CcpA regulates biofilm formation and competence in Streptococcus gordonii

Streptococcus gordonii is an important member of the oral biofilm community. As an oral commensal streptococcus, S. gordonii is considered beneficial in promoting biofilm homeostasis. CcpA is known as the central regulator of carbon catabolite repression in Gram‐positive bacteria and is also involved in the control of virulence gene expression. To further establish the role of CcpA as central regulator in S. gordonii, the effect of CcpA on biofilm formation and natural competence of S. gordonii was investigated. These phenotypic traits have been suggested to be important to oral streptococci in coping with environmental stress. Here we demonstrate that a CcpA mutant was severely impaired in its biofilm‐forming ability, showed a defect in extracellular polysaccharide production and reduced competence. The data suggest that CcpA is involved in the regulation of biofilm formation and competence development in S. gordonii.     

Mutational analysis of the adcCBA genes in Streptococcus gordonii biofilm formation

Streptococcus gordonii, a primary colonizer, is part of the pioneer biofilm consortium that initiates dental plaque development on tooth surfaces. An insertion of Tn917-lac transposon into the adcR gene produced a biofilm-defective phenotype. S. gordonii adcR is a regulatory gene and is part of an operon (adc) that includes three other genes, adcCBA. AdcC contains a putative consensus-binding site for adenosine triphosphate, AdcB is a putative hydrophobic membrane protein, and AdcA is a putative lipoprotein permease. Mutants were constructed by insertional inactivation in each of the three adcCBA genes and their effects on biofilm formation examined. The adcC::spec(R) and adcB::spec(R) mutations displayed a biofilm-defective phenotype, whereas the adcA::spec(R) mutant was biofilm-positive in a static polystyrene microtiter plate biofilm assay. All three mutants formed poor biofilms in a flow-cell system and were competence-defective, suggesting the adc operon plays an important role in S. gordonii biofilm formation and competence.     

Inhibition of Streptococcus mutans biofilm formation using extracts from Assam tea compared to green tea

ObjectiveStreptococcus mutans, a gram-positive oral bacterium, has been identified as one of the principal etiological agents of human dental caries. To clarify the nature of the difference anti-biofilm effect against S. mutans between Assam tea from Camellia sinensis var. assamica, partially fermented, and green tea from Camellia sinensis, non-fermented, active agents from the teas were purified.MethodsEffects of Assam tea and green tea samples on biofilm were assessed by using the conventional titer plate method and the human saliva-coated hydroxyapatite discs. The purification and identification of inhibitors were performed by using ultrafiltration with centrifugal filter devices and high performance liquid chromatography.ResultsAssam tea has stronger biofilm inhibition activity against S. mutans than green tea. A substance of <10 kDa in mass in Assam tea had a high concentration of galloylated catechins and a stronger biofilm inhibiting activity than green tea. In contrast, substances >10 kDa in mass from green tea included higher concentrations of polysaccharides composed of galacturonic acid, such as pectin, that enhance biofilm formation.ConclusionsThe higher concentrations of galloylated catechins in Assam tea may assist in prevention of dental caries, whereas in green tea, this mode of inhibition was likely offset by the presence of pectin. Purification of catechins in partially fermented Assam tea with lower-molecular-weight polysaccharide than pectin may be useful for developing oral care products such as toothpaste and oral care gel pastes.     

呋喃C-30对变异链球菌生物膜早期形成的影响

目的：探讨密度感应拮抗剂呋喃C-30对变异链球菌生物膜早期形成的影响。方法：将体外合成的密度感应拮抗剂呋喃C-30按终浓度10、100μmol/L分别配制于含变异链球菌的牛心脑浸液培养基,37℃微需氧培养24h,形成生物膜后,用生物膜定量分析仪检测生物膜形成的量。结果：100μmol/L呋喃C-30组变异链球菌生物膜的形成受到显著抑制,磁珠成像开始减弱和完全消失的时间均迟于对照组;生物膜开始形成后,其生物膜形成指数低于对照组,差异有统计学意义（P〈0.05）。结论：呋喃C-30在100μmol/L浓度时能有效抑制变异链球菌生物膜的形成,其应用可能为龋病防治提供新的思路。     

A review on the characterization of a novel oral Veillonella species,V. tobetsuensis,and its role in oral biofilm formation

The genus Veillonella consists of small, strictly anaerobic, gram-negative cocci that lack flagella, spores, and a capsule. Although 11 species have been established in the genus Veillonella, five species, namely V. atypica, V. denticariosi, V. dispar, V. parvula, and V. rogosae, are recognized as oral Veillonella. The main habitats of oral Veillonella are the tongue, dental biofilm, and the buccal mucosa, and correspondingly it has been suggested that oral Veillonella contributes to oral biofilm formation and may lead to the formation of dental disease such as periodontitis.Recently, unknown strains displaying all phenotypic characteristics of the genus Veillonella were isolated from human tongue biofilm. The production of a major cellular fatty acid (C_13:0 and C_17:1ω8) was consistent with that of other members of the genus Veillonella. Based on the comparative analysis of 16S rDNA, dnaK, and rpoB gene sequences, the unknown strains represent a novel species, and were named Veillonella tobetsuensis.V. tobetsuensis was detected by PCR using specific primers based on the dnaK gene sequence in 5 out of 27 subjects with other oral Veillonella species. The prevalence of V. tobetsuensis ranged from 7.6 to 20.0% in these subjects.Using the wire method, all six oral Veillonella species stimulated the formation of biofilm by Streptococcus gordonii. The greatest amount of biofilm was formed by S. gordonii in the presence of V. tobetsuensis. These results suggest that among the oral Veillonella species, V. tobetsuensis plays an important role in biofilm formation by S. gordonii.     

Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro

OBJECTIVE: Sodium benzoate (SB), potassium sorbate (PS) and sodium nitrite (SN) are commonly used food preservatives. In this in vitro study, the effects of these substances on biofilm formation of Streptococcus mutans were analysed. METHODS: In addition to the microtiter plate test (MPT), a biofilm reactor containing bovine enamel slabs (BES) was used to study the influence of food preservatives on biofilm formation in 5 independent periods of 4 days each. These included one period with chlorhexidine digluconate (CHX) as a positive control as well as a period with growth medium alone as a negative control. The vitality of the biofilm on BES was detected using live/dead staining and confocal laser scanning microscopy. Additionally, the number of colony forming units (CFU) was determined. RESULTS: In MPT 0.12% SN significantly reduced the biofilm formation. PS at a concentration of 0.4% tended to inhibit biofilm formation, whereas the inhibition for 0.8% PS was significant. Less inhibition was caused by 0.8% SB. In the biofilm reactor 0.06% of SN, 0.1% of SB and 0.1% PS significantly reduced the covering grade as well as the CFU of the biofilm. Biofilm vitality was reduced significantly by CHX to a level of 32.5% compared to the control. Only SB reduced the vitality to a level of 19.1%. SN and PS showed no influence on biofilm vitality. CONCLUSION: This study indicates the potential of food preservatives as inhibitory agents in S. mutans biofilm formation, which should be kept in mind when studying the effects of conserved food on dental plaque biofilm in situ.     

Synthetic bromated furanone inhibits autoinducer-2-mediated communication and biofilm formation in oral streptococci

INTRODUCTION: Autoinducer-2 (AI-2) is a widespread communication-signal molecule that allows bacteria to sense and react to environmental factors. In some streptococci AI-2 is reported to be involved in virulence expression and biofilm formation. It has earlier been shown that the alga Delisea pulchra produces bromated furanones, which prevent bacterial colonization of the algae. METHODS AND RESULTS: We have previously published a novel and simple synthesis of (Z)-5-bromomethylene-2(5H)-furanone. In this study we showed that our synthesized furanone inhibited biofilm formation and bioluminescence induction by Streptococcus anginosus, Streptococcus intermedius, and Streptococcus mutans, as well as bioluminescence induction by Vibrio harveyi BB152. CONCLUSION: We suggest that the effect is linked to interference with the AI-2 signaling pathway because adding furanone to the medium had no effect on the ability of the AI-2-defective S. anginosus luxS and S. intermedius luxS mutants to form biofilms.     

In vitro Streptococcus mutans adhesion and biofilm formation on different esthetic orthodontic archwires

ObjectivesTo evaluate the ability of different esthetic archwires to retain oral biofilms in vitro.Materials and MethodsSeven different brands of coated orthodontic archwires were tested: two epoxy coated, two polytetrafluoroethylene coated, two rhodium coated, and one silver plus polymer coated. Conventional uncoated metallic archwires were used as controls. Streptococus mutans adherence to archwires was quantified by colony count following 24 hours of biolfilm growth, and total wire-associated biofilm was measured using a crystal violet staining assay. For both tests, two conditions were used: 0% sucrose and 3% sucrose. For statistical analysis, P < .05 was considered as statistically significant.ResultsFor S. mutans colony forming units per biofilm, there were no statistically significant differences among the various archwires (P = .795 for 0% sucrose; P = .905 for 3% sucrose). Regarding total biofilm formed on archwires in the 3% sucrose condition, there were statistically significant differences in crystal violet staining only for the comparison between Niti Micro Dental White and Copper Ni-Ti wires (P < .05).ConclusionsThe clinical use of esthetic-coated orthodontic wires may be considered to have similar risks as uncoated archwires for biofilm retention.     

Influence of sucrose and xylitol on an early Streptococcus mutans biofilm in a dental simulator

ObjectivesIn vitro methods to study dental biofilms are useful in finding ways to support a healthy microbial balance in the oral cavity. The effects of sucrose, xylitol, and their combination on three strains of Streptococcus mutans and one strain of Streptococcus sobrinus were studied using a dental simulator.MethodsA simulator was used to mimic the oral cavity environment. It provided a continuous-flow system using artificial saliva (AS), constant temperature, mixing, and hydroxyapatite (HA) surface in which the influence of xylitol was studied. The quantities of planktonic and adhered bacteria were measured by real-time qPCR.ResultsCompared against the untreated AS, adding 1% sucrose increased the bacterial colonization of HA (p < 0.0001) whereas 2% xylitol decreased it (p < 0.05), with the exception of clinical S. mutans isolate 117. The combination of xylitol and sucrose decreased the bacterial quantities within the AS and the colonization on the HA by clinical S. mutans isolate 2366 was reduced (p < 0.05). Increasing the concentration (2%–5%) of xylitol caused a reduction in bacterial counts even in the presence of sucrose.ConclusionsThe continuous-culture biofilm model showed that within a young biofilm, sucrose significantly promotes whereas xylitol reduces bacterial colonization and proliferation. The results indicate that xylitol affects the ability of certain S. mutans strains to adhere to the HA. Clinical studies have also shown that xylitol consumption decreases caries incidence and reduces the amount of plaque. This study contributes to the understanding of the mechanism behind these clinical observations.     

戈登链球菌对牙龈卟啉单胞菌生物膜形成的影响

目的 观察戈登链球菌（S.gordonii）对牙龈卟啉单胞菌（P. gingivalis）生物膜超微结构及生物膜中牙龈卟啉单胞菌数量的影响。方法 分别形成P. gingivalis单菌种生物膜和P. gingivalis－S.gordonii混合生物膜,利用扫描电镜观察其超微结构,定量PCR法对生物膜中P. gingivalis数量进行定量分析。采用SPSS 13.0软件包对数据进行统计学分析。结果 P. gingivalis生长72 h,与单菌种生物膜相比,P. gingivalis-S.gordonii混合生物膜形成量明显增多;而且混合生物膜的超微结构更规则、有序、多孔隙。在生物膜形成24、48、72 h后,混合生物膜中P. gingivalis的数量分别是单菌种生物膜中P. gingivalis数量的5.4、3.8和4.4倍。结论 早期定植菌S.gordonii的存在,使P. gingivalis更容易定植、形成生物膜。     

Effects of sub-minimum inhibitory concentrations of lemon essential oil on the acid tolerance and biofilm formation of Streptococcus mutans

ObjectivesLemon essential oil (LEO) is a kind of secondary metabolite from lemon peels and has been found to inhibit cariogenic bacteria for decades. However, its effects on main cariogenic virulence factors are rarely reported. The present study aimed to investigate the effects of sub-minimum inhibitory concentrations (sub-MICs) of LEO on the acid tolerance and biofilm formation of Streptococcus mutans (S. mutans) and preliminarily reveal the possible underlying mechanisms.DesignsEffects of LEO on the acid tolerance and biofilm formation of S. mutans were investigated by the broth dilution method and crystal violet staining method respectively. The expression of luxS, srtA and spaP gene was also determined to explore the underlying mechanism. In addition, Tea polyphenols (TP), a major natural inhibitor of cariogenic virulence factors, and limonene (LIM), the major component of LEO, were selected as comparisons to evaluate the effects of LEO.ResultsSub-MICs of LEO, LIM and TP exhibited a dose-dependent inhibition of growth of S. mutans at pH ranging from 4.0 to 7.0. The formation of S. mutans biofilm was remarkably inhibited and the inhibitory rates of LEO, LIM and TP were 97.87%, 94.88% and 96.01% respectively at 1/2 MIC. Similarly, a down-regulation was observed in the expression of luxS, srtA and spaP gene at sub-MIC levels.ConclusionsEffects of LEO were similar or slightly stronger than LIM and TP, suggesting that LEO might represent a novel, natural anticarious agent that inhibited the specific genes associated with bacterial acid tolerance and biofilm formation without necessarily affecting the growth of oral bacteria.     

Clotrimazole and econazole inhibit Streptococcus mutans biofilm and virulence in vitro

Objective: The aim of this study was to determine the inhibitory effect of eight antifungal drugs on S. mutans growth, biofilm formation and virulence factors.MethodsThe actions of antifungal drugs on S. mutans were determined by recovery plates and survival kinetic curves. Biofilms were observed by scanning electron microscopy and the viable cells were recovered on BHI plates, meanwhile biofilms were stained by BacLight live/dead kit to investigate the biofilm viability. Bacteria/extracellular polysaccharides staining assays were performed to determine the EPS production of S. mutans biofilms. Acidogenicity and acidurity of S. mutans were determined using pH drop and acid tolerance assays, and the expression of ldh gene was evaluated using qPCR.ResultsWe found that clotrimazole (CTR) and econazole (ECO) showed antibacterial activities on S. mutans UA159 and S. mutans clinical isolates at 12.5 and 25 mg/L, respectively. CTR and ECO could also inhibit S. mutans biofilm formation and reduce the viability of preformed biofilm. CTR and ECO affected the live/dead ratio and the EPS/bacteria ratio of S. mutans biofilms. CTR and ECO also inhibited the pH drop, lactate acid production, and acid tolerance. The abilities of CTR and ECO to inhibit S. mutans ldh expression were also confirmed.ConclusionsWe found that two antifungal azoles, CTR and ECO, had the abilities to inhibit the growth and biofilm formation of S. mutans and more importantly, they could also inhibit the virulence factors of S. mutans.     

十二烷基硫酸钠对血链菌生物膜的作用

目的:观察十二烷基硫酸钠(sodiumdoecylsulfate,SDS)促进血链菌生物膜表面脱落的效果,并对其和氯己定、玉洁纯联合应用的效果进行比较。方法:在模拟人口腔环境的人工口腔内,形成4h血链菌生物膜,分别放入300mmol·L-1SDS、300mmol·L-1SDS+2%氯己定和300mmol·L-1SDS+0.3%玉洁纯中,作用时间为3、10、30min,然后应用激光共聚焦扫描显微镜和死菌/活菌荧光染色技术相结合,比较其促进血链菌生物膜表面脱落的作用,对所得数据进行方差分析。结果:300mmol·L-1SDS应用于4h血链菌生物膜,可以有效促进细菌脱落,300mmol·L-1SDS和2%氯己定联合应用的效果较差;300mmol·L-1SDS和0.3%玉洁纯联合应用于生物膜3min,细菌几乎全部脱落。结论:300mmol·L-1SDS和0.3%玉洁纯联合应用,对促进菌斑生物膜脱落的作用最强。     

不同分子量的壳聚糖对血链菌生物膜的脱落作用

目的:观察不同分子量的壳聚糖促进血链菌生物膜表面的脱落作用。方法:在模拟人口腔环境的人工口腔内,形成4h血链菌生物膜,分别放入黏度5cps(小分子量)、80cps(中分子量)和600cps(大分子量)的2%壳聚糖溶液中,作用时间为3、10、30min,然后应用激光共聚焦扫描显微镜和死菌/活菌荧光染色技术相结合,比较其促进血链菌生物膜表面脱落的作用,对所得数据进行方差分析。结果:壳聚糖应用于4h血链菌生物膜,可以有效促进细菌脱落(P<0.05),黏度5cps(小分子量)的壳聚糖的作用效果最好(P<0.01)。结论:黏度5cps(小分子量)的壳聚糖是一种有效促进菌斑生物膜脱落的药物。