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Isolation and mode of action of a cell-free bacteriocin (mutacin) from serotype g Streptococcus mutans MT3791.

A bacteriocin, mutacin MT3791, was isolated from the culture supernatant of a dialysate medium of Tryptose phosphate broth cultures of Streptococcus mutans MT3791 (serotype g), a clinical isolate from a carious lesion of a Japanese child. The mutacin was found to inhibit the growth of most indicator strains of S. mutans and S. salivarius by the drop assay method. The mutacin was purified 1,450 fold from culture supernatant of S. mutans MT3791 by 60% saturated ammonium sulfate precipitation, followed by ultracentrifugation at 300,000 X g for 18 h. Gel filtration studies using Sepharose 4B indicated that the mutacin was highly aggregated, but could be dissociated by addition of 1% (final concentration) Tween 80 or 6M urea. The mutacin was heat stable, but the activity was destroyed after autoclaving at 120 degrees C for 60 min. It was susceptible to the enzymatic action of papain and pronase, but not trypsin, lipase or nucleases. The mutacin was adsorbed to whole cells of most strains of S. mutans. Mutacin MT3791 was bactericidal for an indicator strain, S. mutans MT703R (serotype e). The mutacin inhibited the incorporation of isotope-labelled precursors of protein, DNA, and RNA. Furthermore, mutacin MT3791 selectively and markedly suppressed the growth of S. mutans present in the dental plaque obtained from active carious lesions in the teeth of children. Possible role of mutacin in vivo was discussed with special reference to oral microbial ecology.

Bacteriocins↗

Relationship of bacteriocin-like inhibitor production to the pigmentation and hemolytic activity of mutans streptococci.

An inhibitor production typing (P-typing) scheme originally devised for hemolytic streptococci of Lancefield groups A-G has been successfully applied to 35 mutans streptococcus isolates recovered from plaque cultures of 60 Dunedin schoolchildren. Thirteen different P-type designations were identified. Although 11 (31%) of the isolates failed to produce detectable inhibitory activity on the conventional blood agar medium used for P-typing, four of these isolates were inhibitor-positive on Trypticase Soy agar supplemented with 2% yeast extract and 0.5% calcium carbonate (TSYCa). Four mutans strains displayed strong beta-hemolysis on Columbia agar base containing human blood when incubated in a 5% CO2 in air atmosphere. Three of these also produced weak beta-hemolysis on sheep blood-supplemented medium and were further distinctive in that they were the only inhibitor P-type 767 strains to be detected in the present study. Five mutans isolates were pigment producers and this property seemed to occur independently of both the beta-hemolytic activity and the P-type designation. Upon testing an additional collection of 18 mutans strains of various serotypes, only seven (39%) were inhibitor-positive. However, three of the four serotype c strains were inhibitor producers. Two strains of serotype d and one of serotype g were more hemolytic on sheep than on human blood agar medium. In general, it seems that the most common human mutans streptococci (serotype c strains) are more likely than are other mutans strains to produce bacteriocin-like inhibitory activity and to be hemolytic for human rather than sheep erythrocytes.

Bacteriocins↗

Changes in enamel surface roughness and adhesion of Streptococcus mutans to enamel after vital bleaching.

OBJECTIVES: The purpose of this in vitro study was to observe the influence of vital bleaching on changes to the enamel surface and adhesion of Streptococcus mutans to tooth enamel. METHODS: The coronal part of each of 70 extracted third molars was cut in half, with either the buccal or lingual half used for experiments or controls. Experimental halves were assigned to the following conditions: (A) enamel was bleached 1, 3 or 5 times using a bleaching material with or without etching; or (B) etched condition without bleaching. All control samples were kept intact in physiological saline solution. Surface roughness (Ra; roughness center-line average: microm) of enamel was measured for 35 pairs of specimens. TS broth culture medium containing 3% glucose was inoculated with S. mutans and cultured for 72 h before adding the other 35 pairs of specimens. Under scanning electron microscopy, the number of S. mutans colonies was counted and statistically analysed. RESULTS: Compared to controls, bleached enamel displayed increased colonies of S. mutans. Repeated bleaching further increased bacterial adhesion and maximal colonies counts were found under conditions of five bleaching treatments plus etching (p</=0.01). Compared to controls, roughness increased after etching. However, no linear correlation was found between number of S. mutans colonies and roughness. CONCLUSION: We conclude that both surface roughness and adhesion of S. mutans to the enamel surface increase after bleaching.

Acid Etching, Dental↗

Tracing genotypes of mutans streptococci on tooth sites by random amplified polymorphic DNA (RAPD) analysis.

The aim of this study was to clarify the distribution and persistence of mutans streptococci on different tooth sites in the same oral cavity. Thirteen subjects, aged 20-40 years, were examined. Salivary levels of mutans streptococci, caries prevalence, oral hygiene habits and status of tooth surfaces sampled were recorded. Plaque samples were obtained from four sites, the mesial and buccal surfaces of the first permanent molar on the right side of the lower jaw (46m and 46b), the distal surface of the first permanent premolar (24d) and the mesial surface of the second permanent premolar (25m) on the left side of the upper jaw, using sterile toothpicks on two occasions at 4-7-month intervals. The samples were cultivated on site-specific Strip mutans. Up to 10 colonies/site were isolated when present and genotyped by random amplified polymorphic DNA (RAPD) analysis, after species identification with PCR. Genotyping was also performed by restriction endonuclease analysis (REA) on 148 isolates, and results were consistent with the RAPD results. Most mutans streptococcus-positive samples were obtained from 46m. Within each individual, the same genotype occurred on at least two sites on all but one sampling occasion. A maximum of seven different genotypes were found in an individual. For a particular tooth site, four genotypes occurred simultaneously and taking both sampling occasions together the maximum was six different types. The same genotypes/types were found again after 4-7 months on 25 sites in 12 subjects. Fifteen sites were mutans streptococcus-positive on only one sampling occasion. The results indicate that several different genotypes of mutans streptococci colonize a tooth site, and that the same genotype colonizes several sites in the same oral cavity. Persistence of genotypes on a site for several months and interindividual differences in the occurrence of genotypes were also found.

Adult↗

Different bacteriocin activities of Streptococcus mutans reflect distinct phylogenetic lineages.

Bacteriocins produced by mutans streptococci are known as mutacins. In this study 16 broadly active mutacin-producing Streptococcus mutans strains from New Zealand, North America and Europe were classified into four groups (A-D) on the basis of differences in their activity in deferred antagonism tests against either the homologous producer strain (to test for presence of self-immunity) or indicator strains Staphylococcus aureus 46 and Enterococcus faecium TE1. Two of the strains included in the study (UA140 and UA96) were representatives of the group I and II mutacin producer strains previously described by Caufield and co-workers. One of the New Zealand isolates of group A (S. mutans strain N) appeared to produce inhibitory activity similar to that of the group I prototype strain UA140. Four other New Zealand isolates of group B (S. mutans strains M19, M34, B34 and D14) had mutacin II-like activity. The group B mutacin producers differed from the group A mutacin producers in their additional activity against Staph. aureus 46. Seven S. mutans strains (M46, B46, B57, M12, M28, B28 and 13M) were distinguished from the group A and group B mutacin producers in that they inhibited E. faecium TE1. These were called group C mutacin producers. Strains H7 and H23 resembled the group C strains in their action on both indicator strains TE1 and 46. However, these two strains failed to exhibit immunity to their own inhibitory products in the deferred antagonism test and were separately classified as group D mutacin producers. Phylogenetic analysis of the strains by several genotypic and phenotypic characteristics revealed that the mutacin groups were associated with distinct evolutionary lineages of S. mutans.

Adult↗

Inducible and constitutive formation of fructanase in batch and continuous cultures of Streptococcus mutans.

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.

Bacterial Proteins↗

Construction of NotI restriction map of the Streptococcus mutans genome.

Streptococcus mutans and Streptococcus sobrinus are the major causative organisms of human dental caries. Pulsed-field gel electrophoresis (PFG) showed that the restriction enzyme NotI produced ten and six DNA fragments from the genomes of S. mutans strain MT8148 and S. sobrinus strain 6715, respectively. The sizes of the chromosomes of S. mutans and S. sobrinus were each estimated to be about 2200 kb. The NotI restriction map of S. mutans MT8148 genome was constructed by Southern blot analysis with probes that overlapped two adjacent NotI fragments. Several virulence-associated genes of S. mutans were placed on the NotI restriction map. In addition, unique 'fingerprints' of S. mutans chromosomal DNA digested with NotI were produced by PFG, and these may be useful for epidemiological studies.

Base Sequence↗

Genotypic diversity and virulence traits of Streptococcus mutans in caries-free and caries-active individuals.

The present study evaluated the relationship between clonal diversity and some virulence traits of Streptococcus mutans isolated from eight caries-free and eight caries-active subjects. A total of 155 S. mutans isolates from caries-free subjects and 144 isolates from caries-active subjects were obtained from samples of saliva, dental plaque and tongue surface and identified by PCR. The isolates were submitted to arbitrarily primed (AP)-PCR (OPA-2 and OPA-13) and multilocus enzyme electrophoresis (MLEE) to establish the genotypic diversity. Production of water-insoluble glucan (WIG) (monitored by SDS-PAGE), final pH of cultures and the ability of bacterial cells to adhere to smooth glass in the presence of sucrose were measured. High and comparable abilities of MLEE and AP-PCR were found to distinguish S. mutans genotypes, using Simpson's index of discrimination (0.971 and 0.968, respectively). The results showed a significant difference (P < 0.01) in the number of genotypes when caries-free and caries-active groups were compared by both fingerprinting methods used. Final pH (P = 0.32) and the percentage of adherence to a glass surface (P = 0.62) did not show differences between the two groups; however, the intensities of WIG bands from the caries-active group were greater than those from the caries-free group (P < 0.01). In addition, WIG was positively correlated with the ability of S. mutans to adhere to a glass surface (r = 0.34, P = 0.02) from caries-active subjects. These data showed that AP-PCR analysis and MLEE are both effective methods for assessing the genetic relatedness of S. mutans. Using these techniques, it was found that there is a larger number of genotypes of S. mutans with increased ability to synthesize WIG in caries-active individuals.

Adolescent↗

Frequency of four different mutacin genes in Streptococcus mutans genotypes isolated from caries-free and caries-active individuals.

The ability of Streptococcus mutans to produce mutacins, combined with the production of other virulence factors such as lactic acid, may contribute to the pathogenesis of this bacterium. In the present study, the detection of genes encoding mutacin types I/III, II and IV was performed by PCR with specific primers to each type in a total of 63 S. mutans genotypes isolated from caries-active and caries-free individuals. In the caries-free group, PCR screening for mutacin IV revealed that 31.8% of strains were positive for this mutacin. PCR for the other three mutacins tested (I/III and II) did not yield amplicons in any S. mutans strains in this group. The PCR with primers of mutacin IV showed 68.3% positive genotypes in the caries-active group, on the other hand, the amplicons of mutacins I/III revealed 41.5% positive strains that carried these genes. The chi square test showed significant differences in the number of positive strains to mutacin IV when comparing the caries-free and caries-active genotypes of S. mutans (P = 0.01). All tested S. mutans strains were negative by PCR for mutacin II. The low frequencies of detection of some mutacin genes suggest the existence of high diversity and polymorphism in the production of genetic determinants of mutacin-like substances. In addition, the production of a wide spectrum of mutacins can play an important biological role in colonization by S. mutans strains, mainly in the niche of high-complexity microbial communities.

Adolescent↗

Isolation and characterization of Streptococcus mutans in heart valve and dental plaque specimens from a patient with infective endocarditis.

Streptococcus mutans, known to be an aetiologic agent of dental caries, also causes infective endocarditis (IE), although a comparison of isolates from the oral cavity and infected heart valve of the same patient has not been reported. In the present study, infected heart valve and dental plaque samples from a patient with IE were analysed. Broad-range PCR with DNA sequencing revealed that 50 clones from the dental plaque isolates were composed of oral streptococci and periodontopathic bacteria, whereas only Streptococcus mutans was detected in 50 clones from the heart valve. Eighteen strains of Streptococcus mutans were isolated from dental plaque and seven from the heart valve, and the biochemical properties of each were in accordance with those of Streptococcus mutans. DNA fingerprinting analysis revealed that all the oral isolates of Streptococcus mutans had similar patterns, which were different from those of the isolates from the infected heart valve. Western blotting using glucosyltransferase (GTF)-specific antiserum showed that the seven strains from the heart valve lacked the three types of intact GTF. In addition, the sucrose-dependent adhesion rates of these isolates were significantly lower than those of the oral isolates (P<0.001). Furthermore, the isolates from the heart valve were less susceptible to erythromycin and kanamycin. These results indicate that the properties of the Streptococcus mutans strains isolated from the infected valve were different from those of typical oral strains, which may be related to the effects of IE.

Anti-Bacterial Agents↗

Enhancement of murine immune responses to orally administered haptenated Streptococcus mutans.

The induction of immune responses to orally administered trinitrophenyl (TNP)-haptenated Streptococcus mutans and its enhancement with muramyldipeptide (MDP), peptidoglycan (PG), and concanavalin A (Con A) were investigated in lipopolysaccharide (LPS)-non-responsive C3H/HeJ mice and the syngeneic, LPS-responsive C3H/HeN strain. Both mouse strains manifested similar immune responses, primarily of the IgM isotype, after a single gastric intubation (GI) with TNP-S. mutans. However, when groups of animals were first carrier-primed by GI with S. mutans for 2 consecutive days, followed by a single GI with TNP-S. mutans 1 week later, C3H/HeJ mice gave a significantly higher (P less than or equal to 0.01) splenic IgA anti-TNP plaque-forming cell (PFC) response than identically treated C3H/HeN mice. Furthermore, saliva, urine and serum from these C3H/HeJ mice possessed high levels of IgA anti-TNP antibodies as determined by the enzyme-linked immunosorbent assay, whereas C3H/HeN mice exhibited low antibody levels. Oral administration of Con A (either 250 micrograms or 500 micrograms/mouse) or purified PG (1 mg/mouse) at the time of TNP-S. mutans immunization resulted in significantly (P less than or equal to 0.01) enhanced splenic IgA anti-TNP PFC responses, especially in C3H/HeJ mice. On the other hand, MDP promoted IgA anti-TNP PFC responses in LPS-responsive C3H/HeN mice but did not augment responses in C3H/HeJ animals. A similar immune response pattern was seen when antibody levels were measured in serum, saliva, and urine of both mouse strains. These results demonstrate that haptenated S. mutans is a good antigen for the induction of high IgA responses in orally immunized C3H/HeJ mice and that this high response can be enhanced with the adjuvants Con A and PG. However, MDP is ineffective in C3H/HeJ mice but enhances IgA responses in normal LPS-responsive C3H/HeN animals.

Acetylmuramyl-Alanyl-Isoglutamine↗

Effect of a bacteriocin-producing strain of Streptococcus sobrinus on infection and establishment of Streptococcus mutans on tooth surfaces in rats.

The effect of bacteriocin produced by Streptococcus sobrinus MT6223 on infection and establishment of Streptococcus mutans MT6222 was studied in specific pathogen-free rats. These strains were isolated from a carious lesion of a single subject. S. mutans MT6222 was found to be susceptible to the growth inhibitory action of S. sobrinus MT6223. When simultaneously inoculated into the oral cavity of rats, even a small inoculum (10(5) CFU) of S. sobrinus MT6223 completely inhibited colonization of S. mutans MT6222 on the tooth surface. Also, S. sobrinus MT6223 eliminated S. mutans MT6222 when MT6223 (10(8) CFU) was inoculated 2 days after the inoculation of 10(8) CFU cells of MT6222. Similar results were obtained in dental plaque samples from the tooth surface and the fissures of the upper molars at the end of the experiment. However, when S. sobrinus MT6223 (10(8) CFU) was inoculated 2 weeks after the inoculation of S. mutans MT6222 (10(8) CFU), MT6223 coexisted with MT6222. However, the plaque samples showed that MT6223 inhibited the establishment of MT6222 on smooth surfaces, but not in fissures. In addition, MT6223 protected against subsequent infection with MT6222. However, a nonbacteriocinogenic mutant of S. sobrinus MT6223 did not inhibit the infection and establishment of S. mutans MT6222.

Animals↗

Relationship between mutans streptococci in saliva and their colonization of the tooth surfaces.

The relationship between the salivary concentration of mutans streptococci and their prevalence on different tooth surfaces was studied in 114 subjects. Plaque samples were obtained from all tooth surfaces in the dentition and the infection magnitude of mutans streptococci was determined. The salivary concentrations of mutans streptococci correlated significantly with the number of colonized tooth surfaces and with the infection level of mutans streptococci for individual teeth or groups of tooth surfaces. The highest correlation values were found for buccal and approximal surfaces and for molars followed by premolars and anterior teeth. The 10 tooth surfaces best reflecting the salivary levels of mutans streptococci were 5 buccal and 5 approximal sites, 6 of them localized on maxillary posterior teeth. A significant positive relationship was noted between the prevalence of mutans streptococci in saliva and on the dorsum of the tongue.

Adolescent↗

Streptococcus mutans, not detected?

The growth of Streptococcus mutans was followed for three years in 24 healthy 12-17 year-old children, who were selected for the study on the basis of undetectable levels of salivary S. mutans. The saliva samples were cultured by a dip-slide method based on mitis-salivarius agar supplemented with sucrose and bacitracin. At the final occasion, additional examination of plaque and saliva of 10 children using TSY20B agar was performed. During the 3-year study period, S. mutans was found in saliva samples from 15 children on one or more of the 4 test occasions, suggesting that most 'S. mutans negative individuals' were those in whom S. mutans could not be temporarily detected. The additional examination of plaque did not increase the number of 'S. mutans-positive individuals'.

Adolescent↗

Mutans streptococci in saliva and dental caries in children living in a high and a low fluoride area.

The aim of this study was to obtain information about the quantitative distribution of salivary mutans streptococci and the relation to dental caries in children with contrasting levels of natural water fluoride. A total of 698 schoolchildren aged 12 yr were selected from areas with high (1.2 ppm) or low (0.1 ppm) fluoride concentration in the drinking water. They were all examined for the presence and number of mutans streptococci in saliva by a specially prepared plastic strip, the "Strip mutans" method, cultivated in a selective broth. Data on the caries experience were obtained from the dental records and from bite-wing radiographs. Mutans streptococci were identified in 82% of all children with no difference between the two areas. The number of mutans streptococci was however significantly (p less than 0.05) lower among the children from the high fluoride area than those from the low fluoride area. Children with no detectable or low levels (0-10 CFU) of mutans streptococci had less caries experience than children with moderate or high levels (11- greater than 500 CFU) in both areas investigated.

Child↗

Human strains of mutans streptococci show different cariogenic potential in the hamster model.

The cariogenic potential of fresh humans strains of Streptococcus mutans and Streptococcus sobrinus was examined in hamsters maintained on a high sucrose diet. The strains 1B 16 (S. mutans) and B13 (S. sobrinus) isolated 20-25 yrs ago served as positive controls. Three series of experiments were run. Some strains were tested once and some strains were tested in all 3 experiments. The animals infected with S. sobrinus strains generally showed lower caries scores than those infected with S. mutans strains. Among the groups infected with the different strains of S. mutans the caries scores varied. In one of the experiments the caries score of the animals infected with one of the Icelandic S. mutans strains was significantly higher than that of the positive control group and the group infected with one of the fresh Swedish isolates. Thus, different strains of mutans streptococci where shown to induce varying caries activity in the hamster model.

Adolescent↗

Crevicular IgG antibodies and recovery of locally implanted Streptococcus mutans in humans.

This study was undertaken with the aim of examining the effect of highly different IgG antibody activities in gingival crevicular fluid (GCF) on the elimination of a streptomycin-resistant strain of Streptococcus mutans (S. mutans) topically implanted on the tooth. Two groups of people were selected for the study, one with high and one with low specific IgG antibody activity (IgG antibody activity against S. mutans) in GCF. During an experimental period of 15 d the specific IgG antibody activity in GCF in the same individual stayed on a relatively constant level. Between individuals, however, the specific IgG antibody activity varied considerably. The number of indigenous mutans streptococci and the number of implanted bacteria on the same tooth surface were strongly correlated. The implanted S. mutans strain was rapidly eliminated in all subjects. No difference in the elimination of the implanted strain of S. mutans could be demonstrated between the groups of high and low specific IgG antibody activity.

Adult↗

Serum antibody responses to Streptococcus mutans antigens in humans systemically infected with oral streptococci.

Sera from patients with subacute bacterial endocarditis (SBE) due to Streptococcus mutans or other oral streptococci and from normal subjects were assayed by enzyme-linked immunosorbent assay for antibodies to defined S. mutans antigens. Antibodies of IgG and IgA isotypes to Ag I/II and Ag III were greatly elevated in S. mutans-SBE sera, and the IgA antibodies in 3 sera included both polymeric and monomeric forms. Elevated IgM and IgG anti-lipoteichoic acid and IgG and IgA anti-serotype c polysaccharide antibodies were also found. The sera of 4 of 6 patients infected with other oral streptococci also displayed antibodies to S. mutans Ag I/II. Sera of 3 patients infected with Streptococcus mitis or Streptococcus oralis, but none of the S. mutans-infected cases, showed elevated antibodies to human heart sarcolemma, and all SBE sera had elevated rheumatoid factor. These results suggest that the known surface protein antigens of S. mutans are immunodominant in humans, and are not likely to be heart cross-reactive.

Antibodies, Bacterial↗