the sucrase and transferase component activities of S. mutans Gtf-B. Enzyme-linked immunosorbent assay (ELISA) using anti-Gtf-B antibody verified the interaction of rAbpA and amylase with Gtf-B. A S. gordonii abpA-deficient mutant showed greater biofilm growth under static conditions than wild-type in the presence of sucrose. Interestingly, biofilm formation by every strain was inhibited in the presence of saliva.The results suggest that an extracellular protein network of AbpA-amylase-Gtf may influence the ecology of oral biofilms, likely during initial phases of colonization.Saliva-bacterial interactions influence the establishment and maintenance of the microflora of dental plaque [1-3]. Amylase is the most abundant enzyme in human saliva and is a constituent of the complex glycoproteinacious acquired pellicle that immediately forms on cleaned teeth [4-7]. Dental plaque forms on the pellicle and is responsible for the most common oral diseases, dental caries and periodontitis [8,9]. Amylase specifically binds with high affinity to several oral commensal streptococcal species, including Streptococcus gordonii, Streptococcus mitis, Streptococcus parasanguinis, Streptococcus cristatus, and Streptococcus salivarius, but not to Streptococcus sanguinis or to cariogenic streptococci including Streptococcus mutans and other mutans streptococci [10-13]. Amylase-binding bacteria constitute a substantial proportion of the total cultivable flora on human teeth a
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