Preliminary characterization of four bacteriocins from Streptococcus mutans

The various properties of the inhibitory substances produced by Streptococcus mutans strains C67-1, Ny257-S, Ny266, and T8, and the fact that inhibitory zones produced could not be associated with lactic acid (or other organic acids), bacteriophages, or hydrogen peroxide indicate that these substances can be classified as mutacins. The substances produced by strains C67-1, Ny266, and T8 possessed similar properties. They were shown to be thermoresistant (100 degrees C, 30 min), low molecular weight (less than 3500) substances sensitive to proteolytic enzymes (chymotrypsin, papain, pronase E, proteinase K, and trypsin) and they were active against most of the Gram-positive bacteria tested but not against most of the Gram-negative bacteria. The substance produced by strain Ny257-S differs from the other three by its narrower activity spectrum, its lower thermoresistance (80 degrees C, 30 min), and its higher molecular weight (8,000-14,000). The gene or the genes coding for the mutacins are probably located on the chromosome since no plasmid DNA could be detected in these four producing strains. Restriction-fragment patterns of C67-1 and T8 suggest that these strains are closely related, as supported by the strong similarity observed between the properties of their mutacins.     

Incidence of bacteriocinogeny among fresh isolates of Streptococcus mutans

Among 139 Streptococcus mutans fresh isolates tested, using the deferred-antagonism test and Streptococcus sanguis Ny 101 as the indicator strain, we observed that the frequency of detection of inhibition zones was reduced by 19% (from 53 to 34%) when arginine (1%) was used in the overlay agar. Among pigmented strains, the frequency of mutacin-like production was 70%. The frequency with which inhibition zones were detected varied from 7 to 91%, depending on the indicator strain used. These results indicate that the ability to detect the presence of mutacin-like substances varies widely and is dependent to a great extent on the methodology used.     

Effects of dietary carbohydrates on mutacin production and activity

Although mutacins (bacteriocins produced by Streptococcus mutans) were shown to be active in vivo, their ecological role in the oral cavity is still controversial. In the present paper, the effect of dietary carbohydrates, one of the ecological parameters which influences oral bacterial populations, on the activity and the production of mutacins from four S. mutans strains (C67-1, Ny257, Ny266 and T8) is described. Results obtained by the deferred antagonism test in solid media and by the mixed cultivation of the mutacinogenic strains with a sensitive indicator strain in liquid batch cultures, indicate that a minimal fermentable sugar concentration is needed for mutacin production. Among all the fermentable carbohydrates tested (fructose, glucose, lactose, mannitol and sucrose), none significantly affected the production and the activity of the four mutacinogenic strains used, in concentrations up to 5%. Although the results do not discount the possibility of mutacin inactivation in vivo, they indicate that they are not affected by dietary carbohydrates.     

Competition between oral Streptococcus species in the chemostat under alternating conditions of glucose limitation and excess

Abstract Oral Streptococcus species experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. To investigate the competitiveness of various streptococcal species under fluctuating carbohydrate supply, 2-membered chemostat cultures were run.
Under continuous limitation of glucose or sucrose, all 6 Streptococcus mutans test strains were outcompeted by Streptococcus sanguis P4A7 or Streptococcus milleri B448. This indicated that S. mutans had a lower affinity for glucose and sucrose than S. sanguis and S. milleri .
Mixed cultures were then subjected to hourly pulses with glucose. Under these conditions S. mutans Ny344 competed successfully with S. milleri B448, but still lost the competition against S. sanguis P4A7. The streptococci responded to pulses by taking up glucose at the maximum rate almost instantaneously. S. sanguis P4A7 had the highest rate of glucose uptake while the q _max value of S. mutans Ny344 was higher than that of S. milleri B448. This suggested a causal relationship between q _max and competitiveness.     

Interactions between oral bacteria: inhibition of Streptococcus mutans bacteriocin production by Streptococcus gordonii

Streptococcus mutans has been recognized as an important etiological agent in human dental caries. Some strains of S. mutans also produce bacteriocins. In this study, we sought to demonstrate that bacteriocin production by S. mutans strains GS5 and BM71 was mediated by quorum sensing, which is dependent on a competence-stimulating peptide (CSP) signaling system encoded by the com genes. We also demonstrated that interactions with some other oral streptococci interfered with S. mutans bacteriocin production both in broth and in biofilms. The inhibition of S. mutans bacteriocin production by oral bacteria was stronger in biofilms than in broth. Using transposon Tn916 mutagenesis, we identified a gene (sgc; named for Streptococcus gordonii challisin) responsible for the inhibition of S. mutans bacteriocin production by S. gordonii Challis. Interruption of the sgc gene in S. gordonii Challis resulted in attenuated inhibition of S. mutans bacteriocin production. The supernatant fluids from the sgc mutant did not inactivate the exogenous S. mutans CSP as did those from the parent strain Challis. S. gordonii Challis did not inactivate bacteriocin produced by S. mutans GS5. Because S. mutans uses quorum sensing to regulate virulence, strategies designed to interfere with these signaling systems may have broad applicability for biological control of this caries-causing organism.     

乳杆菌代谢产物对化脓性链球菌的抑制作用

【目的】分析乳杆菌代谢产物对化脓性链球菌的抑制作用。【方法】基于双层平板打孔法,通过测量抑菌圈大小来检测乳杆菌代谢产物对化脓性链球菌的抑菌作用;然后分别采用高效液相色谱法和4-氨酰安替比林法检测乳杆菌代谢产物中的有机酸和H2O2含量;最后,检测乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)、最小杀菌浓度(MBC)。【结果】对化脓性链球菌的抑菌效果以植物乳杆菌KLDS1.0667最好,副干酪乳杆菌KLDS1.0342-1次之,瑞士乳杆菌KLDS1.0203抑菌效果最差;乳酸和乙酸产量KLDS1.0667>KLDS1.0342-1>KLDS1.0203;H2O2产量KLDS1.0203>KLDS1.0667>KLDS1.0342-1。在抑菌试验中,乳杆菌的发酵上清液经去除H2O2处理后抑菌圈直径都减小;将发酵上清液的p H调至7.0后均检测不到抑菌圈。结果表明,乳杆菌代谢产物中对化脓性链球菌起抑制作用的主要物质为有机酸和H2O2,其中乳酸是产生抑菌作用的最主要物质。乳酸、乙酸和H2O2对化脓性链球菌的最小抑菌浓度(MIC)分别为1.28、0.64和0.008 g/L,对化脓性链球菌的最小杀菌浓度(MBC)分别为5.12、2.56和0.032 g/L。【结论】乳杆菌可利用其代谢产物对化脓性链球菌产生抑制作用,主要抑菌物质为有机酸和H2O2。     

赤藓糖醇对主要致龋链球菌及耐氟菌株生长和产酸的影响

目的通过赤藓糖醇对变形链球菌、远缘链球菌及其耐氟菌株混合菌生长和产酸影响的体外研究,为赤藓糖醇防龋作用的机理提供制论依据。方法采用最小抑菌浓度递增法对变形链球菌（S.mutans ATCC 25175,S.m）、远缘链球菌（S.sobrinus 6715,S.s）进行氟化钠体外诱导耐氟菌株（S.m-FR、S.s-FR）,利用液体稀释法配制赤藓糖醇TSB液8个浓度,分别加入含有变形链球菌、远缘链球菌及其耐氟菌株的细菌混悬液48 h,用比浊法观察其对混合菌生长的影响,并用pH计测定培养前后上清液的△pH值。结果吸光度A值和△pH值实验前后与对照组相比最低浓度为12%时差异均有统计学意义（P〈0.05）,且随着浓度的升高A值和△pH值均下降。结论赤藓糖醇能抑制变形链球菌、远缘链球菌及耐氟菌株混合菌生长和产酸,并且随着浓度的升高抑制作用增强。     

一株产右旋糖苷酶海洋细菌的筛选鉴定

【目的】筛选鉴定产右旋糖苷酶的海洋细菌,并对其所产右旋糖苷酶的酶学性质及在变异链球菌牙菌斑生物膜中的应用进行初步研究。【方法】利用平板透明圈法从海洋环境中筛选产右旋糖苷酶的细菌,根据菌株形态特征、生理特征及16S rDNA序列确定其分类学地位,采用体外生物膜模型研究该酶对变异链球菌牙菌斑生物膜形成的抑制作用。【结果】从海泥中筛选出一株产右旋糖苷酶的细菌KQ11,初步鉴定为节杆菌(Arthrobacter sp.)。该菌株的最适生长温度为30°C,最适生长pH 7.5,最适生长NaCl浓度为0.4%。右旋糖苷酶的最适作用温度为45°C,最适作用pH为5.5。该酶能有效地抑制变异链球菌牙菌斑生物膜的形成。【结论】菌株KQ11右旋糖苷酶能够抑制变异链球菌牙菌斑生物膜的形成,可望用于漱口液等口腔护理产品中。     

Comparison of extracellular protein profiles of seven serotypes of mutans streptococci grown under controlled conditions

Extracellular proteins produced by the four human commensal species of mutans streptococci were analysed. The organisms used were Streptococcus mutans, serotypes c, e and f, Streptococcus cricetus, serotype a, Streptococcus rattus, serotype b, and Streptococcus sobrinus, serotypes d and g. They were grown in continuous culture at different generation times and pH values in media containing either glucose or fructose to determine the extent of variation in extracellular protein production that could occur for an individual strain. The results for different organisms grown under the same conditions were then compared. The total amount of protein of molecular mass greater than or equal to 60 kDa varied considerably with the growth conditions and with the strain. Generally more protein was present at a higher pH, conditions under which the organisms also form more lipoteichoic acid. With respect to individual protein components SDS-PAGE proved better than isoelectric focusing for detecting phenotypic responses by a particular strain to environmental changes and differences between the different strains. Differences in the molecular masses of protein components were particularly pronounced in the regions designated P1 (185-200 kDa), P2 (130-155 kDa) and P3 (60-95 kDa). Every strain produced at least one component in the P1 region that cross-reacted with antiserum to the purified protein from S. mutans serotype c, a protein which is indistinguishable from antigens B and I/II. Two components in the P2 region were dominant in the case of S. cricetus and S. sobrinus strains and showed glucosyltransferase (GTF) activity. GTF activity was also detected in the P3 region, particularly with S. mutans strains.     

大蒜素抗变异链球菌的致龋效果

目的研究大蒜素对口腔变异链球菌生长及其菌斑生物膜粘附的抑制作用。方法二倍稀释法梯度稀释测最小抑菌浓度(minimum inhibitory concentration,MIC),将MIC以上2个梯度浓度对应的培养物涂布于BHI培养基上进行次代培养获得最低杀菌浓度(minimum bactericidal concentration,MBC);酶标仪测A值观察不同浓度大蒜素抑菌效应;抑制产酸试验观察抑制细菌产酸效应;结晶紫法研究亚抑菌浓度提取物对变异链球菌粘附能力及生物膜总量的影响;采用激光共聚焦荧光显微镜(laser scanning confocal microscopy,LSCM)观察常态牙菌斑生物膜生长过程中及药物处理后牙菌斑生物膜中死菌和活菌的构成,研究其对牙菌斑生物膜结构和活性的影响。结果抑菌试验中,得到大蒜素MIC为12.8 mg/L,MBC为25.8 mg/L。MIC及亚抑菌浓度抑菌试验显示均有一定的抑菌性,抑制率为2.17%～67.12%,并且抑菌性与浓度梯度成正相关。产酸试验显示24 h内大蒜素明显抑制细菌产酸(P0.01),细菌粘附试验结果显示大蒜素在MIC时生物膜的生成速度最慢,生物膜的总量最低(P0.01)。共聚焦荧光显微镜可见大蒜素组随药物浓度增加,菌斑生物膜较薄,绿色的活菌及团块明显减少,抑制生物膜的生长。结论大蒜素对变异链球菌生长、产酸与粘附有一定抑制作用。     

Examine growth inhibition pattern and lactic acid production in Streptococcus mutans using different concentrations of xylitol produced from Candida tropicalis by fermentation

Twenty clinical isolates of Streptococcus sp. were isolated from six clinical samples of dental caries on MSFA. Amongst these isolates, five clinical isolates were identified as S treptococcus mutans on the basis of morphological, biochemical and 16S rDNA sequencing. The isolated strains of S. mutans were exposed to fermented and purified xylitol (0.25-15.0%) and tested for its anti-microbial effects against control medium (Brain Heart Infusion without xylitol) after 12 h. The plate assay was developed using bromocresol green as an indicator dye in order to study the relative growth inhibition pattern of clinical sample at different concentrations of an anti-microbial compound in a single petriplate. The morphology of S. mutans cells in brain heart infusion (BHI) medium containing xylitol resulted in a diffused cell wall as observed using gram staining technique. The minimum inhibitory concentration (MIC) is 0.25% for S. mutans obtained from different clinical samples. The MIC(50) and MIC(90) is 5.0% and 10.0% xylitol respectively of the selected S. mutans being designated as clinical isolate B (6). The zone of inhibition was 72 mm and lactic acid production was 0.010 g/l at 10% xylitol concentration in Brain Heart Infusion Broth.     

Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.     

黄连素对变异链球菌抑制作用的体外研究

目的分析黄连素对体外变异链球菌生长、产酸、粘附的影响,探讨其防龋作用。方法采用微量肉汤稀释法进行最小抑菌浓度(MIC)的测定;然后通过试管粘附法测定不同浓度药液对变异链球菌粘附作用的影响;最后计算不同浓度药液作用24h后pH值的变化。结果黄连素对变异链球菌的MIC为1.25mg/mL,MBC为5.00mg/mL。实验组对变异链球菌的粘附及产酸的抑制作用随着药物浓度的增加而增强,与阴性对照组相比差异有统计学意义(P0.05)。结论黄连素可抑制体外变异链球菌的生长、产酸及粘附。     

Isolation and characterization of the mutans streptococci from the dental plaques in Koreans

Mutans streptococci have been implicated as cariogenic bacteria in dental caries because they can produce high levels of dental caries-causing lactic acid and extracellular polysaccharide. The aim of this study was to isolate and characterize the mutans streptococci from the dental plaque obtained from Koreans. The dental plaque samples were collected from the anterior and molar teeth of both jaws in 155 subjects (aged 2 to 33.2 years, average age 13.7+/-4.7 years). The samples were diluted by 100-fold in 1x PBS and plated on mitis-salivarius bacitracin (MSB) agar plates. The mutans streptococci grown on MSB plates were screened by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dextranase gene (dex). The mutans streptococci were identified at the species level using a 16S rDNA sequencing comparison method. The biochemical tests were carried out to biotype the mutans streptococci. Ninety-five strains of the mutans streptococci out of 358 colonies, which were derived from 141 subjects, were isolated. Of them, 77 strains and 18 strains were Streptococcus mutans and Streptococcus sobrinus, respectively. The biotyping data showed that 62, 1, 20, 10, and 2 strains were biotypes I, II, IV, V and variant, respectively. Of the two strains of variant biotype, one strains was similar to biotype IV except that it was positive to the arginine hydrolysis test. We considered this one strain a new biotype, and classified it as biotype VII. In conclusion, S. mutans and its biotype I was most frequently isolated in Korean dental plaque. The mutans streptococci strains isolated in this study might be useful for the study of the pathogenesis and the prevention of dental caries.     

Effect of utilization of substrates on the composition of dental plaque

Abstract The microbiota in the mouth is subjected to substrate limitations. In this study we have evaluated the role of competition for carbon and energy substrates on the proportions of 2 microbial species in a simplified plaque ecosystem in gnotobiotic rats. Germ-free rats were inoculated with a combination of Streptococcus sanguis and Streptococcus mutans , or with a combination of Streptococcus milleri and S. mutans . The available carbon and energy sources were varied through the host's diet. 3 Experimental diets were tested: (i) a basal diet low in soluble carbohydrates; (ii) an arginine-supplemented diet; (iii) a sucrose-supplemented diet. Arginine is used for growth by S. sanguis and S. milleri , but not by S. mutans . Sucrose is rapidly fermented by all 3 species.
The total number of viable organisms on the dentition increased when arginine or sucrose were supplied in the diet. With the arginine-supplemented diet, S. sanguis and S. milleri increased while S. mutans decreased. With the sucrose-supplemented diet, S. mutans increased while S. sanguis and S. milleri decreased. These results were explained by assuming that the organism with the highest growth rate on the supplementary substrate competes most favourably. Changes in the environmental pH, due to breakdown of sucrose and arginine, might also have affected the competition between the streptococci. In addition, production of extracellular glucans from sucrose could be a competitive advantage for S. mutans .     

Growth inhibition of Streptococcus mutans and Leuconostoc mesenteroides by sodium fluoride and ionic tin.

Sodium fluoride caused inhibition of growth rate and growth levels of Streptococcus mutans with glucose as the primary energy and carbon source. Stannous fluoride increased growth lag nad caused a much greater inhibition of growth rate than did sodium fluoride. Neither compound was found to be bactericidal when culture viability was measured after 6 days of incubation. Leuconostoc mesenteroides, which lacks a phosphotransferase system for sugar transport, showed less inhibition of growth rate with both inhibitors than did S. mutans, which possesses a phosphotransferase system. Metabolism of glucose or lactose which requires enolase activity shoed sodium fluoride inhibition, whereas metabolism of arginine or pyruvate does not involve enolase activity and showed no inhibition of growth.     

Growth Inhibition of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> with Low Xylitol Concentrations

No studies on the concentration dependency of the inhibition of Streptococcus mutans with xylitol are available. We studied xylitol-induced growth inhibition of two type strains, S. mutans NCTC 10449 and Ingbritt, and three clinical isolates of S. mutans. The strains were grown in Brain Hearth Infusion Medium in the presence of 0.001% (0.066 mM), 0.005% (0.33 mM), 0.01% (0.66 mM), 0.1% (6.6 mM), and 1% (66 mM) xylitol. Growth was followed by measuring the absorbance at a wavelength of 660 nm. The highest xylitol concentration tested in this study, 1%, showed mean inhibition percentages ranging from 61% to 76% when the growth inhibition of the five strains was compared to the control without xylitol at log-phase. For 0.1% xylitol, the inhibition percentages ranged from 22% to 59%. A concentration dependency was seen in the growth inhibition, with 0.01% xylitol being the lowest xylitol concentration inhibiting all five strains significantly (p < 0.001). The growth inhibition percentages determined for 0.01% xylitol, however, were low, and the inhibition was significantly weaker as compared to 0.1% and 1% xylitol. Our results suggest that low xylitol concentrations of 0.1% (6.6 mM) could inhibit mutans streptococci in vivo but even lower xylitol concentrations may be inhibitory.     

一株白芍内生放线菌的分离、活性及系统发育分析

目的:分离白芍中拮抗农作物致病菌和人类常见病原菌的内生放线菌,并进行系统发育分析。方法:采用3种分离培养基,从白芍根部分离内生放线菌;通过滤纸片法筛选具有拮抗活性的菌株,观察菌丝形态,并进行16S rDNA序列系统发育分析。结果:从白芍中分离得到16株内生放线菌,其中从FYSCA培养基中分离到9株;16株内生放线菌中有6株具有拮抗作用,菌株S-BS033004对5种病原菌有拮抗活性,尤其是对棉花黄萎病菌和小孢拟盘多毛孢菌和耐青霉素类金黄色葡萄球菌的拮抗作用显著,抑菌圈≥20mm。经16S rDNA系统发育分析表明该菌株与Streptomyces anulatus NBRC13369T等6株链霉菌模式菌株亲缘关系较近,相似性均为99.7%。结论:白芍内生放线菌S-BS033004是一株杀菌谱较广的链霉菌,具有很好的开发潜力。     

Inhibitory effect of oolong tea polyphenols on glycosyltransferases of mutans Streptococci.

Oolong tea extract (OTE) was found to inhibit the water-insoluble glucan-synthesizing enzyme, glucosyltransferase I (GTase-I), of Streptococcus sobrinus 6715. The GTase-inhibitory substance in the OTE was purified successive adsorption chromatography on Diaion HP-21 and HP-20 columns; this was followed by further purification by Sephadex LH-20 column chromatography. A major fraction that inhibited GTase activity (fraction OTF10) was obtained, and the chemical analysis of OTF10 indicated that it was a novel polymeric polyphenol compound that had a molecular weight of approximately 2,000 and differed from other tea polyphenols. Catechins and all other low-molecular-weight polyphenols except theaflavin derived from balck tea did not show significant GTase-inhibitory activities. It was found that OTE amd PTF10 markedly inhibit GTase-I and yeast alpha-glucosidase, but not salivary alpha-amylase. Various GTases purified from S. sobrinus and Streptococcus mutans were examined for inhibition by OTE and OTF10. It was determined that S. sobrinus GTase-I and S. mutans cell-free GTase synthesizing water-soluble glucan were most susceptible to the inhibitory action of OTF10, while S. sobrinus GTase-Sa and S. mutans cell-associated GTase were moderately inhibited; no inhibition of S. sobrinus GTase-Sb was observed. Inhibition of a specific GTase or specific GTases of mutants streptococci resulted in decreased adherence of the growing cells of these organisms. The inhibitory effect of OTF10 on cellular adherence was significantly stronger than that of OTE.