Analysis of Streptococcus mutans biofilm proteins recognized by salivary immunoglobulin A

Introduction:  The purpose of this study was to examine the Streptococcus mutans biofilm cellular proteins recognized by immunoglobulin A (IgA) in saliva from various caries-defined populations.
Methods:  Biofilm and planktonic S. mutans UA159 cells were prepared. The proteins were extracted, separated by two-dimensional gel electrophoresis, transferred to blotting membranes, and probed for IgA using individual saliva samples from three groups of subjects; those who developed 0 caries (no active caries), 5–9 caries (medium), or more than 10 caries (severe) over a 12-month interval.
Results:  Several proteins were recognized by salivary IgA in all groups of saliva but spot distribution and intensity varied greatly between the groups, and some proteins were recognized more strongly in biofilm cells than in planktonic culture, and vice versa. Furthermore, 15 proteins were only recognized by saliva from the 'no active caries' group, and four proteins were recognized by saliva samples from subjects in all three groups. Specifically, antigen I/II was recognized less in biofilm cells by caries-free saliva compared with planktonic cells. However, salivary IgA antibody to antigen I/II was absent in blots using saliva from the 'medium caries' and 'severe caries' groups.
Conclusion:  The bacterial molecules recognized by caries-free saliva are significant factors for S. mutans caries formation, and their inhibition could be a therapeutic target. In addition, saliva of caries-free subjects includes significant IgA antibody against antigen I/II of S. mutans , indicating a protective mechanism. However, microorganisms may protect themselves from host immune attack by forming biofilms and decreasing expression of antigen I/II.     

Influence of starvation and biofilm formation on acid resistance of Streptococcus mutans

The aim of this study was to investigate acid resistance induced by starvation or biofilm formation in Streptococcus mutans ATCC 25175. The artificial biofilms were made on cover glasses, starved for 24 h and immersed in 0.1 M lactate buffer at pH 3.8 for 10 min. The biofilms were also exposed to 5% sucrose solution for 20 min to simulate acid shock produced by sucrose metabolism. Confocal laser scanning microscopy with fluorescein isothiocyanate staining measured the resultant minimum pH in biofilms. Live and dead organisms in biofilms were differentiated by confocal laser scanning microscopy with proidium iodide and SYTO9 staining. The same processes were used to treat planktonic organisms. The results showed that starved biofilms or planktonic cells showed significantly more viable bacteria after acid shock induced either by lactic acid or during sucrose consumption than non-starved biofilms or planktonic cells. In addition, biofilms showed greater resistance to acid shock induced by lactic acid than planktonic cells, whereas similar results were obtained where sucrose was used as a carbon source to reduce pH in biofilms and planktonic cells. Thus, it is suggested that starvation protects both biofilm and planktonic S. mutans from acid shock induced either by lactic acid or during sucrose consumption, while biofilm formation seemed to protect bacteria only from acid shock induced by pH 3.8 lactate buffer but not the acid shock of a slightly higher pH produced during sucrose consumption.     

表面蛋白抗原P及其在变异链球菌生物膜形成中的作用

变异链球菌表面蛋白抗原(SPA)P是一类介导细菌黏附和生物膜形成的重要的黏附毒力因子,具有高度保守性.SPAP含有1 561个氨基酸残基,其线性结构氨基酸序列由前导肽区、N端、A区、V区、P区、C端和细胞壁锚着端组成.SPAP具有的淀粉样纤维特性,在细菌生物膜形成中十分重要.SPAP可与牙面获得性膜中的唾液成分结合,介导变异链球菌对牙面的初始黏附.SPAP通过分选酶转移肽共价结合到细菌胞壁表面,在内源性的表面蛋白释放酶作用下又可从细菌胞壁表面释放出来,从而使变异链球菌生物膜降解.本文就SPAP的结构、淀粉样纤维特性,变异链球菌黏附,SPAP各区在生物膜形成中的作用等研究进展作一综述,明确其生物学特性,对于龋病病因学和龋病防治的意义不言而喻.     

变异链球菌LuxS基因缺陷突变株的构建及其生物膜结构的初步观察研究

目的:通过同源重组法构建变异链球菌LuxS基因缺陷突变菌株,比较其与变异链球菌UA159标准株在生物膜形成上的差异。方法:运用基因同源重组方法将氯霉素抗性基因(Cmr)与LuxS基因上下游区域的2个基因片段按一定顺序重组到PUC载体的多克隆位点中,构建出带氯霉素抗性标志的重组质粒,将载体质粒转化到含完整LuxS基因的变异链球菌UA159中,利用氯霉素抗性筛选出LuxS基因缺陷的突变株,并利用聚合酶链式反应(PCR)和哈氏弧菌发光实验进行检测。以釉质磨片为载体,在扫描电镜下观察上述两菌株含有20 g/L葡萄糖、20 g/L蔗糖的乳酪消化胨酵母(TPY)液体培养基中形成24 h的生物膜。结果:PCR基因扩增结果显示:突变株LuxS基因已被Cmr基因完全替换,成功的构建了变异链球菌LuxS基因突变株,并且突变株不能诱导哈氏弧菌BB170的生物发光。当用葡萄糖作为补充糖源时,变异链球菌UA159标准株和LuxS基因突变株所形成的生物膜表现型未见明显差异;而用蔗糖作为补充糖源时,两菌株所形成的生物膜有明显不同,UA159生物膜相对平滑均质,而LuxS基因突变株生物膜呈松散的蜂房状,基质间有较大的间隙,形成较大的团簇状菌落。结论:成功构建出LuxS基因缺陷的变异链球菌突变株,与变异链球菌UA159标准株其生物膜结构发生改变,提示LuxS会影响变异链球菌生物膜的形成。     

Effect of fluoride on glucose incorporation and metabolism in biofilm cells of Streptococcus mutans

The aim of this study was two-fold: firstly, to study the effect of high fluoride concentrations on carbohydrate metabolism in Streptococcus mutans present in biofilms on hydroxyapatite; and, secondly, to evaluate the effect of fluoride-bound hydroxyapatite on lactic acid formation in growing biofilms of Strep. mutans. Biofilms of a clinical strain of Strep. mutans on saliva-coated hydroxyapatite beads were incubated with sodium fluoride over a wide range of concentrations. At high fluoride concentrations (>10 mM) the incorporation of [14C]-labeled glucose decreased by 80-85%, at both pH 7.0 and 5.6. At lower fluoride concentrations, the effect of fluoride on the incorporation of labeled glucose was pH-dependent in both biofilm cells and in planktonic cells. At pH 7.0, fluoride at concentrations < 10 mM had little or no effect. Pretreatment of hydroxyapatite discs with fluoride varnish (Fluor Protector) or fluoride solutions caused a statistically significant reduction of lactic acid formation in associated, growing biofilms of Strep. mutans. Fluoride varnish and 0.2% (47.6 mM) sodium fluoride solution exhibited a statistically significant inhibitory effect on lactate production.     

十肽对变异链球菌生物膜生长和结构影响的实验研究

目的：评价人工合成抗菌肽（十肽）对口腔变异链球菌生物膜生长、结构及活性的影响能力。方法???人工合成十肽（氨基酸序列：KKVVFKVKFK-NH2）通过对细菌生物膜药物最低抑制生物膜形成浓度（MBIC）和药物最低清除已形成生物膜浓度（MBRC）2个指标的测定,研究和评估十肽对变异链球菌单菌生物膜的抑制和清除作用;在激光共聚焦扫描显微镜（CLSM）下观察十肽作用下变异链球菌生物膜的形态、结构及活性变化。结果???当十肽浓度为62.5~125μg?mL-1时,能够抑制变异链球菌生物膜的形成;当十肽浓度为250~500μg?mL-1时,可破坏已形成24?h后的生物膜,CLSM下观察到十肽作用后生物膜结构残存,死菌增多,生物膜厚度明显降低。结论??新型人工合成抗菌肽（十肽）不仅可抑制主要致龋菌变异链球菌单菌生物膜的生长形成,而且可破坏已经形成24?h后的生物膜结构。     

The effects of orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva

Abstract

Objective. To investigate the effects of various orthodontic bonding steps on biofilm formation of Streptococcus mutans in the presence of saliva. Materials and methods: Hydroxyapatite (HA) and orthodontic adhesive (AD) disks were prepared to a uniform size. HA disks were etched with 37% phosphoric acid gel in the etched group (HE). In the primed group (HP), Transbond XT primer was applied to the etched HA surface and light-cured. For biofilm formation, Streptococcus mutans was grown on each specimen in a biofilm medium with either glucose or sucrose in the presence of fluid-phase UWS (F-UWS) or surface adsorbed saliva (S-UWS). The adherent bacteria were quantified by enumeration of the total viable counts of bacteria. Biofilms formed on each surface were examined by scanning electron microscopy. Results. When glucose was used, both F-UWS and S-UWS suppressed biofilm formation of S. mutans. Compared to HA and HE, biofilm formation was significantly inhibited on HP and AD in the presence of glucose. Biofilm-forming patterns that were inhibited by saliva were restored in a sucrose-containing medium. F-UWS promoted biofilm formation on HA and HE, while S-UWS significantly promoted biofilm formation on HP. S. mutans developed biofilm better on HA and HE than on AD when sucrose was used as the sole carbohydrate source. Conclusions. This study suggests that the biofilm development by S. mutans is significantly influenced by the orthodontic bonding procedure. Biofilm formation of S. mutans was inhibited on AD more than other surfaces, irrespective of the presence of saliva or a carbohydrate source.     

套式PCR检测唾液中变形链球菌及远缘链球菌

目的 探索一种快速、简便地从人类唾液中同时检测变形链球菌和远缘链球菌的方法。方法 分别以变形链球菌gtfI和远缘链球菌gtfB基因设计两组成套引物，首先用套式PCR（二次PCR）检测变形链球菌和远缘链球菌标准株和临床株，然后用套式PCR直接从唾液中检测这两种细菌。结果 变链菌（血清型c,e,f)的标准株及临床株第1次PCR扩增产物为517bp，第2次扩增产物为468bp；远缘链球菌（血清型d,g)及道勒链球菌（血清型h)的标准株及临床株第1次PCR扩增产物为712bp，第2次扩增产物为663bp;其他异种菌均不能扩增出产物，因此该PCR检测具有高度的特异性。细菌纯培养物及唾液PCR检测的敏感性分别是：第1次PCR为10^5CFU，第2次PCR为10^3CFU。结论 套式PCR能快速在人类唾液中同时检测变形链球菌和远缘链球菌。该检测方法有望运用于临床检测，对揭示两种细菌与龋病发生关系的研究具有一定价值。     

The inability of Streptococcus mutans and Lactobacillus acidophilus to form a biofilm in vitro on dentine pretreated with ozone

Background: The use of ozone therapy in the treatment of dental caries is equivocal. The aim of this study was to use an in vitro model to determine the effects of prior ozone application to dentine on biofilm formation and to measure any associated reduction in bacteria viability. Methods: Twenty dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials. Ten dentine discs were infused with ozone for 40 seconds, 10 samples remained untreated as a control. The vials were filled with nutrient medium, sterilized and placed into the outflow from a continuous chemostat culture of Streptococcus mutans and Lactobacillus acidophilus for four weeks. At the conclusion of the experiment bacterial growth was monitored by taking optical density readings of the growth medium in each vial and the outer surface of the dentine specimens were examined by scanning electron microscopy as shown by SEM analysis. Results: Ozone infusion prevented biofilm formation on all the treated samples while there was substantial biofilm present on the control specimens. While the average optical density of the control specimens was almost twice that of the ozone infused dentine (0.710 for the control with a SD of 0.288 and 0.446 for the ozonated samples with a SD of 0.371), the results were not significant (p > 0.05). Conclusions: This preliminary study has shown that the infusion of ozone into non‐carious dentine prevented biofilm formation in vitro from S. mutans and L. acidophilus over a four‐week period. The possibility exists that ozone treatment may alter the surface wettability of dentine through reaction with organic constituents.     

变形链球菌(血清型C)临床分离株AP-PCR基因分型

目的 探讨变形链球菌（血清型C）菌珠基因型与龋发生的关系。方法 用Chelex法撮细菌染色体DNA,经多次实验自行确立AP－PCR最佳反应条件,并对278株来自不同龋敏感个体的临床分离株进行AP－PCR基因型分析。结果 278株变链菌临床分离株共区分出105种基因型,无龋个体84．21％携带1种基因型,而高龋患者95％携带2种或2种以上基因型。结论 变链菌菌株间存在明显的遗传多态性,个体携带变链球菌基因型的种数与其致龋性密切相关。     

Deletion of gtfC of Streptococcus mutans has no influence on the composition of a mixed-species in vitro biofilm model of supragingival plaque

Glucosyltransferases from Streptococcus mutans are thought to play an important role in bacterial adherence to the tooth surface. The goal of the present study was to determine the effect of the deletion of the gtfC gene, which encodes a glucosyltransferase that catalyses primarily the formation of insoluble glucan (mutan), on colonization of S. mutans in a mixed-species biofilm model of supragingival plaque. A gtfC deletion mutant of S. mutans UA159 grew poorly in biofilms on a polystyrene surface in Todd–Hewitt medium containing sucrose, but biofilm formation in the semi-defined fluid universal medium (FUM) was not affected. The S. mutans gtfC mutant colonized with the same efficiency as the wild-type strain when grown together with five other species in a mixed-species biofilm on hydroxyapatite in a mixture of FUM and saliva with pulses of sucrose and showed the same ability to demineralize enamel in vitro . Colonization of mutant and wild-type strains was also equal in an association experiment in specific-pathogen-free rats. However, the gtfC mutant gave rise to more dentinal fissure lesions and smooth surface caries than the wild-type strain; this could be caused by a change in diffusion properties as a result of to the lack of mutan.     

环境氧条件影响寡发酵链球菌抑制变形链球菌作用的研究

目的 探讨外界环境氧气条件的改变对寡发酵链球菌(Streptococcus oligofermentans,So)与变形链球菌(Streptococcus mutans,Sm)之间抑制作用的影响,以及对So产过氧化氢能力的影响,以期探讨这两种细菌在不同口腔环境条件下的共存模式.方法 通过二氧化碳培养法建立细菌培养的有氧和厌氧环境;运用平板培养法观察So与Sm之间的抑制作用;运用4-氨基安替吡啉-辣根过氧化物酶法测定有氧和厌氧环境下So对数生长期各时间段过氧化氢的产量和So生长周期过氧化氢初始产生速率.结果 同时接种So和Sm,厌氧环境下未见So抑制Sm,有氧环境下So轻度抑制Sm,受抑制区面积约为菌膜面积的1/5;先接种So再接种Sm,两种环境下均可见So抑制Sm,厌氧环境下受抑制区面积约为菌膜面积的1/5,有氧环境下约为4/5;先接种Sm再接种So,两种环境下均未见So生长;有氧环境下So对数生长期各时间段过氧化氢产量明显高于厌氧环境(P<0.05);厌氧环境下So生长周期过氧化氢初始产生速率为每分钟(11.84±3.97) μmol/L,仅为有氧环境时[(24.13±4.46) μmol/L]的49%(P<0.05).结论 环境氧条件影响So对Sm的抑制作用,有氧环境下抑制作用更强;有氧环境下So产过氧化氢的能力强于厌氧环境.

Abstract：

Objective To investigate the effect of environmental oxygen on the inhibition between Streptococcus oligofermentans(So) and Streptococcus mutans(Sm) and the producibilities of hydrogen peroxide by So. Methods The aerobic and anaerobic environment was established by the carbon dioxide cultivation. The inhibition between So and Sm was observed by plating method. The production and synthesis rates of hydrogen peroxide by So were determined in both aerobic and anaerobic environment by 4-ATTP-horseradish peroxidase method at A510. Results When both Sm and So were inoculated at the same time, Sm was not inhibited under the anaerobic environment, vice versa. Sm was slightly inhibited by So under the aerobic environment, the inhibition area was 1/5 of all bacterial membrane. When So was cultivated first and then Sm applied, So could inhibite Sm growth under both anaerobic and aerobic conditions. The inhibition area was 1/5 of bacterial membrane under the anaerobic environment, and 4/5 under the aerobic environment. When Sm was cultivated first and then So applied, So was unable to proliferate under both conditions. During the logarithmic phase, the production of H2O2 by So under the aerobic environment was higher than under the anaerobic environment (P<0.05).The initial synthesis rate of H2O2 by So during growth cycle under the anaerobic condition was (11.84±3.97) μmol/L per minute, which was only 49% of that under the aerobic environment[(24.13±4.46) μmol/L per minute].Conclusions The oxygen has the effect on the inhibition between So and Sm, and the inhibition in the aerobic environment is much stronger than in the anaerobic environment. The synthesis ability of hydrogen peroxide by So under the aerobic environment is higher than under the anaerobic environment.     

Antineoplastic agents inhibit the growth of Streptococcus mutans and Streptococcus sanguis in vitro

The effect of methotrexate (MTX) and doxorubicin on the growth, metabolism and ultrastructure of Streptococcus mutans and Streptococcus sanguis was studied in vitro. Both anticancer drugs exerted an inhibitory effect on the oral streptococci. MTX was more inhibitory than doxorubicin. The minimum inhibitory concentrations (MICs) of MTX to S. mutans were 0.25-2.5 micrograms/ml and that of doxorubicin 0.2 mg/ml. The MICs of MTX and doxorubicin to S. sanguis were 0.025 micrograms/ml and 2.0-0.02 mg/ml, respectively. When saliva samples of patients with malignant tumors receiving various doses of MTX were analyzed, MTX was found to be secreted into the oral cavity at concentrations ranging from 0.014 to 4.486 micrograms/ml. The saliva of these patients was also found to inhibit the growth of S. mutans, and the inhibition zones were in accordance with the MIC values observed. The results suggest that anticancer therapy must be taken into account when the salivary microbiological findings of cancer patients are interpreted.     

The oligopeptide (opp) gene cluster of Streptococcus mutans: identification, prevalence, and characterization

INTRODUCTION: The Opp system is an ATP-binding cassette-type transporter formed by membrane-associated proteins required for the uptake of oligopeptides in bacteria. In gram-positive bacteria, the Opp system, and particularly the oligopeptide-binding protein (OppA), has been shown to be involved in different aspects of cell physiology, including intercellular communication and binding to host proteins. METHODS: In the present study we began to investigate the Opp system of Streptococcus mutans, the main etiological agent of dental caries. RESULTS: Five opp genes (oppABCDF) organized in a single operon were identified in the genome of the S. mutans UA159 strain. Amino acid sequence analyses showed that the S. mutans OppA is closely related to an ortholog found in Streptococcus agalactiae. Incubation of S. mutans UA159 cells with an anti-OppA-specific serum did not inhibit biofilm formation on polystyrene plates. Moreover, S. mutans UA159 derivatives carrying deletions on the oppA or oppB genes did not show significant growth impairment, increased sensitivity to aminopterin, or defective capacity to form biofilms on polystyrene wells in the presence or not of saliva. Remarkably, only two out of three laboratory strains and one out of seven clinical strains recovered from tooth decay processes harbored a copy of the oppA gene and expressed the OppA protein. CONCLUSION: Collectively, these results indicate that, in contrast to other Streptococcus species, the S. mutans Opp system, and particularly the OppA protein, does not represent an important trait required for growth and colonization.     

Isolation and characterization of Lactobacillus species inhibiting the formation of Streptococcus mutans biofilm

Oral bacterium S11 was isolated from the saliva of young children without dental caries and with little or no visible supragingival plaque. The S11 strain showed 99.5% similarity with Lactobacillus fermentum, and was identified on the basis of biochemical characteristics and a 16S rDNA sequence. S11 strain and its culture supernatant significantly inhibited the formation of the insoluble glucan produced by Streptococcus mutans Ingbritt. S11 did not affect the multiplication of S. mutans Ingbritt, but the adherence of S. mutans Ingbritt onto cuvette walls was inhibited completely.     

蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响

目的探讨蔗糖环境对寡发酵链球菌与变异链球菌双菌种生物膜形成的影响,并与血链球菌和变异链球菌的双菌种生物膜形成进行比较。方法运用菌落计数法观察蔗糖环境下唾液包被的玻璃生物模型中单/双菌株寡发酵链球菌,变异链球菌和血链球菌24 h生物膜形成情况;运用激光共聚焦显微镜观察蔗糖环境下寡发酵链球菌,变异链球菌和血链球菌单/双菌株生物模型中24h生物膜形成厚度。结果无糖环境下,单菌株模型中,菌落数:血链球菌(55.67±5.36)>变异链球菌(53.48±2.63)(P>0.05)>寡发酵链球菌(46.24±2.34)(P<0.05);生物膜厚度:血链球菌(17.23±3.82)>变异链球菌(15.16±4.21)(P>0.05)>寡发酵链球菌(10.54±4.37)(P<0.05)。双菌株模型中,变异链球菌菌落数降低幅度:血链球菌组>寡发酵链球菌组(P<0.05)。生物膜厚度:血链球菌组(8.12±2.82)<寡发酵链球菌组(11.27±3.55)(P<0.05)。蔗糖环境下,单菌株模型中,菌落数:变异链球菌(58.54±2.74)>血链球菌(51.87±5.35)>寡发酵链球菌(48.57±3.05)(P<0.05)生物膜厚度:变异链球菌(20.63±5.71)>血链球菌(13.37±4.93)>寡发酵链球菌(12.45±4.62)(P<0.05)双菌株模型中;变异链球菌菌落数降低幅度,寡发酵链球菌组>血链球菌组(P<0.05),生物膜厚度:寡发酵链球菌组(6.67±2.19)<血链球菌组(10.45±2.72)(P<0.05)。结论外界糖环境影响寡发酵链球菌和血链球菌对变异链球菌的抑制作用,蔗糖环境下,寡发酵链球菌的抑制作用强于血链球菌。     

变异链球菌对人唾液腺细胞系免疫球蛋白受体影响的初步研究

目的:观察变异链球菌(S.mutans)对人唾液腺细胞系(HSG)中多聚免疫球蛋白受体(Pigr)表达的影响.方法:用免疫荧光染色验证细胞系来源与Pigr在HSG中的表达.将对数生长中期的S.mutans水热灭活后与HSG细胞共培养,CCK-8法观察细胞增殖,qPCR与Western blot检测S.mutans对Pi...     

变形链球菌耐氟菌株、远缘链球菌耐氟菌株与亲代菌株基因组AP-PCR的比较

目的：利用随机引物聚合酶链反应(AP-PCR)技术，对变形链球菌、远缘链球菌和耐氟菌株的基因组进行比较，判定变形链球菌耐氟突变、远缘链球菌耐氟突变后，遗传型是否发生改变方法：采用AP-PCR技术，经多次实验自行确立AP-PCR反应条件，对变形链球菌、远缘链球菌及耐氟闲株的基因组进行比较结果：变形链球菌耐氟菌株、远缘链球蒲耐氟菌株的基因组已发生改变。结论：变形链球菌耐氟突变、远缘链球菌耐氟突变后，已具有自身的遗传性。     

Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model

There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar‐overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited.