Susceptibility of planktonic versus attached<Emphasis Type="Italic"> Streptococcus sanguinis</Emphasis> cells to chlorhexidine

The effect of chlorhexidine (CHX) on the viability of Streptococcus sanguinis was investigated in a preclinical biofilm model separately on cells in the planktonic or attached life form. Saliva-coated human enamel and glass slides were exposed to the streptococci suspended in sterile saliva for 30 min and 60 min in the flow chamber system. The CHX exposition was performed in two parts: pretreatment of the planktonic bacteria before their attachment to enamel or glass, and treatment of bacteria already attached to enamel. The susceptibility measured by vitality percentages was determined by fluorescence microscopy using vital/dead cells. After CHX pretreatment of planktonic cells, the mean values of the vitality percentages after adhesion were 14-18% (enamel) and 24-25% (glass). In contrast, the mean vitality percentages of untreated attached streptococci reached 70-75% (enamel) and 68% (glass). The vitality percentages of CHX-exposed bacteria dropped markedly to 2-5%, whereas those of untreated attached cells remained at 65-66%. The exposure of initially attached streptococci to CHX resulted in greater reduction of bacterial viability than with the planktonic counterparts. This preclinical biofilm model allows the investigation of various bacterial life forms and can furthermore be used to select efficient antiplaque therapeutics which might be beneficial for clinical plaque control.     

Evaluation of ultrasonic scaling unit waterline contamination after use of chlorine dioxide mouthrinse lavage

BACKGROUND: An infection control problem in dental operatories which is not fully controlled is waterline contamination by heterotrophic mesophilic bacteria. These bacteria are present in water supplies as a planktonic phase and adhere to the lumen of tubings as a biofilm comprised of their external cell surface glycocalyx and by production of extracellular carbohydrate polymers. The adherent film is most difficult to remove. The accumulated planktonic phase can be reduced significantly by flushing water from the lines before use in patient treatment, but will return when the equipment is idle through the accumulation of more planktonic phase and by slough of the biofilm surface-adsorbed phase not yet enmeshed in the carbohydrate matrix. Chlorine dioxide has antimicrobial activity against many bacteria, spores, and viruses. It is used in water supply treatment as a disinfectant and slime preventive and has an advantage over chlorine in that carcinogenic trihalomethanes are not generated. METHODS: This study compared use of phosphate buffer-stabilized chlorine dioxide (0.1%) mouthrinse as a lavage in ultrasonic dental scaler units with the use of tap water as a control. Sterile water flushed through the units onto heterotrophic plate count (HPC) sampler plates was cultured 7 days at room temperature and colonies were counted at 12x. One test and one control unit were used for biopsy of internal tubing and scanning electron microscopy imaging. RESULTS: The HPC counts, in colony forming units (CFU)/ml, were reduced 3- to 5-fold by flushing tap water through the units, but they returned after units were idle overnight. When phosphate-buffered chlorine dioxide mouthrinse was used as a lavage, CFU/ml were reduced 12- to 20-fold. Holding chlorine dioxide in waterlines overnight reduced recurrent buildup compared to water (P <0.05). Scanning electron microscopy images indicated a significant reduction of biofilm coverage by chlorine dioxide as compared to water (P<0.001). CONCLUSIONS: Phosphate-buffered chlorine dioxide mouthrinse was effective in these short-term trials for control of waterline contamination in ultrasonic dental scaling units. It should prove as useful in dental professional waterline applications as it has in industrial uses for biofilm control.     

Effectiveness of ozone against periodontal pathogenic microorganisms

Ozone has been proposed as an adjunct antiseptic in periodontitis therapy. The aim of this study was to investigate the antimicrobial effectiveness of gaseous/aqueous ozone, in comparison with that of the established antiseptic chlorhexidine digluconate (CHX), against periodontal microorganisms. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Parvimonas micra in planktonic or biofilm cultures were exposed, for 1 min, to gaseous ozone, aqueous ozone, CHX, or phosphate-buffered saline (control). None of the agents was able to substantially reduce the A. actinomycetemcomitans count in biofilm cultures. In contrast, P. gingivalis, T. forsythia, and P. micra could be eliminated by 2% CHX or by ozone gas at 53 gm(-3) . Significantly greater antimicrobial effects were observed against planktonic cultures than against biofilm-associated bacteria. The rate of killing was influenced by the species of bacteria, and by the type and concentration of agent. There were no significant differences in the effectiveness of aqueous ozone (20 μg ml(-1) ) or gaseous ozone (≥ 4 gm(-3) ) compared with 2% CHX but they were more effective than 0.2% CHX. Therefore, high-concentrated gaseous and aqueous ozone merit further investigation as antiseptics in periodontitis therapy. A safe system for applying gaseous ozone into the periodontal pocket that avoids inhalation still needs to be developed.     

没食子鞣质和联合氟化钠对不同状态变链菌葡萄糖基转移酶活性的影响

目的：实验采用析因设计方法研究天然维药没食子和没食子联合氟化钠对变形链球菌浮游及生物膜状态下葡萄糖基转移酶（GTF）活性的影响,探讨实验药物防龋的作用机制。方法：用脑心浸液液体培养基分别培养浮游和生物膜状态下的变形链球菌,根据析因实验的分组将配置好的没食子鞣质、氟化钠、没食子鞣质联合氟化钠加入相应的菌液中厌氧培养18h。硫酸铵沉淀法提取粗酶,考马斯亮蓝法和蒽酮法分别测定总蛋白和还原糖含量,计算酶活性和药物对酶活性的抑制率。实验结果采用SPSS17．0软件包进行数据分析。结果：GTF酶活性在细菌不同培养状态下有统计学意义（P〈0．05）,浮游状态下GTF酶活性高于生物膜状态;没食子、氟化钠、没食子联合氟化钠对细菌不同状态下GTF酶活性的抑制率有差异（P〈0．05）,浮游状态下的葡萄糖基转移酶比生物膜状态对药物作用敏感,没食子的抑制作用最为显著。抑制率没食子〉没食子联合氟化钠〉氟化钠。结论：没食子鞣质可能是通过抑制葡萄糖基转移酶活性抑制变形链球菌的粘附,从而达到防龋的作用。     

没食子鞣质和联合氟化钠对不同状态变链菌葡萄糖基转移酶活性的影响

目的：实验采用析因设计方法研究天然维药没食子和没食子联合氟化钠对变形链球菌浮游及生物膜状态下葡萄糖基转移酶（GTF）活性的影响,探讨实验药物防龋的作用机制。方法：用脑心浸液液体培养基分别培养浮游和生物膜状态下的变形链球菌,根据析因实验的分组将配置好的没食子鞣质、氟化钠、没食子鞣质联合氟化钠加入相应的菌液中厌氧培养18h。硫酸铵沉淀法提取粗酶,考马斯亮蓝法和蒽酮法分别测定总蛋白和还原糖含量,计算酶活性和药物对酶活性的抑制率。实验结果采用SPSS17．0软件包进行数据分析。结果：GTF酶活性在细菌不同培养状态下有统计学意义（P〈0．05）,浮游状态下GTF酶活性高于生物膜状态;没食子、氟化钠、没食子联合氟化钠对细菌不同状态下GTF酶活性的抑制率有差异（P〈0．05）,浮游状态下的葡萄糖基转移酶比生物膜状态对药物作用敏感,没食子的抑制作用最为显著。抑制率没食子〉没食子联合氟化钠〉氟化钠。结论：没食子鞣质可能是通过抑制葡萄糖基转移酶活性抑制变形链球菌的粘附,从而达到防龋的作用。     

Endodontic and Salivary Isolates of Enterococcus faecalis Integrate into Biofilm from Human Salivary Bacteria Cultivated In Vitro

IntroductionThe aim of this study was to examine whether Enterococcus faecalis isolates from endodontic patients (from saliva and from a root canal) are able to prevail against salivary bacteria when grown in coculture in a biofilm reactor.MethodsSaliva that was tested to be free of E. faecalis was used as the inoculum. The fate of E. faecalis was examined by using culture techniques and fluorescence in situ hybridization (FISH).ResultsThe root canal isolate accounted for 37.4% of the biofilm and 31.9% of the planktonic phase when examined by the culture technique, whereas the proportions examined by FISH showed 15.3% in the biofilm and 11.7% in the planktonic phase. The saliva isolate (as examined by the culture technique) accounted for 32.4% in the biofilm and 27.1% in the planktonic phase, respectively, compared with 14.1% in the biofilm and 9.5% in the planktonic phase when examined by FISH analysis.ConclusionsThese results led to the suggestion that E. faecalis could persist in the biofilm of the human oral cavity. Because of the ubiquitous presence of E. faecalis, root canal infections may arise from different sources.     

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.     

Electrical enhancement of Streptococcus gordonii biofilm killing by gentamicin

This electrical enhancement was demonstrated in an in vitro model. Streptococcus gordonii biofilms were grown for 6 days in continuous-flow reactors on one-tenth strength trypticase peptone broth. The biofilms attained a mean areal cell density of 2.4 x 10(8) c.f.u./cm2 and a thickness of approx. 19 microm. Biofilms exhibited characteristic resistance to killing by an antibiotic. When treated with 2 microg/ml gentamicin for 24 h, they exhibited a 0.84 log reduction in viable cell numbers; a 4.7 log reduction was measured in a planktonic culture. Killing of planktonic bacteria by this treatment was reduced to 1.2 log when an oxygen-scavenging enzyme was added to the medium. When a 2-mA direct current was applied during antibiotic treatment, biofilm killing increased to a 4.3 log reduction. Electrical current alone caused a 1.9 log reduction in biofilm cell counts. It is suggested that gentamicin was less effective against Strep. gordonii under anaerobic conditions than it was under aerobic conditions and that this can explain both the reduced susceptibility of the biofilm (due to oxygen depletion) and electrical enhancement of efficacy (due to oxygen generation by electrolysis).     

Dental unit waterline contamination and its possible implications during periodontal surgery

BACKGROUND: Dental unit waterline contamination has become a concern to clinical dentistry. This concern arises from the fact that bacteria sloughed from established biofilms in dental unit waterlines increase heterotrophic bacteria counts in water exiting these units. METHODS: Scanning microscopy and bacterial viability staining were used to examine the sessile and planktonic biofilm present in dental unit waterlines and water samples, respectively. In addition, the limulus amebocyte assay was used to measure the lipopolysaccharide (LPS) levels in water samples. RESULTS: All dental unit waterlines were coated with a well-established biofilm made up of filamentous and bacillus-like microorganisms. Water samples collected from these dental units contained high numbers of individual bacteria and bacterial aggregates. A viability staining technique identified significantly more bacteria in water than could be cultured, and 64% of the total bacterial population stained as nonvital. Since the bacterial load (viable and nonviable) was high, we examined the LPS in dental unit water samples. The mean LPS levels in water collected from high-speed and air/water lines in use were 480 and 1,008 endotoxin units (EU)/ml. This was significantly higher than the mean level of 66 EU/ml found in water samples collected from adjacent clinic sinks. The LPS level at the start of the day (2,560 EU/ml) was reduced by 70% with 1 minute of flushing (800 EU/ml). Flushing times of 5 and 10 minutes were not able to reduce LPS levels to zero. CONCLUSION: The presence of high heterotrophic bacterial counts, sloughing biofilm, and high LPS levels are discussed in relation to patient risk and periodontal wound healing biology.     

Susceptibility of Porphyromonas gingivalis in biofilms to amoxicillin,doxycycline and metronidazole

The susceptibility of Porphyromonas gingivalis to amoxicillin, doxycycline and metronidazole was determined by a standardized method taking into account the biofilm mode of growth of subgingival bacteria. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 48-h biofilms of P. gingivalis established on membrane filters in a Modified Robbins Device were determined by agar dilution. The results were compared to (i) conventional MIC determinations, (ii) the susceptibility of planktonic cultures with cell numbers equal to those of the biofilms, and (iii) results for detached biofilm cells. The MICs of the biofilms of the six reference strains and clinical isolates containing 107-8 cells/filter were much higher than the conventional MIC values. However, the MIC of planktonic cultures of equal cell numbers also increased, indicating that an inoculum effect is part of the explanation of the increased resistance of biofilms. Still, the MBCs of biofilms were 2-8 times, and those for doxycycline up to 64 times, greater than the MBC values for planktonic cultures.     

厚朴酚、小檗碱与锌离子对口臭致病菌的抑菌研究

目的评价厚朴酚、小檗碱、锌离子单品及三者联合应用对两种口臭致病菌在生物膜状态下的抑菌效果。方法采用厚朴酚（厚朴酚组）、小檗碱（小檗碱组）、锌离子（锌离子组）的单一成分药液及各成分混合药液分别作用于培养12h、24h的牙龈卟啉单胞菌、具核梭杆菌双菌种生物膜,去离子水滴加于生物膜作为对照组。激光共聚焦显微镜下观察各药物单品及联合应用对双菌种生物膜的抑制效果,计算各组生物膜活菌百分比。结果在加入厚朴酚、小檗碱、锌离子及3种成分的混合液之后,培养12h双菌种生物膜中的活菌百分比均下降,与对照组活菌百分比（70．03％±4．98％）相比,差异有统计学意义（P〈0．05）,混合液组活菌百分比明显减少（21．51％±3．31％）。在加入厚朴酚、小檗碱、锌离子及三者混合液之后,培养24h双菌种生物膜中的活菌百分比均下降,与对照组活菌百分比（84．23％±4．07％）相比,差异有统计学意义（P〈0．05）,混合液组活菌百分比明显减少（38．71％±4．25％）。结论厚朴酚、小檗碱、锌离子均能抑制口臭致病菌生物膜状态下的生长,3种成分联合应用抑菌效果更为显著。     

Impact of growth conditions on susceptibility of five microbial species to alkaline stress

The effects of different growth conditions on the susceptibility of five taxa to alkaline stress were investigated. Enterococcus faecalis ATCC 29212, Streptococcus sobrinus OMZ 176, Candida albicans ATCC 90028, Actinomyces naeslundii ATCC 12104, and Fusobacterium nucleatum ATCC 10953 were grown as planktonic cells, allowed to adhere to dentin for 24 hours, grown as monospecies or multispecies biofilms on dentin under anaerobic conditions with a serum-enriched nutrient supply at 37 degrees C for 5 days. In addition, suspended biofilm microorganisms and 5-day old planktonic multispecies cultures were used. Microbial recovery upon direct exposure to saturated calcium hydroxide solution (pH 12.5) for 10 and 100 minutes was compared with control exposure to physiologic saline. Planktonic microorganisms were most susceptible; only E. faecalis and C. albicans survived in saturated solution for 10 minutes, the latter also for 100 minutes. Dentin adhesion was the major factor in improving the resistance of E. faecalis and A. naeslundii to calcium hydroxide, whereas the multispecies context in a biofilm was the major factor in promoting resistance of S. sobrinus to the disinfectant. In contrast, the C. albicans response to calcium hydroxide was not influenced by the growth condition. Adherence to dentin and interspecies interactions in a biofilm appear to differentially affect the sensitivity of microbial species to calcium hydroxide.     

Effect of two antimicrobial agents on early in situ biofilm formation

OBJECTIVES: The aim of this observer-blind, controlled, three-cell cross-over study was to evaluate the influence of an amine fluoride/stannous fluoride (Meridol, 250 ppm; ASF) and a chlorhexidine mouthrinse (CHX; Chlorhexamed forte, 0.2%) compared with water on in situ biofilm growth. MATERIAL AND METHODS: After a professional toothcleaning seven volunteers had to wear a special acrylic appliance, in which six specimens each were inserted to allow the build-up of intra-oral biofilms. The volunteers had to rinse twice daily for 1 min. with 10 ml of the allocated mouthrinse. After 48 h of wearing, the specimens with the adhering biofilms were removed from the splints and stained with two fluorescent dyes, which selectively stain vital bacteria green and dead bacteria red. Under the confocal laser scanning microscope biofilm thickness (BT) was evaluated. To examine bacterial vitality (BV%) the biofilms were scanned (1 microm sections) and digital images were made. An image analysis program was used to calculate the mean BV as well as the BV of the single sections. After a wash-out period of 14 days a new test cycle was started. RESULTS: The use of CHX and ASF resulted in a BT of 8.4+/-4.4 mum and 15.7+/-9.9 compared with 76.7+/-29.4 mum using water. The mean vitality (in %) was reduced from 66.1+/-20.4 to 23.3+/-11.6 and 23.9+/-12.4 using CHX and ASF, respectively. Both active solutions reduced BT and BV significantly compared with water (p<0.001). Differences between the two active solutions were not significant (p>0.05). CONCLUSION: Both mouthrinses showed antibacterial and plaque-reducing properties against the in situ biofilm. The study design enables the examination of an undisturbed oral biofilm and for the first time shows the influence of antibacterial components applied under clinical conditions regarding biofilm formation.     

口腔致病菌在生物膜状态和浮游态中的特性比较

在人类口腔中存在着种类繁多的天然菌群,这些细菌以牙菌斑生物膜的形式生长.菌斑生物膜作为人类龋病和牙周病的主要致病因素,有诸多代谢特点和生理现象明显不同于浮游态的细菌.下面就数种主要的口腔致病菌在生物膜状态和浮游态下的特性,如对抗菌剂的敏感性、基因表达和信号传导、耐酸性等方面的研究进展作一综述.     

变形链球菌中LuxS介导的群体感应系统的研究进展

LuxS介导的群体感应系统以自体诱导物-2（AI-2）分子作为通用信号,可介导不同细菌的交流。作为口腔致龋生物膜中主要致病菌的变形链球菌中也存在这种感应系统。本文就LuxS信号系统在变形链球菌生物膜形成和毒力因子分泌方面的作用以及它所介导的变形链球菌与口腔中其他细菌的交流等内容作一综述。     

口腔微生物生物膜分散物质的研究进展

生物膜是黏附在固体表面,包裹在自身产生的胞外多聚基质中的细菌群体。生物膜的形成和发展包括细菌的黏附、繁殖和分散。附着于某表面的生物膜将其中的细菌释放、分散到周围环境以传播到新的位置形成新的群落即生物膜的分散。生物膜分散是生物膜生长发展周期中一个重要的阶段,起到重要的传播作用。对许多致病菌而言,生物膜的分散能使生物膜的细菌转化为浮游状态,促进感染的扩散。生物膜的形成能提高细菌对抗微生物剂及宿主防御反应的抵抗力。在口腔中,口腔微生物能附着于口腔组织及修复体表面形成生物膜。人类龋病、牙周病是口腔的慢性感染性疾病,它们的发生与生物膜密切相关。生物膜分散机制是近年的研究热点,促进生物膜分散的新制剂可能成为攻克生物膜感染又一靶点。分散物质的临床意义和可能的临床应用具有广阔的前景。本文对口腔中能促进生物膜分散的分散物质作一综述。     

Bactericidal effect of delmopinol on attached and planktonic Streptococcus sanguinis cells

The aim of this investigation was to determine the antibacterial effect of varying concentrations of delmopinol-HCl on attached as well as on planktonic Streptooccus sanguinis cells in vitro. In addition, a possible antiadhesive effect on attached micro-organisms was to be investigated. S. sanguinis cells were allowed to attach to glass surfaces. These as well as planktonic cells were exposed to delmopinol-HCI in concentrations ranging from 0.2% to 0.00005% for 2 min. The percentage of vital bacteria was calculated by means of a fluorescence staining method. Total counts of attached bacteria were performed to determine any possible detaching effect by the delmopinol-HCl. The CFU were determined for the planktonic bacteria. Attached as well as planktonic bacteria showed a marked decrease in vitality following exposure to 0.2% delmopinol-HCl. After exposure to 0.05% this was only the case with the attached microorganisms. The total number of attached bacteria was not reduced by the delmopinol treatment. During initial dental biofilm formation, delmopinol-HCl causes a bactericidal effect when applied in concentrations of 0.05% and higher.     

Antibacterial Activity of Endodontic Sealers against Planktonic Bacteria and Bacteria in Biofilms

Introduction

The aim of this study was to investigate the antibacterial activity of 4 endodontic sealers against bacteria planktonic grown or in biofilms commonly detected from persistent and secondary endodontic infections.

Fluorescence microscopic visualization and quantification of initial bacterial colonization on enamel in situ

OBJECTIVE: The acquired salivary pellicle has been defined as proteinaceous film free of bacteria. However, due to the large numbers of microorganisms existent in the oral fluids, it is conceivable that adherent bacteria are already present in the initial pellicle. The aim of this in situ study was to visualize and to quantify these bacteria. DESIGN: Initial biofilm formation was performed on bovine enamel slabs mounted buccally on individual splints and carried in situ by six subjects for 3, 30 and 120 min, respectively. After intraoral exposure, the slabs were rinsed with saline solution and the adherent bacteria were investigated with the following fluorescence microscopic methods: staining with 4',6-diamidino-2-phenylindole (DAPI), staining of vital and nonvital bacteria with fluoresceinediacetate and ethidiumbromide (live/dead staining) and fluorescence in situ hybridization (FISH) of eubacteria and streptococci, respectively. In addition, determination of colony forming units after ultrasonically induced detachment of bacteria was performed. RESULTS: With all the methods, bacteria were detected in the initial in situ biofilm irrespective of the formation time. The numbers of bacteria revealed high intraindividual and interindividual variability and the microorganisms were distributed randomly in small aggregates. The results of the epifluorescence microscopic techniques corresponded well. The mean number of adherent bacteria detected was in the range of 10-20x10(4)cm(-2). CONCLUSION: Already after 3 min, adherent bacteria are present in the initial pellicle. For the first time, DAPI-staining as well as FISH have proven success for visualization of initial intraoral colonization of enamel specimens.