Biofilm–substrate interaction: from initial adhesion to complex interactions and biofilm maturity

The bacteria‐related diseases of dental caries, pulp and periapical infections, and diseases of the periodontium are biofilm diseases. Dental plaque exhibits the general properties of biofilms and an understanding of biofilm development in health and disease is crucial to the understanding of dental disease, and will unlock effective targeted treatment strategies. Biofilm development requires complex interactions between microorganisms, and with the host. Initially planktonic cells in the aqueous environment of the oral cavity attach to host surfaces, first by physical forces though ultimately by specific adhesive reactions between host and bacteria molecules. Initial bacterial colonizers alter the immediate environment and offer additional binding sites to other bacteria that cannot react directly with the host surface. These co‐aggregation reactions allow various bacterial species to join the evolving community and participate in its development. Development of the microbial community is also dependent on the nutritional source. Early colonizing bacteria are adept at utilizing the immediate nutritional source at the host site, primarily carbohydrates. With the accumulation of bacteria, nutritional demands become more complex and proteins, amino acids, glycoproteins, and vitamins must be supplied from either the host tissues or other bacteria. The biofilm community must co‐operate to effectively achieve metabolism of these complex molecules and perform other functions, while other interbacterial inhibitory reactions such as bacteriocin production or quorum sensing occur in the biofilm, influencing the development of the bacterial community. As such, oral bacteria do not exist independently but function as a co‐ordinated, spatially organized, and metabolically integrated microbial community.     

Antibiotic resistance in an in vitro subgingival biofilm model

INTRODUCTION: The purpose of this study was to utilize an in vitro biofilm model of subgingival plaque to investigate resistances in subgingival biofilm communities to antibiotics commonly used as adjuncts to periodontal therapy. METHODS: Biofilms were grown on saliva-coated hydroxyapatite supports in trypticase-soy broth for 4 h-10 days and then exposed for 48 h to either increasing twofold concentrations of tetracycline, amoxicillin, clindamycin, and erythromycin or therapeutically achievable concentrations of tetracycline, doxycycline, minocycline, amoxicillin, metronidazole, amoxicillin/clavulanate, and amoxicillin/metronidazole. RESULTS: Concentrations necessary to inhibit bacterial strains in steady-state biofilms were up to 250 times greater than the concentrations needed to inhibit the same strains grown planktonically. In the presence of therapeutically available antibiotic concentrations, significantly higher proportions of the biofilms remained viable as the biofilms reached steady-state growth. The combinations of amoxicillin/clavulanate and amoxicillin/metronidazole were the most effective in suppressing growth. These combinations were particularly effective against biofilms up to and including 7 days of age and inhibited 90% or more of the bacteria present relative to untreated controls. As the biofilms approached steady state, these combinations were less effective with 50-60% of the bacteria retaining viability. CONCLUSION: Most, but not all, species of subgingival bacteria are considerably more resistant in biofilms than in planktonic cultures. Resistance appeared to be age-related because biofilms demonstrated progressive antibiotic resistance as they matured with maximum resistance coinciding with the steady-state phase of biofilm growth.     

Recalcitrance of Streptococcus mutans biofilms towards detergent-stimulated detachment

The biofilm mode of growth protects the plaque microorganisms against environmental attacks, such as from antimicrobials or detergents. Detergents have a demonstrated ability to detach initially adhering bacteria from enamel surfaces, but the ability of detergent components to detach plaque bacteria is not always obvious from in vivo experiments and reports on their clinical efficacy are inconsistent. It is likely that antimicrobials or detergents are unable to penetrate the plaque and reach the bacteria that actually link the plaque mass to the substratum surface. Attenuated total reflectance/Fourier transform infrared spectroscopy was used to measure the transport of sodium lauryl sulphate (SLS) through Streptococcus mutans HG 985 biofilms. The transport of SLS to the base of the S. mutans biofilms was not hindered, while moreover an accumulation of SLS near the base of the biofilms was found, suggesting that SLS was adsorbed to biofilm components. X-ray photoelectron spectroscopy confirmed the ability of S. mutans, grown on sucrose supplemented medium, to adsorb SLS, and simultaneously indicated that exposure of cells to SLS might lead to a loss of surface proteins. Furthermore, experiments in a parallel-plate flow chamber demonstrated that initially adhering S. mutans HG 985 could be stimulated to detach by SLS, but that, depending on the growth stage of the biofilm, only maximally 27% of biofilm bacteria could be stimulated to detach by a 4% (w/v) SLS solution.     

Biofilms, a new approach to the microbiology of dental plaque

Dental plaque has the properties of a biofilm, similar to other biofilms found in the body and the environment. Modern molecular biological techniques have identified about 1000 different bacterial species in the dental biofilm, twice as many as can be cultured. Oral biofilms are very heterogeneous in structure. Dense mushroom-like structures originate from the enamel surface, interspersed with bacteria-free channels used as diffusion pathways. The channels are probably filled with an extracellular polysaccharide (EPS) matrix produced by the bacteria. Bacteria in biofilms communicate through signaling molecules, and use this “quorum-sensing” system to optimize their virulence factors and survival. Bacteria in a biofilm have a physiology different from that of planktonic cells. They generally live under nutrient limitation and often in a dormant state. Such “sleepy” bacteria respond differently to antibiotics and antimicrobials, because these agents were generally selected in experiments with metabolically active bacteria. This is one of the explanations as to why antibiotics and antimicrobials are not as successful in the clinic as could be expected from laboratory studies. In addition, it has been found that many therapeutic agents bind to the biofilm EPS matrix before they even reach the bacteria, and are thereby inactivated. Taken together, these fundings highlight why the study of bacteria in the oral cavity is now taken on by studying the biofilms rather than individual species.     

Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system

The aim was to establish defined-species consortium plaque biofilms to investigate enamel and root caries in an artificial mouth. Strains of the putative enamel and root caries pathogens, Streptococcus mutans, Strep. sobrinus, Actinomyces naeslundii and Lactobacillus rhamnosus, were screened in batch culture for potential cariogenic properties: a low terminal pH, ability to aggregate, and catabolic diversity. The strains selected were grown as monoculture biofilms and as consortium plaque biofilms in a multiplaque artificial mouth. The biofilms were supplied with a constant flow of a simulated oral fluid and were given periodic sucrose (and in some instances glucose) to simulate meals. All the bacteria except L. rhamnosus formed large, monospecies biofilms with resting pH in the range 5.3-5.8. The consortia biofilms were larger and had a resting pH of 4.9-5.3. The consortia biofilms supplied with 8-hourly carbohydrate comprised mainly 'mutans' streptococci (58, SD 5.5%) and L. rhamnosus (42, SD 5.7%). A. naeslundii characteristically was absent or present in a low percentage (up to 4% colony-forming units). All biofilms demineralized polished bovine enamel and dentine blocks, as assessed by microradiography and enamel-surface microhardness measurement. The consortia also demineralized intact enamel and tooth roots; they were more cariogenic to enamel than any of the monoculture biofilms, as measured by enamel-surface softening, but variation in lesion depth was proportional to biofilm wet weight irrespective of acidogen composition (r = 0.93, p < 0.05). Enamel lesions had a well-mineralized intact surface and a zone of subsurface demineralization, typical of early natural lesions. Dentine and root lesions showed extensive demineralization but lacked a pronounced surface mineralized zone. Substitution of glucose for sucrose had no effect on the cariogenicity of the consortium to bovine enamel or human roots and had no major effect on the plaque composition. Continuously supplied fluoride (19 parts/10(6)) resulted in a substantially reduced enamel surface softening and subsurface demineralization of intact roots. It was concluded that consortia biofilms of selected caries pathogens generate realistic caries lesions in all tooth hard tissues under controlled growth conditions in the artificial mouth. This in vitro caries experimental model may prove useful for the study of interrelations between the plaque biofilm, tooth tissues and the oral environment, and for the development of procedures to modify the course of caries development.     

The antibacterial effect of photodynamic therapy in dental plaque-derived biofilms

Background and Objective:  Photodynamic therapy has been advocated as an alternative to antimicrobial agents to suppress subgingival species and to treat periodontitis. Bacteria located within dense biofilms, such as those encountered in dental plaque, have been found to be relatively resistant to antimicrobial therapy. In the present study, we investigated the ability of photodynamic therapy to reduce the number of bacteria in biofilms by comparing the photodynamic effects of methylene blue on human dental plaque microorganisms in the planktonic phase and in biofilms.
Material and Methods:  Dental plaque samples were obtained from 10 subjects with chronic periodontitis. Suspensions of plaque microorganisms from five subjects were sensitized with methylene blue (25 μg/mL) for 5 min then exposed to red light. Multispecies microbial biofilms developed from the same plaque samples were also exposed to methylene blue (25 μg/mL) and the same light conditions as their planktonic counterparts. In a second set of experiments, biofilms were developed with plaque bacteria from five subjects, sensitized with 25 or 50 μg/mL of methylene blue and then exposed to red light. After photodynamic therapy, survival fractions were calculated by counting the number of colony-forming units.
Results:  Photodynamic therapy killed approximately 63% of bacteria present in suspension. By contrast, in biofilms, photodynamic therapy had much less of an effect on the viability of bacteria (32% maximal killing).
Conclusion:  Oral bacteria in biofilms are affected less by photodynamic therapy than bacteria in the planktonic phase. The antibacterial effect of photodynamic therapy is reduced in biofilm bacteria but not to the same degree as has been reported for treatment with antibiotics under similar conditions.     

Dental plaque as a microbial biofilm

New technologies have provided novel insights into how dental plaque functions as a biofilm. Confocal microscopy has confirmed that plaque has an open architecture similar to other biofilms, with channels and voids. Gradients develop in areas of dense biomass over short distances in key parameters that influence microbial growth and distribution. Bacteria exhibit an altered pattern of gene expression either as a direct result of being on a surface or indirectly as a response to the local environmental heterogeneity within the biofilm. Bacteria communicate via small diffusible signalling molecules (e.g. competence-stimulating peptide, CSP; autoinducer 2); CSP induces both genetic competence and acid tolerance in recipient sessile cells. Thus, rates of gene transfer increase in biofilm communities, and this is one of several mechanisms (others include: diffusion-reaction, neutralization/inactivation, slow growth rates, novel phenotype) that contribute to the increased antimicrobial resistance exhibited by bacteria in biofilms. Oral bacteria in plaque do not exist as independent entities but function as a co-ordinated, spatially organized and fully metabolically integrated microbial community, the properties of which are greater than the sum of the component species. A greater understanding of the significance of dental plaque as a mixed culture biofilm will lead to novel control strategies.     

牙周致病菌和致龋菌间生长关系的体外动态观察

目的研究6种代表性牙周致病菌和致龋菌在菌斑生物膜和悬浮液中的消长关系。方法将双菌组（致龋菌＋牙周致病菌）接种于改良恒化器中,连续培养1h、24h、48h和96h,然后取在羟基磷灰石表面形成的生物膜和悬浮液再进行细菌培养。结果与血链球菌混合培养时,菌斑生物膜和悬浮液中牙周致病菌均增多,而血链球菌明显减少（P〈0.05）;具核梭杆菌与变形链球菌培养时,菌斑生物膜和悬浮液中具核梭杆菌明显增多,而变形链球菌减少（P〈0.05）。菌斑生物膜中致龋菌占优势,相应的悬浮液中牙周致病菌96h开始占优势。悬浮液中细菌量波动更明显。结论2类致病菌在菌斑生物膜与相应的悬浮液中的生长是不一致的。2类致病菌之间相互作用也许是决定菌斑生物膜内部生态环境发展方向的主要因素。     

Effects of sublethal exposure to an antiseptic mouthrinse on representative plaque bacteria

Abstract Although the mechanism responsible for the clinical antiplaque efficacy of oral antiseptics is generally considered to be primarily one of bactericidal activity, it has been suggested that oral antiseptics may have additional effects on bacteria exposed to sublethal levels. Studies reported herein, investigated the effects of sublethal levels of an essential oil-containing antiseptic mouthrinse (Listerine Antiseptic, Warner-Lambert Co., Morris Plains. NJ) on selected activities of representative plaque microorganisms using in vitro models. These studies demonstrated that sublethal exposure to the tested oral antiseptic can have significant effects in reducing intergeneric coaggregation, increasing bacterial generation time, and extracting endotoxin from Gram-negative bacteria. These in vitro activities can be correlated with features of plaque formation and pathogenicity seen in vivo: however, additional studies will be necessary to confirm that these mechanisms are, in fact, operative clinically.     

Spatial distribution of vital and dead microorganisms in dental biofilms

To examine the spatial structure of dental biofilms a vital fluorescence technique was combined with optical analysis of sections in a confocal laser scanning microscope (CLSM). Enamel slaps were worn in intraoral splints by three volunteers for five days to accumulate smooth-surface plaque. After vital staining with fluorescein diacetate and ethidium bromide the specimens were processed for CLSM examination. Optical sections 1 microm apart were analysed in the z-axis of these dental biofilms. One of the films was 15 microm high, sparse and showed low vitality, i.e. <16%, while the others were taller (25 and 31 microm) and more vital, i.e. up to 30 and 69%, respectively. In all instances the bacterial vitality increased from the enamel surface to the central part of the plaque and decreased again in the outer parts of the biofilm. The spatial arrangement of the microorganisms in the biofilm showed voids outlined by layers of vital bacteria, which themselves were packed in layers of dead material.     

Bacterial biofilm: structure,function, and antimicrobial resistance

Biofilms are microbial communities attached to surfaces and encased in an extracellular matrix of microbial origin. They represent the predominant form of microbial life. Biofilms are everywhere and can develop on virtually every natural and man‐made surface. Biofilms are also ubiquitous in both normal and pathogenic human processes. Biofilm formation has been demonstrated for numerous pathogens and is clearly one of the main strategies for bacterial survival in a variety of sites within the human body. In almost all instances, the biofilm lifestyle helps bacteria survive and persist within the environment. This review discusses the fundamental biology of microbial biofilm and how biofilms impact the pathogenesis of human infections. The different mechanisms involved in the reduced antimicrobial susceptibility of microorganisms in pathogenic biofilms are discussed in detail in this review. Possible approaches that could be explored in the search for new anti‐biofilm strategies to eradicate medically relevant biofilms are also presented.     

Application of the Zürich biofilm model to problems of cariology

The term biofilm is increasingly replacing 'plaque' in the literature, but concepts and existing paradigms are changing much more slowly. There is little doubt that biofilm research will lead to more realistic perception and interpretation of the physiology and pathogenicity of microorganisms colonizing plaques in the oral cavity. There is clear evidence that the genotypic and phenotypic expression profiles of biofilm and planktonic bacteria are different. Several techniques are available today to study multispecies biofilms of oral bacteria, each having its particular advantages and weaknesses. We describe a biofilm model developed in Zürich and demonstrate a number of applications with direct or indirect impact on prophylactic dentistry: spatial arrangement and associative behavior of various species in biofilms; multiplex fluorescent in situ hybridization analysis of oral bacteria in biofilms; use of the biofilm model to predict in vivo efficacy of antimicrobials reliably; mass transport in biofilms; de- and remineralization of enamel exposed to biofilms in vitro. The potential of biofilm experimentation in oral biology has certainly not yet been fully exploited and dozens of possible interesting applications could be investigated. The overall physiological parameters of multispecies biofilms can be measured quite accurately, but it is still impossible to assess in toto the multitude of interactions taking place in such complex systems. What can and should be done is to test hypotheses stemming from experiments with planktonic cells in monospecies cultures. In particular, it will be interesting to investigate the relevance to biofilm composition and metabolism of specific gene products by using appropriate bacterial mutants.     

口腔细菌生物膜对抗菌剂的敏感性

许多研究证实生物膜中的细菌对抗菌剂的敏感性比其处于浮游状态时要低得多 ,而口腔细菌却常以生物膜方式致病 ,对许多抗菌剂具有很强的抵抗力 ,这就造成实验室的药敏结果用于临床效果不佳。因此详细了解处于生物膜状态的口腔细菌对抗菌剂的敏感性 ,对于临床上治疗和预防许多口腔疾病具有重要意义     

Development of in vitro Biofilm Model: Artificial Food Supplementation in Chemostat-type System

We developed a biofilm model with artificial food supplementation (AFS), which was more similar to food intake in the human oral cavity, for the elucidation of pathogenic mechanisms and biofilm formation, and the evaluation of active agents. A five-organism bacterial consortium containing Streptococcus mutans was grown in a chemostat-type system with continuous supplementation of basal medium containing mucin (BMM). Biofilms formed on the surfaces of saliva-coated hydroxyapatite discs. As periodic application of AFS, the entire volume of culturing media was replaced with a sucrose-supplemented broth and re-incubated for 30 min. Then, the media were refilled with fresh BMM every 12 h. For comparison with the AFS biofilm, the sucrose pulses (SP) biofilm model, which is without any intermittent replacement cultures, was established by referring to previously reported biofilm models. The biological and physical properties of the biofilms generated by AFS and SP were evaluated. The results showed that the bacterial composition, amount of carbohydrate, pH responses, and structure of the AFS biofilm were more similar to those of in vivo or in situ dental plaque biofilms, compared with those of the SP biofilm. Additional experiments showed that the same bacterial growth patterns were reproducible in the AFS biofilm. Consequently, utilization of this newly developed biofilm model will lead to a better understanding of the pathogenic mechanisms in biofilm formation, and will be useful for the evaluation of active agents.     

Current and future strategies for the control of mature oral biofilms—Shift from a bacteria-targeting to a matrix-targeting approach

Oral biofilms, unlike those formed at most other sites in the human body, are unique because surgical intervention is usually unnecessary for their removal. The control of oral biofilms relies mainly on mechanical elimination. Chemical controls are used as alternative or adjunctive methods when elimination using dental instruments proves difficult. For many years, researches have focused on how microorganisms could be rapidly killed using high concentrations of antimicrobials or new antibacterial agents.New strategies that target the matrices of mature biofilms are needed because biofilm matrices inhibit the effect of antimicrobial agents through absorption and degradation. These strategies include the following possibilities: (1) use of antimicrobial agents with superior penetration properties and (2) degradation or detachment of matrix polymers.In order to investigate the penetration properties of antimicrobial agents, time-lapse microscopy can be used for noninvasive visualization of antimicrobial action inside models of oral biofilms. Factors that affect drug penetration properties include molecular weight, charge, hydrophilic–hydrophobic balance, and malabsorption/modification by the biofilm matrix.However, strategies that rely on antimicrobial agents have an important limitation. Even if the microorganisms are eradicated, their biofilm structure remains intact and may promote secondary biofilm and tartar formation. Therefore, future strategies should shift their focus to degradation and/or removal of matrix polymers.Some detachment-promoting agents have been reported. These agents have the potential to control biofilm accumulation effectively. Since the clinical efficacy remains unclear, and in addition to limitations of targeting a specific polysaccharide component and retention properties, further investigations are warranted.     

Involvement of periodontopathic biofilm in vascular diseases

Oral bacteria inhabit biofilms, which are firm clusters adhering in layers to surfaces and are not easily eliminated by immune responses and are resistant to antimicrobial agents. Dental plaque is one such biofilm. In the past 10 years, subgingival plaque bacteria forming biofilms have been increasingly reported to be involved in systemic diseases. A close relationship between microbial infections and vascular disease has also been reported in the past two decades. The present review discusses the significance of the ecologic characteristics of biofilms formed by periodontopathic bacteria in order to further clarify the associations between periodontal disease and systemic disease. We focus on the relationships between periodontal disease-associated bacteria forming biofilms and vascular diseases including atherosclerosis and carotid coronary stenotic artery disease, and we discuss the direct and indirect effects on vascular diseases of lipopolysaccharides as well as heat shock proteins produced by periodontopathic bacteria.     

Evaluation of the Effect of Two Chlorhexidine Preparations on Biofilm Bacteria In Vitro: A Three-Dimensional Quantitative Analysis

IntroductionMicroorganisms are essential in the development of periradicular diseases and are the major causative factors associated with endodontic treatment failures. Microbial biofilms are communities of bacteria that attach to surfaces and form heterogeneous three-dimensional structures. The purpose of this study was to develop a biofilm model that closely mimicked in vivo biofilm and to determine its susceptibility to endodontic antimicrobial irrigants by three-dimensional quantitative analysis.MethodsCollagen-coated hydroxyapatite (C-HA) and uncoated hydroxyapatite (HA) disks were inoculated with dispersed subgingival plaque for 3 weeks. Thick biofilms rich in spirochetes were formed on both substrates. Biofilms were subjected to 1-, 3-, and 10-minute exposures to CHX-Plus (Vista Dental Products, Racine, WI) and 2% chlorhexidine (CHX). After treatment, the volume ratio of dead bacteria to all bacteria in biofilms, indicated by the ratio of red and (red + green) fluorescence, was analyzed by confocal laser scanning microscopy for each medicament.ResultsThe proportion of killed bacteria was depended on the type of irrigant and the time of exposure in both C-HA and HA biofilm models (p = 0.00). CHX-Plus showed higher levels of bactericidal activity at all exposure times than 2% CHX (p < 0.001). The C-HA biofilm was thicker than the HA biofilm. Less bacteria were killed in C-HA biofilm than in the HA model.ConclusionsThis multispecies biofilm model and quantitative analysis methodology may be useful for the evaluation of the antimicrobial effectiveness of endodontic disinfecting agents.     

The effects of histatin-derived basic antimicrobial peptides on oral biofilms.

Susceptibility of bacteria to antimicrobial agents is strongly reduced by the formation of complex biofilms. We investigated whether synthetic histatin analogs with broad-spectrum antibacterial activity in vitro were also active against these complex mixtures of bacteria, as present in saliva and plaque. In a simplified model system for dental plaque, hydroxyapatite discs were placed in a continuous culture system comprised of Streptococcus mutans, S. sanguis, S. salivarius, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, and Prevotella intermedia. Ex situ treatment of the biofilms formed on these discs with 100 microg/mL of peptide dhvar4 significantly reduced facultative anaerobic, total anaerobic, and obligate anaerobic Gram-negative counts with 0.8, 0.5, and 0.5 log units, respectively. Ex vivo treatment of salivary bacteria gave reductions of 0.4, 0.7, and 1.5 log units, respectively. For ex vivo treatment of plaque bacteria, reductions of 0.4, 0.4, and 1.4 log units, respectively, were found. In both saliva and plaque samples, obligate anaerobic Gram-negative bacteria were significantly more susceptible to dhvar4 than facultatively anaerobic or anaerobic bacteria as a whole (p=0.013 and p=0.018, for salivary bacteria, and p=0.021 and p=0.020 for plaque bacteria, respectively). Although the oral bacteria are protected by biofilm formation, the synthetic histatin analog caused a significant reduction of viable counts in a model for oral biofilm as well as in isolated oral biofilms.     

The anticariogenic effect of amine fluorides on Streptococcus sobrinus and glucosyltransferase in biofilms

Dental caries is a chronic infectious disease caused by the accumulation of bacterial plaque (biofilm) on tooth surfaces. Antibacterial agents, in addition to other preventive measures, can control dental plaque accumulation. Amine fluorides (AmF) are known anticaries agents for over 30 years. The purpose of our study was to assess the adsorption and desorption of AmF to experimental dental biofilm and to evaluate the effect of AmF on Streptococcus sobrinus 6715 and glucosyltransferase (GTF) activity in experimental dental biofilms. The experimental plaque model used in this study consists of hydroxyapatite beads coated with human saliva (sHA), followed by adsorption of S. sobrinus and synthesis of in situ polysaccharides. Our results show that the viability of S. sobrinus in biofilm decreased as the concentration of AmF and chlorhexidine (CHX) increased. The concentration of AmF and CHX required to kill S. sobrinus adherent to sHA is about 100 times greater than the concentration required to kill the same amount of planktonic bacteria. Adsorption of AmF to surfaces was more than 90% and the desorption of AmF from our experimental model was limited. Pre-adsorption of AmF on the surface increased adhesion of S. sobrinus but also resulted in surface killing of the adsorbed bacteria. At low concentrations AmF increased GTF activity in solution by about 10%, but at concentrations above 0.1 mM it inhibited GTF activity. Inhibition of GTF on the surface required about 100 times more AmF than in solution. Our results show that AmF retains its anticariogenic effects in solution and in biofilm systems.     

A comparison of the antibacterial efficacies of essential oils against oral pathogens

Cariogenic bacteria and periodontopathic bacteria are present in dental plaque as biofilms. In this study, we investigated the antibacterial effects of essential oils on the following oral bacteria: Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sobrinus. We tested manuka oil, tea tree oil, eucalyptus oil, lavandula oil, and romarinus oil and determined their minimum inhibitory concentration and minimum bactericidal concentration. The essential oils inhibited the growth of the bacteria tested, manuka oil being the most effective. Minimum bactericidal concentration values showed that lavandula oil acts bacteriostatically, and the remaining oils, bactericidally. Periodontopathic bacterial strains tested were killed completely by exposure for 30 s to 0.2% manuka oil, tea tree oil or eucalyptus oil. Tea tree oil and manuka oil showed significant adhesion-inhibiting activity against P. gingivalis. All the essential oils tested inhibited the adhesion of S. mutans. This study showed that, among the essential oils tested, manuka oil and tea tree oil in particular had strong antibacterial activity against periodontopathic and cariogenic bacteria. From the viewpoint of safety, we also examined the effects of these essential oils on cultured human umbilical vein endothelial cells and found that, at a concentration of 0.2%, they had little effect on cultured cells.