Determination of mutacin activity and detection of mutA genes in Streptococcus mutans genotypes from caries-free and caries-active children

Relationships between genetic diversity, mutacin production and sensitivity to mutacins in Streptococcus mutans were evaluated in 19 clinical isolates from caries-free and caries-active children. Mutacin production was tested against 30 indicator strains; results showed significant variations in the inhibitory spectra of the clinical isolates. There was no association between the inhibitory spectrum of the infecting strain and the caries experience or the level of mutans streptococci infection of the host. Homology to the mutA gene coding for mutacin II was detected in one clinical isolate; none of the clinical isolates showed homology to the mutA genes coding for mutacins I or III. Genotyping by random amplified polymorphic DNA (RAPD) reactions grouped the isolates into three clusters, but no correlation was found between any of the clusters and mutacin activity, caries experience or level of mutans streptococci in the host.     

Determination of mutacin activity and detection of mutA genes in Streptococcus mutans genotypes from caries‐free and caries‐active children

Relationships between genetic diversity, mutacin production and sensitivity to mutacins in Streptococcus mutans were evaluated in 19 clinical isolates from caries‐free and caries‐active children. Mutacin production was tested against 30 indicator strains; results showed significant variations in the inhibitory spectra of the clinical isolates. There was no association between the inhibitory spectrum of the infecting strain and the caries experience or the level of mutans streptococci infection of the host. Homology to the mutA gene coding for mutacin II was detected in one clinical isolate; none of the clinical isolates showed homology to the mutA genes coding for mutacins I or III. Genotyping by random amplified polymorphic DNA (RAPD) reactions grouped the isolates into three clusters, but no correlation was found between any of the clusters and mutacin activity, caries experience or level of mutans streptococci in the host.     

Latex agglutination test for detection of mutans streptococci in relation to dental caries in children.

A simple and rapid system based on a latex agglutination (LA) reaction was devised for the detection of mutans streptococci in dental plaque. Latex particles were sensitized with antibodies against whole cells of Streptococcus mutans strains MT8148 (serotype c), MT703R (e) and OMZ175 (f) and Strep. sobrinus strains B13 (d) and 6715 (g). These sensitized particles were agglutinated within a few minutes after addition of 1.0-10 ng serotype-specific antigen from the homologous organisms or the nitrous acid extract of whole cells at 10(5)-10(6) c.f.u. The LA test specifically differentiated not only mutans streptococci from the other oral streptococci but also Strep. sobrinus from Strep. mutans. The LA test was also applicable to extracts of plaque from 206 human subjects who harboured mutans streptococci. In clinical trials, the outcome of the LA test correlated significantly with the number of mutans streptococci found in plaque (p less than 0.0001), which was quantified by the selective cultivation of mutans streptococci. Furthermore, the LA test discriminated between Strep. mutans and Strep. sobrinus from human dental plaque. The sensitivity and the specificity of the LA test for detection of mutans streptococci were 78.9 and 100%. The degree of reactivity in the LA test correlated significantly with the number of decayed tooth surfaces (p less than 0.0001) and decayed and filled tooth surfaces (p less than 0.0001). These results suggest that the LA test could be useful clinically for the detection of mutans streptococci in dental plaque as well as serving as a caries-activity test.     

Production and properties of bacteriocins (Mutacins) from Streptococcus Mutans

Studies using the stab culture technique determined that almost all of the reference Streptococcus mutans strains of serotypes c and e produced bacteriocins (mutacins) against more than one of the 10 indicator organisms which included strains of Strep. mutans, Strep. salivarius, Strep. sanguis and group A Strep. pyogenes. However, reference strains of Strep. mutans types a and d never elaborated mutacins against more than one indicator strain. Among 113 clinical isolates of Strep. mutans obtained from Japanese children, 84 strains (74 per cent) inhibited at least one of the indicator strains. Type c strains accounted for 85 of the 113 clinical isolates, and these strains produced mutacins more frequently than strains of other serotypes. Very few strains produced a cell-free form of mutacin in Trypticase Soy broth enriched with 2 per cent yeast extract. Mutacins were generally heat-stable, but some were destroyed by treatment with proteolytic and/or lipolytic enzymes. Part of the mutacin activity diffused through cellophane paper. These results suggest that mutacins contain at least 2 kinds of inhibitory substances ranging from low molecular weight materials to higher molecular weight, protein-lipid complexes. Mutacinogeny was not due to the accumulation of hydrogen peroxide. Production of mutacin was influenced markedly by the culture media employed.     

Suppression of Streptococcus sobrinus 6715 (g) in plaques by Streptococcus mutans 32K (c)

The dental plaque of 96 healthy donors was screened for the production of such antibacterial substances as mutalipocin and bacteriocin and 192 strains of mutans streptococci isolated: 28 produced mutalipocin and 22 produced bacteriocin. Mutalipocin produced by these 28 S. mutans strains possessed similar biochemical and biological characteristics of well-characterized mutalipocin-producing strain S. mutans 32K (serotype c). When equal amounts of S. mutans 32K and S. sobrinus 6715 (g) were cultured together, cells of S. sobrinus 6715 were completely killed in 18 h. In addition, S. mutans 32K inhibited in vitro plaque formation by S. sobrinus 6715, and S. mutans 32K also eliminated in vitro plaque preformed by S. sobrinus 6715. In rat experiments, S. mutans 32K could preemptively colonize in plaque preformed by S. sobrinus 6715. On the other hand, S. sobrinus 6715 could not colonize in plaque preformed by S. mutans 32K. The results indicate that S. mutans serotype c which produces antibacterial substances is able to invade denatl plaque and replace the other mutans streptococci. This investigation offers one of the possible explanation why S. mutans serotype c is a predominant species among mutans streptococci in human plaque.     

Suppression of Streptococcus sobrinus 6715 (g) in plaques by Streptococcus mutans 32K (c)

The dental plaque of 96 healthy donors was screened for the production of such antibacterial substances as mutalipocin and bacteriocin and 192 strains of mutans streptococci isolated: 28 produced mutalipocin and 22 produced bacteriocin. Mutalipocin produced by these 28 S. mutans strains possessed similar biochemical and biological characteristics of well-characterized mutalipocin-producing strain S. mutans 32K (serotype c). When equal amounts of S. mutans 32K and S. sobrinus 6715 (g) were cultured together, cells of S. sobrinus 6715 were completely killed in 18 h. In addition, S. mutans 32K inhibited in vitro plaque formation by S. sobrinus 6715, and S. mutans 32K also eliminated in vitro plaque preformed by S. sobrinus 6715. In rat experiments, S. mutans 32K could pre-emptively colonize in plaque preformed by S. sobrinus 6715. On the other hand, S. sobrinus 6715 could not colonize in plaque preformed by S. mutans 32K. The results indicate that S. mutans serotype c which produces antibacterial substances is able to invade dental plaque and replace the other mutans streptococci. This investigation offers one of the possible explanation why S. mutans serotype c is a predominant species among mutans streptococci in human plaque.     

beta-haemolysis and pigment production by the oral bacterium Streptococcus mutans

The prevalence of beta-haemolysis and yellow pigmentation among 89 isolates of Streptococcus mutans from human dental plaque and 59 Strep. mutans isolates from positive cultures of human dental root canals were investigated. beta-Haemolytic strains were more frequent among the root-canal isolates (25.4 per cent) than among the dental plaque isolates (12.4 per cent). The distribution of pigment production among beta-haemolytic strains (22 pigmented strains out of 26 strains) and among non-haemolytic strains (5 pigmented strains out of 122 strains) showed a relationship between haemolysin and pigment production. beta-Haemolytic, pigmented strains (biotype c and e and 35-39 per cent guanine plus cytosine) were common in plaque and were the dominant type of Strep. mutans in some plaques. As beta-haemolysis by Strep. mutans was only demonstrated after strict anaerobic growth, whereas pigment production was demonstrated after growth under less anaerobic conditions (candle jar) on sucrose-containing agar plates, the observation of pigment may facilitate a presumptive identification of beta-haemolytic strains of Strep. mutans.     

Mutans streptococci, lactobacilli and caries experience in 12-year-old Icelandic urban children, 1984 and 1991

Abstract In order to evaluate changes in salivary counts of cariogenic bacteria and relate these to trends in caries experience, stimulated saliva was collected from a 20% random sample of 12-yr-old residents of Reykjavik. Iceland (252 children) in 1991 under conditions consistent with those of a survey conducted in 1984. The mean and median counts of salivary mutans streptococci and lactobacilli remained similar in the two studies. However, the frequency distribution at lower levels of mutans streptococci differed significantly between 1991 and 1984, e.g. in the present study 25.8% of the children had < 10⁵ compared with 13.8% in the study 1984. The mean caries prevalence in the permanent dentition (DFS_tot) was 11.0, which is significantly lower than in 1984 (mean DFS_tot 28.8). A significant difference in caries prevalence was found at various levels of salivary mutans streptococci. Strep. mutans (serotype c/e/f) was carried by all mutans streptococci-positive children, save one child, who carried only Strep. sobrinus. The proportion of 12-yr-olds who carried Strep, sobrinus had decreased significantly to 15.7% from 34.0% in 1984. Significantly more children with Strep. sobrinus showed high levels of total mutans streptococci than children with only Strep. mutans. As the case was in 1984 significantly more Strep. sobrinus carriers had a high level of salivary lactobacilli as well as higher caries prevalence than the children who did not carry this species.     

Colonization of rat molar teeth by mutans streptococci with different salivary agglutination characteristics

The oral implantation of salivary agglutination-positive and -negative mutans streptococci was studied using streptomycin resistant (StrR) organisms. StrR Streptococcus mutans strains Ingbritt and NCTC 10449 are agglutinated by rat saliva and the StrR strains Streptococcus sobrinus 6715-13 and Strep. mutans GS5 are not. Four groups of Sprague-Dawley rats were inoculated orally with each organism (one per group) and fed a sucrose diet. A further two groups of animals were similarly inoculated with either the agglutination-positive Strep. mutans Ingbritt or the agglutination-negative Strep. sobrinus 6715-13 and fed a glucose diet. StrR streptococci were recovered from smooth-surface dental plaque of all animals on the sucrose diet with no significant difference in the recovery of agglutination-positive Strep. mutans strains Ingbritt and NCTC 10449 and agglutination-negative Strep. mutans GS5. However, the recovery of agglutination-negative Strep. sobrinus 6715-13 from smooth-surface plaque of animals on either the sucrose or the glucose diets was significantly lower than that of the other strains. Agglutination-positive Strep. mutans Ingbritt colonized smooth enamel surfaces of animals on the sucrose and the glucose diets in numbers that were not significantly different. However, the colonization of such surfaces by agglutination-negative Strep. sobrinus 6715-13 was significantly enhanced by the sucrose diet. Agglutination-positive and -negative StrR mutans streptococci were recovered from fissure plaque of all inoculated sucrose-fed animals in numbers that were not significantly different. Successful colonization of smooth enamel surfaces by the StrR streptococci resulted in increased smooth-surface caries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of NaF and SnF2 on growth, acid and glucan production of several oral streptococci

The effects of low concentrations of these fluorides on Streptococcus sobrinus, Streptococcus mutans, Streptococcus salivarius, Streptococcus mitior and Streptococcus sanguis were investigated. Without fluoride, mutans streptococci (Strep. mutans and Strep. sobrinus) produced more acid than the other strains; both fluorides reduced acid production in all strains. NaF had little effect on growth; SnF2 decreased growth in all strains, most evidently in the Strep. mutans. In all growth conditions, Strep. sobrinus produced the most alkali-soluble glucan. Both fluorides enhanced alkali-soluble glucan production in mutans streptococci while only SnF2 enhanced this in the other streptococci.     

Similarity of bacteriocin activity profiles of mutans streptococci within the family when the children acquire the strains after the age of 5

It has been shown that there is a window of infectivity for mutans streptococci between the ages of 19 and 31 months, when many children acquire mutans streptococci transmitted from their mothers. Part of the children that escape this window acquire mutans streptococci at a later age. In this group, maternal transmission is expected to be less prevalent. The present study compared the bacteriocin activity profiles of mutans streptococci isolated from mothers, fathers and children when the children acquired the mutans streptococci between the ages of 5 and 11. Twelve families were randomly selected from a group of 11-year-old children who were known to have acquired mutans streptococci during this age period. From the saliva of the mothers (n = 12), fathers (n = 8) and children (n = 12) approximately 30 mutans streptococci strains were isolated. All isolates were tested twice for bacteriocin activity against 21 indicator strains with a double-layer technique. Bacteriocin activity of strains was considered to be different when the number of strains against which bacteriocin was produced differed >1 or when the width of one or more inhibition zones differed > or =4 mm. In 7/12 mother-child pairs similar profiles were found. In the 8 father-child pairs similar profiles were only found on 2 occasions. In these 2 families, all 3 ( mother, father and child) harboured strains with a similar profile. In 4/8 father-mother pairs similar profiles were found. There was no correlation between the prevalence of mutans streptococci strains, the number of indicator strains against which the strains made bacteriocin, nor the mean size of the inhibition zones and the presence of similarity of bacteriocin activity profiles of mutans streptococci within the family members. The results show that even when a child acquires mutans streptococci after the age of 5, there may be similarity between mutans streptococci in mother, father and child, indicating that transmission between the family members occurs.     

Mutacin production in Streptococcus mutans genotypes isolated from caries-affected and caries-free individuals

Relationships between genetic diversity and mutacin production in Streptococcus mutans were evaluated in 319 clinical isolates from eight caries-affected and eight caries-free individuals. The isolates were submitted to mutacin typing and AP-PCR (arbitrarily primed polymerase chain reaction) assay. The mutacin production was detected for 12 Streptococcus sp. indicator strains. Results showed significant variations in the mutacin production profiles and the inhibitory spectra of both groups. A possible association was seen between mutacin activity and the distinct patterns of Streptococcus sp. colonization in the two groups. Genotyping by AP-PCR using the primers OPA-02 and OPA-13 revealed 101 distinct genotypes against 48 phenotypes identified by mutacin typing. No correlation was observed between the inhibitory spectra of mutacin and genotypic similarities based on AP-PCR analyses. According to our results, strains of the same S. mutans genotype showed different mutacin profiles, suggesting a high degree of interstrain diversity. In conclusion, mutacin production seems to be of clinical importance in the colonization of S. mutans and is highly diversified in the S. mutans species.     

Infection with the bacterium Streptococcus mutans and salivary IgA antibodies in mothers and their children

Salivary IgA-antibody titres against different streptococcal antigens were measured in 64 mothers and their first-born children. Their presence and the level of Streptococcus mutans had been monitored for several years. The children had carried this bacterium for various lengths of time and now had varying numbers of salivary Strep. mutans. The subjects had participated in a longitudinal study completed one and a half years before, concerning prevention of early colonization of the child's mouth by Strep. mutans through measures undertaken by the mother. Whole-saliva samples were collected from all subjects and analysed by ELISA techniques for total IgA, and for IgA antibodies to whole cells of Strep. mutans serotype c and d, whole cells of Strep. sanguis, purified polysaccharide and protein antigens of Strep. mutans serotype c and d; a pool of Escherichia coli antigens was used as a control. No significant differences were found in the level of any of the specific IgA antibodies in children with different levels of indigenous Strep. mutans. A tendency to higher IgA antibody activity was found in children who had levels of Strep. mutans first detectable after three years of age and who had carried the bacterium for six months or less. The IgA antibody activity to Strep. mutans specific serotype c antigen and to whole cells of Strep. mutans serotype c (JC 2) was significantly higher in children with no caries experience when compared to children with more than two df-surfaces. This study offers no clear evidence for a protective role of salivary IgA antibodies against Strep. mutans colonization.     

Cariostatic activity of cacao mass extract

Chocolate is suspected to contain some caries-inhibitory substances. The cariostatic activity of cacao mass extract (CM), the main component of chocolate, was examined in vitro and in experimental animals. CM showed no detectable effects on the cellular growth and acid production of mutans streptococci. On the other hand, the cell-surface hydrophobicity of mutans streptococci was significantly reduced by the presence of CM. Furthermore, insoluble glucan synthesis by the glucosyltransferases from either Streptococcus mutans MT8148R or Strep. sobrinus 6715 was inhibited by CM, but not significantly. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CM. Finally, CM in both a 40% sucrose diet and drinking water resulted in reductions of caries development and plaque accumulation in rats infected with Strep. sobrinus 6715, but not significantly. These results indicate that cacao mass extract possesses some anticariogenic potential, but its anticaries activity is not strong enough to suppress significantly the cariogenic activity of sucrose.     

Lysis of Streptococcus sanguis by an extracellular enzyme from the bacterium Streptococcus mutans from human dental plaque

The ability of crude extracellular enzyme produced by the oral bacterium Streptococcus mutans AL7-1 to lyse living cells of Streptococcus sanguis ATCC 10556, 10557 and 10558 was examined. This enzyme showed lytic activity of living cells and cell walls of only Strep. sanguis ATCC 10558 strain and severed at random the long chains of this strain of living cells. Early log phase cells of this strain were more sensitive to this lytic enzyme than were late-log phase cells. In view of these results, the relationship between this lytic enzyme from Strep. mutans and a decrease in the number of serotype III strains of Strep. sanguis in dental plaque is discussed.     

Isolation frequency and serotype distribution of mutans streptococci and Actinobacillus actinomycetemcomitans, and clinical periodontal status in Finnish and Vietnamese children

The isolation frequency and serotype distribution of mutans streptococci and A. actinomycetemcomitans (A.a. ) were investigated in a group of Finnish ( n =16) and Vietnamese ( n =16) children, matched by sex, age, and caries status. In the Vietnamese children, the isolation frequencies were higher than in the Finnish children: 100%/62% for mutans streptococci and 78%/13% for A.a . Isolates ( n =3–8) from plaque and saliva were serotyped by immunodiffusion technique using serotype-specific antisera against serotypes c, e, f, d , and g for mutans streptococci and a, b, c, d , and e for A.a . The distribution of mutans streptococci serotypes in Finnish/Vietnamese children was: c 100%/50%; e 10%/31%; d 0%/56%; g 20%/38%. The frequency of plural serotypes was 30%/75%, respectively. In the Vietnamese group the serotype distribution of A.a . was: a 36%, b 27%, and c 63%; 45% of children carried two serotypes. One Finnish child harbored serotype a and one serotype b . The mean percentage of bleeding gingival sites was 7.4 in the Finnish and 15.1 in the Vietnamese group. Calculus and clinically deepened gingival pockets were more frequent findings in the Vietnamese children. The results indicate considerable differences in bacteriologic status and in clinical periodontal status between these Finnish and Vietnamese children.     

变链素的纯化

目的从变异链球菌临床株中分离纯化变链素,为进一步从分子水平研究变链素奠定基础。方法通过抑菌活性检测,从变异链球菌临床株中选择出抑菌活性较强的菌株。用氯仿抽提法从该菌株培养液中粗提变链素,经固相萃取和反相高效液相色谱（RP-HPLC）对粗提物进行纯化。结果获得变链素活性较强的菌株1G。从其200 mL液体培养基中粗提出变链素约15 μg,经固相萃取柱洗脱,再经过RP-HPLC的2次纯化,得到有抑菌活性的成分,此为纯化的变链素。结论变链素相对分子质量小,分离提纯步骤复杂,本实验得到纯化的变链素,为下一步研究变链素的氨基酸序列和基因序列奠定了基础。     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Igarashi T, Yamamoto AA, Goto N. Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction.
Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

Effect of removable orthodontic appliances on oral colonisation by mutans streptococci in children

Little is known about the effect of removable orthodontic appliances on oral colonisation by mutans streptococci (MS). In the present study, the frequency of isolation and serotype distribution of MS were evaluated in two groups of children, one undergoing therapy with removable appliances and the other not subjected to any kind of orthodontic treatment, respectively. Streptococci isolated from dental plaque samples from both groups of children were identified as mutans streptococci on the basis of their morphological and biochemical properties and were then serotyped in an enzyme immuno-assay using monoclonal antibodies. The number of subjects harbouring MS in their dental plaque was statistically higher in the group of orthodontic children without caries experience (CF) in comparison with CF children of the control group (10/12, 83.3% vs. 15/44, 34%). No clear difference was observed in the distribution of the different MS serotypes between the experimental and control group: S. mutans c,f serotype was the most frequently isolated in both groups of children followed by S. mutans serotype e and S. sobrinus serotype g. Such results suggest that the use of removable appliances may lead to the creation of new retentive areas and surfaces, which favour the local adherence and growth of MS. The data obtained stress the importance of a careful monitoring of patients treated orthodontically for risk of caries development.     

Caries inhibitory activity of cacao bean husk extract in in-vitro and animal experiments

Cacao bean husk extract (CBH) was examined for inhibitory effects on the caries-inducing properties of mutans streptococci in vitro and on caries development in specific pathogen-free Sprague-Dawley rats infected with mutans streptococci. CBH reduced the growth rate of almost all oral streptococci examined, which resulted in the reduction of acid production. Furthermore, insoluble glucan synthesis by the glucosyltransferases from Streptococcus mutans MT8148R and Streptococcus sobrinus 6715 was significantly inhibited by CBH. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CBH. The administration of CBH in drinking water resulted in significant reductions of caries development and dental plaque accumulation in rats infected with either Strep. sobrinus 6715 or Strep. mutans MT8148R, and the minimum cariostatic concentration was 1.0 mg/ml. These results indicate that CBH possesses powerful anticariogenic potential.