Xylitol inhibition of anaerobic acid production by Streptococcus mutans at various pH levels

Xylitol inhibits the glycolysis and growth of Streptococcus mutans. We studied the inhibitory effect of xylitol on the acid production of S. mutans at several pH levels under the strictly anaerobic conditions found in the deep layer of dental plaque. Xylitol inhibited the rate of acid production from glucose and changed the profile of acidic end products to formate-acetate dominance, with a decrease in the intracellular level of fructose 1,6-bisphosphate and an intracellular accumulation of xylitol 5-phosphate (X5P). These results were notable at pH 5.5-7.0, but were not evident at pH 5.0. Since the activity of phosphoenolpyruvate phosphotransferase for xylitol was greater at higher pH, it is suggested that xylitol could be incorporated more efficiently at higher pH and that the resultant accumulation of X5P could inhibit the glycolysis of S. mutans more effectively.     

Effect of sodium and potassium ions on intracellular pH and proton excretion in glycolyzing cells of Streptococcus mutans NCTC 10449 under strictly anaerobic conditions

The effect of sodium and potassium ions on intracellular acid production and acid excretion by glycolyzing cells of Streptococcus mutans was examined. S. mutans NCTC 10449 grown under glucose-limited and strictly anaerobic conditions in a continuous culture system was loaded with bis(carboxyethyl)-carboxyfluorescein, a pH-sensitive fluorescent dye, washed and suspended in 0.00–0.30 M NaCl/KCl solution. The dye allowed for the continuous monitoring of intracellular pH while proton excretion was measured simultaneously with a pH-stat. Sodium ions inhibited and potassium ions, at low pH, accelerated the amount of measurable acid excreted extracellularly. In the presence of both NaCl and KCl, proton excretion following the addition of glucose was slightly higher or similar to that observed in the presence of 0.15 M KG alone. Sodium and potassium ions did not affect the proton-ATPase enzyme or the intracellular level of ATP, suggesting that these ions did not directly effect proton pumping activity itself. The inhibition of proton excretion by sodium ions was considered to have probably occurred as a result of an indirect inhibition of proton-ATPase activity by the low intracellular pH induced by sodium ions.     

Simultaneous monitoring of intracellular pH and proton excretion during glycolysis by Streptococcus mutans and Streptococcus sanguis: effect of low pH and fluoride

A system was developed by which 2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein could be used to monitor intracellular pH at the same time that proton excretion was being measured. Streptococcal cells were loaded with the dye, and after the addition of glucose protons were excreted and the intracellular pH increased quickly and remained higher than the extracellular pH of 7.0. The excretion of protons stopped and the intracellular pH returned to the original level when glucose was depleted. The intracellular level of ATP remained high during glucose metabolism and decreased with the depletion of glucose. At extracellular pH of 5.5, and 5.0, the intracellular pH of fasting cells was higher than the extracellular pH value. After addition of glucose there were initial lags of proton excretion and of increases in intracellular pH at the acidic extracellular pH values. In the presence of fluoride, a lag in proton excretion and a simultaneous decrease in intracellular pH were observed, indicating a partial and transient inhibition of proton-ATPase activity.     

Mechanism of inhibition of acid production in Streptococcus mutans by sodium ions under strictly anaerobic conditions

Acids excreted and intracellular levels of glycolytic intermediates during glucose metabolism in Streptococcus mutans NCTC 10449 under strictly anaerobic conditions were quantified in an attempt to understand the effect of sodium ions on bacterial acid production. In the presence of NaCl (0.15–0.30 M), the total amount of individual carboxylic acids excreted was inhibited by up to 31%. The intracellular level of fructose 1,6-bisphosphate increased by 58% and levels of 3-phosphoglycerate and pyruvate decreased by 46% and 12%, respectively. Sodium ions directly inhibited the activities of fructose 1,6-phosphate aldolase and triose phosphate isomerase. This indicated that the glycolytic enzymes responsible for the catalysis of fructose 1,6-bisphosphate to 3-phosphoglycerate were inhibited. However, in spite of the expected reduction in acid production intracellularly, the intracellular pH actually decreased in the presence of sodium ions. It is possible that the low intracellular pH inhibits the activity of the glycolytic enzymes involved in the breakdown of fructose 1,6-bisphosphate to 3-phosphoglycerate.     

High prevalence of mutans streptococci in a population with extremely low prevalence of dental caries

The quantitative distribution of the mutans streptococci was investigated in populations with very low caries prevalence. Three hundred and seven school children from rural Sudan with an estimated age of 12 years were examined for dental status and salivary mutans streptococci. Forty-two isolates from the salivary cultures were determined to species level by studying the serogroup and biotype. Denial caries in permanent teeth was diagnosed in 12% of the children and the mean DMFT was 0.17 (range 0-6 DMFT). Caries was confined to fissures. Mutans streptococci were found in 96% of the children and high counts were obtained in 45%. Of 42 isolated strains, 37 belonged to Streptococcus mutans , 5 to Streptococcus sobrinus and none to Streptococcus rattus. The observations show that the mutans streptococci can be widespread in human populations with extremely low prevalence of dental caries and consequently that these bacteria may be readily propagated in human populations without association to a caries-promoting life style.     

不同pH对变异链球菌体外生物膜形成的影响

目的：了解变异链球菌临床株体外生物膜形成规律以及不同pH对生物膜形成的影响。方法：采用微孔板培养,染色、分光光度测定法（A630）绘制体外不同pH条件下（pH=7.0～5.0）593号、18号菌株以及变异链球菌标准株（ATCC25175）的生物膜生长曲线。结果：体外变异链球菌各株在pH=5.0时均不能形成生物膜;pH=7.0时细菌生物膜形成表现为缓慢的非线性生长,12～24h生物膜开始成熟,24～36h出现一相对的生长停滞期;pH=5.0时对已形成12h的变链菌生物膜生长有明显的抑制作用,但经历12h的酸休克后各菌株的生物膜均能恢复生长。结论：变异链球菌在体外pH=7.0时于12～24h形成稳定的生物膜,该生物膜能抵抗一定程度的酸（pH=5.0）攻击,而浮游状态的细菌则不能。     

Effects of pH on the glucose and lactate metabolisms by the washed cells of Actinomyces naeslundii under anaerobic and aerobic conditions

Effects of pH on the glucose and lactate metabolism by the washed cells of Actinomyces naeslundii genospecies 1 and 2 under anaerobic and aerobic conditions were studied. The rate of acid production from glucose was the highest at pH 7.0 and decreased as the pH lowered to 4.5, irrespective of atmospheric conditions. The anaerobic end-product in the absence of bicarbonate was mainly lactate, while in the presence of bicarbonate the rate of acid production increased 1.8-2.5 times with the production of formate, acetate and succinate in addition to lactate. Under aerobic conditions, the cells produced acids from glucose along with oxygen consumption and the end-product was mainly acetate. In contrast to the glucose metabilism, the cells produced base from lactate along with oxygen consumption. The rates of base production and oxygen consumption were the highest at pH 5.5. The end-products from lactate were acetate and pyruvate. These results indicate that oral actinomyces has a various activity of glucose and lactate metabolism at a wide range of environmental pH and suggest its flexibility in surviving in dental plaque, where the environmental factors fluctuate.     

Mechanism of inhibition of glycolysis in Streptococcus mutans NCIB 11723 by chlorhexidine

Inhibition of the rate of acid production from glucose by the cells of Streptococcus mutans was directly related to the concentrations of 0.075 to 0.20 mM chlorhexidine. Lactate production was inhibited to a greater extent than acetate and formate. Quantification of glycolytic intermediates revealed that the steps in glycolysis inhibited by chlorhexidine were the reactions catalyzed by phosphofructokinase and glyceraldehyde 3-phosphate dehydrogenase and/or phosphoglycerate kinase. However, the activities of these enzymes were not decreased in cells treated with the inhibitor. It was demonstrated that chlorhexidine caused leakage of metabolites from the cells. Our results indicate that the decreased rate of glycolysis caused by chlorhexidine is due to the leakage of metabolic intermediates and not to direct effects on enzymes involved in glycolysis by S. mutans NCIB 11723.     

Intracellular and extracellular pHs of Streptococcus mutans after addition of acids: loading and efflux of a fluorescent pH indicator in streptococcal cells

A pH-sensitive fluorescent dye, 2', 7'-bis-(2-carboxyethyl)-5 and 6-carboxyfluorescein (BCECF), was used to determine intracellular pH (pH(in)). The efflux of BCECF loaded into oral streptococcal cells was determined after incubation of the cells at 35 degrees C for 20 min in the presence and absence of glucose. In the absence of glucose, the fluorescence of intracellular BCECF in Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius and Streptococcus sobrinus decreased only very slightly, indicating that the dye could be useful for pH(in) determination. In the presence of glucose, however, the fluorescence decreased by 57%. Thus, the pH(in) of S. mutans cells was measured by the BCECF method in the absence of glucose at various acidic pH levels by adding lactic, acetic and hydrochloric acids to the cell suspensions. The pH(in) was almost equal to the extracellular pH (pH(out)) for pH(out) values of between 8 and 5, indicating that protons permeated easily across the S. mutans cell membrane. For pH(out) between 5 and 4, pH(in) was constant at around 5, suggesting that the cell membrane was impermeable to protons, or that a cytoplasmic buffering system functioned. pH(in) decreased at pH(out) values of < 4. The constant pH(in) at acidic pH(out) levels could protect intracellular components, such as proteins, against acidification by sugar fermentation.     

Intracellular flux of glucose metabolism in streptococcal cells by simultaneous monitoring of fluorescence dependent on reduced nicotinamide adenine nucleotide and acid excretion under strictly anaerobic conditions

Reduced nicotinamide adenine nucleotide (NADH)-dependent fluorescence and acid excretion during glucose pulse to washed Streptococcus mutans cells were monitored simultaneously at pH 7.0 with a fluorescence spectrophotometer and a pH-stat. Acid excretion started with addition of glucose. At the same time, the fluorescence dropped quickly to a minimum level and increased to a plateau level, suggesting that pyruvate metabolism started immediately after addition of glucose, then the rate of the pyruvate metabolism became almost equal to the rate of glycolysis. When the acid excretion stopped, the fluorescence increased rapidly from the plateau to the maximum level, suggesting that the pyruvate metabolism stopped first, and then began to decrease to the original level. The system used in this study for simultaneously monitoring the level of NADH and acid excretion gives us a crucial tool to clarify a biochemical mechanism of the control of sugar metabolism by streptococci.     

The earlier the colonization by mutans streptococci, the higher the caries prevalence at 4 years of age

Seventy-eight 4-year-old children, examined earlier for the presence of mutans streptococci at 4-month intervals from 15 months of age, were recalled for recording of dental caries and salivary sampling. The saliva samples were analysed for the presence of mutans streptococci, including the species Streptococcus mutans and Streptococcus sobrinus and lactobacilli. The results showed that the earlier the mutans streptococci had been detected in the children, the higher the caries experience. Eighty-nine percent of the children colonized at 2 years of age had experienced caries and had a mean dfs of 5.0 compared with 25% of the non-colonized 4-year-olds with a mean dfs of 0.3. S. mutans was the predominant species. S. sobrinus was usually found in combination with S. mutans , except in 2 children where S. sobrinus was the only species detected. More children with multiple species had higher numbers of total mutans streptococci and a tendency to higher caries prevalence than children with only S. mutans.     

Metabolism of intracellular polysaccharide in the cells of Streptococcus mutatis under strictly anaerobic conditions

Streptococcus mutans, which had accumulated glycogen-like iodophilic intracellular polysaccharide (IPS), produced large amounts of formate, acetate and ethanol from the IPS by pyruvate formate-lyase (PFL) under strictly anaerobic conditions without exogenous sugar. Under aerobic conditions, the same S. mutans produced exclusively lactate and pyruvate from the IPS because of the inactivation of PFL by oxygen. The total amount of acid produced under anaerobic conditions was larger than that under aerobic conditions. The analysis of intracellular glycolytic intermediates revealed that levels of fructose 1,6-bisphosphate (lactate dehydrogenase (LDH) activator) and glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (PFL inhibitors) were low when IPS was used as a glycolytic substrate, implying that PFL functions more efficiently than LDH in IPS metabolism. These findings suggest that the PFL pathway contributes to the acid production from IPS, and may explain partially why the acids in starved dental plaque are mainly acetate and formate.     

不同龋敏感儿童口腔不同基因型变异链球菌临床分离株在不同pH条件下产生GTF能力的比较研究

目的:探讨不同龋敏感儿童口腔变异链球菌不同基因型菌株在不同pH条件下产生GTF的能力.方法:从不同龋敏感儿童口腔变异链球菌中选取66株临床分离株,常规复苏、增菌并配制相同密度的菌悬液后,接种于不同pH值(以0.5为间隔,pH 5.0～7.0)的含2.5 g/L葡萄糖和0.05％ Tween 80的TYC培养基中厌氧培养18h;采用Somogyi法测定还原糖含量,计算酶活性.结果:在同一pH条件下,不同龋敏感儿童口腔变链菌GTF活性的差异具有统计学意义(P＜0.05),龋敏感性越高,GTF活性越大;同一龋敏感儿童口腔变链菌不同基因型菌株GTF活性的差异也具有统计学意义(P＜0.05),GTF活性随基因型的增加而增加;当pH5.5时,高龋、中龋儿童携带一种基因型菌株GTF活性显著下降(P＜0.05).结论:不同龋敏感儿童口腔变异链球菌不同基因型菌株在不同pH条件下GTF活性不同,携带基因型越多的菌株GTF活性越高.     

不同pH值对变异链球菌ffh基因表达的影响

目的 检测变异链球菌（S. mutans）ffh基因在不同pH值条件下的表达水平,分析pH值对S. mutans ffh基因表达的影响,分析调控ffh表达的因素。方法 在不同培养时段（4 h和18 h）和不同pH值（pH值4.0~7.0）条件下培养S. mutans标准菌株UA159,提取样本,用荧光定量聚合酶链反应（qRT-PCR）法检测样本中目的基因ffh的mRNA转录水平的变化情况,分析S. mutans ffh基因在不同培养时段和pH值条件下的表达变化趋势。结果 培养4 h时,ffh基因相对表达量随着pH值的降低而减少,培养18 h时,ffh基因相对表达量随着pH值的降低而增加;相同pH值条件下,ffh基因相对表达量在培养4 h与18 h时的差异有统计学意义（P<0.05）。结论 S. mutans ffh基因的表达受细菌培养时段和pH值的影响。     

Polysaccharide production by cell free transferases in saliva in relation to salivary microflora

Abstract – The supernatant of centrifuged whole saliva was incubated with radiolabeled sucrose to measure polysaccharide production by cell free transferases and to examine whether water-insoluble polysaccharides were produced. Amounts of polysaccharides were considered to reflect the level of cell free transferases in saliva. Plating samples on blood agar, MS and MSB plates gave salivary counts of total CFU, total streptococci, S. salivarius and S. mutans. The capability of the cell free portion of saliva to produce polymers was confirmed and it appeared that the cell free transferases were able to produce water-insoluble polysaccharides. Significant correlations were found between the total and the insoluble polysaccharides from ¹⁴C-G’-sucrose and total CFU, total streptococci, S. salivarius and S. mutans, respectively. Heavily S. mutans infected subjects seemed to produce particularly large amounts of water-insoluble polysaccharides from ¹⁴C-G’-sucrose. The apparently water-insoluble ¹⁴C-F’-polysaccharides correlated significantly to the number of S. salivarius. It was thus concluded that the constitution of the oral microflora and particularly the levels of S. mutans and S. salivarius were of importance for the level of cell free transferase activity.     

Effect of a preventive program on dental caries and mutans streptococci in Polish schoolchildren

The effect of a preventive program was studied in Polish schoolchildren. The study was carried out in a group of 133 children, aged 10-12 yr, divided into a test and a control group. Children in the test group were initially treated with temporary restorations and were then given oral health education and preventive treatment by a specially trained chairside assistant. The preventive treatment included prophylaxis and topical fluoride treatment and was carried out at least 10 times a year. The mean 3-yr DFS increment was 1.7 in the test and 10.9 in the control group. The salivary counts of mutans streptococci were similar in test and control group at baseline and after 1 and 2 yr. At the examination after 3 yr, a difference was established between the two groups.     

赤藓糖醇抑制变异链球菌在牛奶中产酸的实验研究

目的:比较变异链球菌在不同浓度赤藓糖醇-牛奶混合液中的产酸情况.方法:配制含赤藓糖醇质量分数为2％、4％、6％、8％的赤藓糖醇-牛奶混合液,设纯牛奶作为阴性对照,磷酸盐缓冲液作为空白对照,各组溶液经灭菌处理,并经涂板检验无细菌生长后,分别接种变异链球菌菌悬液厌氧培养;培养0、2、4、6、8、10、12h测量各组溶液的pH值,数据用SPSS16.0统计软件进行重复测量设计方差分析.结果:培养0～4h各组的pH值均急速下降;6h以后,阴性组和空白组的pH值仍继续下降,而4个浓度赤藓糖醇—牛奶组的pH值变化微小.各时间点的pH值均以空白对照组最高,阴性对照组最低.培养2h时阴性对照组pH值低于8％赤藓糖醇—牛奶组(P ＜0.05);4 h后各时间点均低于赤藓糖醇—牛奶各浓度组(P＜0.05).不同浓度赤藓糖醇—牛奶组相比,4h前各组间均无统计学差异(P＞0.05);6h后各时间点,除6％与8％组相比无统计学差异(P＞0.05)外,其他各组间差异均有统计学意义(P＜0.05).结论:赤藓糖醇能够有效抑制变异链球菌在牛奶中产酸,且高浓度较低浓度效果更强.     

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.