杨梅黄酮对变形链球菌毒力因子及生物膜的抑制作用研究

目的:探讨杨梅黄酮对变形链球菌(Streptococcus mutans,S.mutans)ATCC 25175毒力因子和生物膜的作用。方法:用液体稀释法准确测定杨梅黄酮对变形链球菌的最小抑菌浓度(MIC)和耐酸性;用结晶紫染色法检测药物对变形链球菌生物膜形成的抑制率;用乳酸试剂盒检测药物对变形链球菌产酸的影响;用蒽酮法检测药物对不溶性多糖生成量的影响;利用场发射扫描电子显微镜(FESEM)和激光共聚焦显微镜(CLSM)观察及分析药物对生物膜的抑制作用。结果:杨梅黄酮对变形链球菌的最小抑菌浓度为0.8 mg/mL,0.2～0.4 mg/mL的药物对生物膜的生成抑制作用明显,同时乳酸、不溶性多糖的生成量明显降低,0.4 mg/mL的药物对细菌耐酸性以及成熟生物膜的影响有显著性差异。结论:杨梅黄酮对于变形链球菌毒力因子和生物膜有显著的抑制作用。     

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。     

变异链球菌荧光报告株在树脂、玻璃离子表面形成生物膜能力的初步研究

目的：本研究通过报告基因技术与激光共聚焦显微镜（Confocal Laser Scanning Microscopy,CLSM）研究变异链球菌荧光报告株（UA140-mrfp）在不同修复材料表面形成生物膜能力,比较树脂与玻璃离子的抗菌性能,为研究双菌、多菌生物膜在材料表面的黏附打下基础,探索CLSM技术在研究生物膜领域中的优势。方法：制作（直径15mm,厚度0.5mm）大小相同的树脂片和玻璃离子片,在其表面形成单菌生物膜,用CLSM分别观察4h、8h、12h、24h生物膜生长情况,在不同位置用CLSM沿Z轴进行扫描,测量其厚度和组织结构。结果：CLSM观察结果显示在不同时段玻璃离子表面形成的生物膜量及其厚度均少于同一时间段树脂表面。结论：变异链球菌荧光报告株在玻璃离子表面形成生物膜的能力低于树脂表面。     

抗菌剂作用后变形链球菌生物膜细胞活力观察

目的:观察抗菌剂作用后变形链球菌生物膜细胞活力。方法:将已形成24h变形链球菌生物膜分别用1000μg/ml、500μg/ml、100μg/ml、50μg/ml、0μg/ml红霉素药液作用3h后取出,对这些生物膜分别进行两种不同的处理。A组将生物膜继续培养后,用死菌活菌荧光染色方法结合激光共聚焦扫描显微镜进行观察;B组将生物膜细胞刮下接种到培养基上,革兰染色观察。结果:A组每个标本均重新形成较完整的生物膜;B组每个标本均发现有变形链球菌生长。结论:变形链球菌生物膜被高浓度抗菌剂作用后,膜中存活的细菌仍能分裂繁殖,并可修复已损伤的生物膜。     

粪肠球菌生物膜的药物敏感性研究

目的 建立体外粪肠球菌生物膜模型,评价10%氢氧化钙溶液和2%洗必泰溶液去除粪肠球菌生物膜的效果.方法 在玻片上形成4、8、12、24、48 h粪肠球菌生物膜,荧光染色后,采用激光共聚焦扫描显微镜(CLSM)观察;用10%氢氧化钙溶液和2%洗必泰溶液分别处理24 h生物膜3、10、30 min,采用CLSM观察;ANO...     

luxS基因在变形链球菌生物膜形成中的硫代谢作用

目的 通过比较在不同条件培养基中变形链球菌Ingbritt C国际标准株及luxS基因缺陷株24 h生物膜形成的厚度及平均荧光强度,探讨luxS基因在变形链球菌生膜形成中的硫代谢作用.方法 建立以圆形玻片为载体的生物膜模型,通过营养补充实验培养两菌株生物膜,利用激光共聚焦扫描显微镜观察测定两菌株生物膜厚度和平均荧光强度.结果 当分别加入一定浓度半胱氨酸、蛋氨酸时,缺陷株生物膜厚度有明显增长,但未恢复至标准株水平;加入半胱氨酸,标准株生物膜厚度有所增加;当加入S-腺苷甲硫氨酸时,缺陷株与标准株生物膜的厚度均降低.结论 在变形链球菌生物膜形成中,luxS基因不但具有密度感应功能,在硫代谢中也起着重要作用.     

五倍子对菌斑生物膜内细菌的抑制作用

目的:应用人工口腔观察五倍子对菌斑生物膜内细菌的抑制作用。方法:采用液体二倍稀释法测定与致龋病关系较密切的4种细菌的最小抑菌浓度,并进一步在人工口腔中形成各实验菌的单一细菌菌斑生物膜,应用菌落计数技术观察五倍子水提取物对菌斑生物膜内细菌的抑制作用。结果:五倍子水提取物对变形链球菌、粘性放线菌、血链球菌、口腔链球菌的生长均有抑制作用,其中对变形链球菌、粘性放线菌和血链球菌的最小抑菌浓度(MIC)为64mg/ml,口腔链球菌为8mg/ml;不同浓度的五倍子水提取物对各实验菌形成的单一细菌菌斑生物膜均有一定的抑制作用,并呈浓度依赖性;即使采用大于MIC的浓度,也不能把釉质表面形成的生物膜完全抑制,釉质表面仍有细菌生长。结论:五倍子水提取物对菌斑生物膜内细菌具有良好的抑制作用;与浮游细菌相比,生物膜中的细菌对五倍子水提取物具有较强的抵抗力。     

细菌素免疫蛋白调控变形链球菌抗菌敏感性及生物膜形成的实验研究

目的 观察细菌素免疫蛋白相关基因对变形链球菌抗菌敏感性及生物膜形成的影响,探讨细菌素免疫蛋白与细菌抗菌剂耐受性的关系,为生物膜抗菌敏感性的研究提供基础数据.方法筛选培养细菌素免疫蛋白基因突变株,绘制生长曲线.酶标仪检测不同质量浓度氨苄青霉素(0.04、0.05、0.06、0.07及0.08 mg/L)、氟化钠(50、100、150、200及250 mg/L)及不同质量分数的次氯酸钠(0.078%、0.156%、0.313%、0.625%及1.250%)作用下变形链球菌标准株、△immA-和△immB-突变株菌液的吸光度值.应用最小生物膜清除浓度(minimal biofilm eradicatin concentration,MBEC)桩钉96孔板以连续稀释法检测醋酸氯己定对3种菌株生物膜的MBEC.应用激光共聚焦扫描显微镜(confocal laser scanning microscope,CLSM)定量分析标准株和突变株生物膜结构.结果 △immA-和△immB-突变株的迟缓期和稳定生长期均比标准株延时1 h.氨苄青霉素为0.06 mg/L时,标准株、△immA-突变株和△immB-突变株菌液吸光度值分别为0.334±0.016、0.027±0.016及0.047±0.018;氟化钠质量浓度为150 mg/L时,3种菌株菌液吸光度值分别为0.254±0.018、0.129±0.011及0.167±0.01;当次氯酸钠质量分数为0.313%时,3种菌株菌液吸光度值分别为0.467±0.008、0.017±0.006及0.050±0.006,以上各组抗菌剂中,标准株与突变株吸光度值差异均有统计学意义(P<0.01).醋酸氯己定对3种菌株的MBEC分别为6.25、1.57及3.13 mg/L.标准株生物膜厚度显著高于△immA-和△immB-突变株(P<0.01);标准株各层活菌比例均高于△immA-突变株(P<0.05);标准株中、外层活菌比例高于△immB-突变株(P<0.01),但内层活菌比例差异无统计学意义(P=0.191).结论细菌素免疫蛋白参与调控浮游细菌生长,尤其在生长初期;细菌素免疫蛋白相关基因缺陷使浮游态变形链球菌抗菌敏感性提高、抗菌剂MBEC降低及生物膜结构不成熟.     

姜黄素对变形链球菌UA159生长、粘附的体外实验研究

目的：探索姜黄素对变形链球菌UA159的抗菌能力及姜黄素对变形链球菌UAl59生物膜形成的影响。方法：微量肉汤稀释法检测姜黄素对变形链球菌的最低抑菌浓度（MIC）。分光光度计分析在抑菌浓度范围内姜黄素对变形链球菌生物膜形成的变化。结果：MIC法测得的姜黄素对变形链球菌的抑菌浓度为128wM,而15μM姜黄素即可显示出抑制变形链球菌生物膜形成的特性,且随着姜黄素浓度增加,抑制能力加强。结论：姜黄素具有抑制变形链球菌生物膜形成的特性,且该抑制能力不是建立在抑制变形链球菌生长基础上的。姜黄素可能成为龋病防治的新试剂。     

载银氧化石墨烯对变异链球菌生长及生物膜形成的影响

目的探讨载银氧化石墨烯（GO/Ag）纳米复合物对变异链球菌（S.mutans）生长及生物膜形成的抑制作用。 方法应用梯度稀释法检测GO/Ag、氧化石墨烯（GO）、纳米银（AgNPs）对变异链球菌的最小抑菌浓度（MIC）;菌落形成单位计数法比较GO/Ag、GO、AgNPs对变异链球菌的抑制作用;采用结晶紫染色法和XTT法测定变异链球菌24 h生物膜的生成量和活性;激光共聚焦扫描显微镜（CLSM）观察生物膜形态和计算24 h生物膜的活菌比例。数据以单因素方差分析（One-Way ANOVA）和LSD-t检验进行统计分析。 结果GO/Ag对变异链球菌的MIC为0.16 mg/mL。0.64 mg/mL GO/Ag对变异链球菌的抑菌率为（82.90 ± 4.87）%,与对照组间的差异有统计学意义（t= 30.804,P<0.001）。而GO和AgNPs材料浓度提升至2.56 mg/mL时,肉眼观察细菌仍未见明显减少。在0.16 mg/mL GO/Ag浓度下,变异链球菌生物膜生成量和活性的减少率分别为（25.12 ± 0.01）%和（38.90 ± 3.42）%,活菌比例为（42.76 ± 19.48）%,与空白对照组间的差异均有统计学意义（t_生成量= 7.274,P_生成量<0.001;t_活性= 7.765,P_活性<0.001;t_活菌比例= 10.412,P_活菌比例<0.001）。 结论与AgNPs和GO相比,GO/Ag新型纳米复合物对变异链球菌的生长具有较好的抑制作用,并能抑制其生物膜形成。     

Assessment of gene associated with Streptococcus mutans biofilm formation

Streptococcus mutans, the primary etiological agent of human dental caries and an obligate biofilm-forming bacterium, has developed a variety of mechanisms to colonize the tooth surface. Oral transmission of S. mutans through contact between mother and child is thought to be one of the risks of developing dental caries. In this study, I surveyed oral transmission frequency of S. mutans from mother to a 3-year-old child. In 19 (10.9%) of 174 mother-child pairs, S. mutans was isolated from both mother and chihld. The identities of genomic DNA from S. mutans in 5 (45.5%) of the 11 mother-child pairs were presented. Among those, the biofilms formed by S. mutans 3 c and 4 c, respectively, which had high and low ability of biofilm formation, were examined by confocal laser scanning microscopy (CLSM). Microscopic analysis revealed that the volume of biofilm formation in 3 c was higher than that in 4 c biofilm in the bottom. Furthermore, DNA microarrays were used to study the gene expression profiles of 3 c and 4 c biofilms. In this paper we describe that about 3.8% of genes showed differential expression; about 2.2% of genes were activated and about 1.6% were repressed in 3 c biofilm compared with 4 c biofilm. The present study suggests that biofilm gene expression is strongly associated with differential biofilm formation. Our identification of biofilm-involved genes points to mechanisms of the virulence of S. mutans and provides a first foothold for studying the natural history of S. mutans infections in mother and child.     

Differences between single- and dual-species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine

Streptococcus mutans, considered a primary pathogen in dental caries, thrives in dental plaque, which is a multispecies biofilm. Metabolic interactions between S. mutans and Veillonella parvula have been suggested. In this study we developed a biofilm model to quantify single-species (S. mutans or V. parvula) and dual-species (S. mutans and V. parvula) biofilm formation, and we identified the differences between the respective biofilms in terms of growth, acid formation, and response to chlorhexidine. Polystyrene 96-well microtiter plates were used for biofilm formation. These biofilms were exposed to various chlorhexidine concentrations (0.025-0.4 mg ml(-1)) and treatment conditions. Growth of the biofilms and the effects of chlorhexidine were evaluated by viable counts. Viability of the two species in all biofilm types was similar ( approximately 10(8) colony-forming units per well) after 72 h. Lactic acid accumulation of dual-species biofilms was significantly lower at 48 and 72 h than single-species biofilms of S. mutans. Dual-species biofilms were less susceptible to chlorhexidine than single-species biofilms when a neutralization step was included. These results indicate that bacteria in dual-species biofilms have different properties from bacteria in single-species biofilms.     

Extracellular polysaccharides do not inhibit the reaction between Streptococcus mutans and its specific immunoglobulin G (IgG) or penetration of the IgG through S. mutans biofilm

The present study investigated whether extracellular polysaccharides inhibit reaction between Streptococcus mutans and its specific immunoglobulin G (IgG) and penetration of the IgG through S. mutans biofilm. The planktonic organisms with or without extracellular polysaccharides were prepared, incubated with rabbit IgG against whole cell of S. mutans and fluorescein isothiocyanate (FITC)-conjugated goat affinity purified antibody to rabbit IgG. Biofilms with or without extracellular polysaccharides were formed on cover glasses and incubated with rabbit IgG against S. mutans and FITC-conjugated goat antibody to rabbit IgG. Then, biofilms were stained with propidium iodide. The amount of specific IgG binding on S. mutans was determined by FITC intensity with a fluorescence microplate reader. The penetration of IgG through biofilms was determined by confocal laser scanning microscopy. The results showed that the fluorescence intensity of FITC in planktonic organisms with extracellular polysaccharides was similar to that in planktonic organisms without extracellular polysaccharides, indicating that extracellular polysaccharides did not inhibit the reaction between S. mutans and its specific IgG. Although biofilms of S. mutans with extracellular polysaccharides were much thicker and denser than those without extracellular polysaccharides, the speed with which IgG penetrated through both of the biofilms did not differ significantly, suggesting that penetration of IgG through S. mutans biofilm was not affected by extracellular polysaccharides.     

变形链球菌luxS基因突变对口腔混合细菌生物膜的影响

目的 研究致龋菌变形链球菌luxS基因在口腔细菌混合培养形成牙菌斑生物膜中的作用。方法 将变形链球菌野生株(UA159)及其2种luxS基因突变株（luxS基因高表达株和luxS基因缺陷株）分别与口腔细菌嗜酸乳杆菌（ATCC4356）按照1∶1比例接种于牛心脑浸液培养基,体外混合培养不同时间,包括生物膜形成过程中的初期（4 h）、中期（14 h）、晚期（24 h）,通过MTT法检测混合菌在生物膜形成的量。通过激光共聚焦显微镜观察混合细菌24 h形成的生物膜结构,实时定量PCR检测变形链球菌相关基因（ftf, smu630, brpA, gbpB, gtfB, vicR, comDE和relA）的表达。采用SPSS17.0软件包对数据进行统计学分析。结果 变形链球菌野生株及其2种luxS基因突变株与嗜酸乳杆菌混合培养14 h后,生物膜的量分别为0.481±0.024、0.591±0.023和0.279±0.019;24 h后,混合细菌形成生物膜的量趋势与该时间点一致,变形链球菌高表达株高于野生株,而缺陷株明显降低;但4 h后形成的生物膜组间无显著差异。激光共聚焦显微镜结果表明,高表达株和野生株的集聚程度更高,形成生物膜的结构更加紧密;而缺陷株生物膜菌间结构比较稀疏。以变形链球菌野生株和嗜酸乳杆菌混合形成的生物膜中相关基因的表达为标准,高表达株相关基因的表达均增加,缺陷株表达均降低,且各组间存在显著差异（P<0.05）。结论 变形链球菌luxS基因影响与具核梭杆菌混合培养形成的牙菌斑生物膜,为进一步研究该基因在生物膜中的作用及其调控机制提供了依据。     

Effects of Streptococcus mutans gtfC deficiency on mixed oral biofilms in vitro

The aim of this study was to examine the influence of glucosyltransferase-gene-negative (gtf-) Streptococcus mutans strains unable to synthesize water-insoluble or soluble glucan on the structure and macromolecular diffusion properties of in vitro grown mixed oral biofilms. Biofilms modeling supragingival plaque consisted of Actinomyces naeslundii OMZ 745, Candida albicans OMZ 110, Fusobacterium nucleatum KP-F2, Streptococcus oralis SK 248, Veillonella dispar ATCC 17748T and one of the S. mutans strains UA159, OMZ 966, OMZ 937 or OMZ 977. Biofilms were grown anaerobically on sintered hydroxyapatite disks for 64.5 h at 37 degrees C. To perform confocal laser scanning microscopy analyses, microorganisms were stained with Syto 13 and extracellular polysaccharides (EPS) with Calcofluor. Macromolecular diffusion properties were measured following timed biofilm exposure to Texas-Red-labeled 70-kDa dextran. Results showed that replacing wild-type S. mutans by a gtfC- mutant led to an increase in the volume fraction occupied by cells from 29 to 48% and a decrease of the EPS volume fraction from 51 to 33%. No such changes were observed when the S. mutans wild-type strain was replaced by a gtfB- or gtfD- mutant. The diffusion coefficient of 70-kDa dextran in biofilms containing the gtfC- S. mutans was 16-fold higher than in biofilms with the wild-type strain indicating a strong macromolecular sieving effect of GTF C-generated glucans. Our data demonstrate the influence of EPS on the structure and macromolecular diffusion properties of an oral biofilm model and uncover our still limited knowledge of the function of EPS in biofilms and plaque.     

Effect of penicillin on Streptococcus mutans, Streptococcus sanguis and lactobacilli in hamsters and in man

The effect of penicillin on the number of oral Streptococcus mutans, Streptococcus sanguis and lactobacilli in hamsters and in man was investigated. This is of interest as S. mutans and lactobacilli are involved in the carious process while S. sanguis is not. Hamsters infected with both S. mutans and S. sanguis or only S. sanguis received penicillin in their drinking water for 14 d. The treatment reduced the proportion of S. mutans and S. sanguis in dental plaque to undetectable levels. After the penicillin treatment the population of S. mutans and S. sanguis gradually increased. In man, the effect of oral penicillin therapy was examined in 21 adults with more than 2 X 10(5) S. mutans per ml saliva. The penicillin treatment had almost no effect on the numbers of S. sanguis and lactobacilli, but a pronounced decrease in the number of S. mutans was observed. The duration of this effect, however, was short. Consequently, such treatment alone is of limited value for the control of the oral infection of these microorganisms.     

Genotypic and phenotypic analysis of S. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions

The oral cavity harbors several Streptococcus mutans genotypes, which could present distinct virulence properties. However, little is known about the diversity and virulence traits of S. mutans genotypes isolated in vivo under controlled conditions of high cariogenic challenge. This study evaluated the genotypic diversity of S. mutans isolated from dental biofilms formed in vivo under sucrose exposure, as well as their acidogenicity and aciduricity. To form biofilms, subjects rinsed their mouths with distilled water or sucrose solution 8 times/day for 3 days. S. mutans collected from saliva and biofilms were genotyped by arbitrarily-primed PCR. Genotypes identified in the biofilms were evaluated regarding their ability to lower the suspension pH through glycolysis and their acid susceptibility and F-ATPase activity. Most subjects harbored only one genotype in saliva, which was detected in almost all biofilm samples at high proportions. Genotypes isolated only in the presence of sucrose had higher acidogenicity than those isolated only in the presence of water. Genotypes from biofilms formed with sucrose were more aciduric after 30 and 60 min of incubation at pH 2.8 and 5.0, respectively. The present results suggest that biofilms formed under high cariogenic conditions may harbor more aciduric and acidogenic S. mutans genotypes.     

Photosensitization of in vitro biofilms by toluidine blue O combined with a light-emitting diode

In natural ecosystems, micro-organisms grow preferentially attached to surfaces, forming matrix-enclosed biofilms. The aim of this study was to determine photodestruction levels in biofilms after subjecting them to photodynamic therapy. Biofilms of Streptococcus mutans, S. sobrinus, and S. sanguinis were grown on enamel slabs for 3, 5 or 7 d. Both the number of viable micro-organisms and the concentration of water-insoluble polysaccharides were analysed, and mineral loss (DeltaZ) analyses were performed on the enamel slabs. The antimicrobial potential of toluidine blue O (0.1 mg ml(-1)), associated with 85.7 J cm(-2) of a light-emission diode, was evaluated on the viability of 5-d biofilms. Both the number of micro-organisms and the concentration of water-insoluble polysaccharide increased with the age of the biofilms. A significant reduction ( approximately 95%) in viability was observed for S. mutans and S. sobrinus biofilms following photosensitization, with a > 99.9% reduction in the viability of S. sanguinis biofilms. In conclusion, a biofilm model was shown to be suitable for studying changes in bacterial numbers and enamel mineralization and for demonstrating the potential value of photosensitization in the control of in vitro biofilms.