Die Mundhöhle als Ökosystem

Biomimetic approaches for the dental plaque control Tooth and gum diseases are widespread and are primarily based on the presence of bacterial biofilms. The characterization of biofilms can be carried out by means of state‐of‐the‐art microbiome analysis that can provide information on bacterial composition and diversity. Modern oral care products mostly contain different antimicrobial agents for biofilm control. These include chlorhexidine, metal salts, and quaternary ammonium compounds, which, however, often kill harmful (dysbiotic) and useful bacteria (homeostatic) (unspecific antimicrobial effect). Recent developments show that innovative concepts shift the ecological balance of plaque in the oral cavity to “physiological commensal bacteria” (homeostasis) or minimize the bacterial colonization on enamel surfaces without having pronounced antimicrobial properties. Examples are biomimetic approaches, i.e. based on selected salivary enzymes or hydroxyapatite.     

Microscope-Based Imaging Platform for Large-Scale Analysis of Oral Biofilms

A microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distribution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grown in situ on BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eubacteria, Streptococcus spp., and Fusobacterium nucleatum were stained using specific fluorescence in situ hybridization (FISH) probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and monitored by an automated wide-field microscope-based imaging platform (Scan∧R). Automated image processing and data analysis were conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation of biofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significant differences in the composition of the microflora were recorded across individual as well as between different enamel surfaces varying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel plates that were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of biofilms up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualization and meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation, Scan∧R overcomes the technical limitations of conventional CLSM.     

Genome-genome interactions: bacterial communities in initial dental plaque

The usual context for genome-genome interactions is DNA-DNA interactions, but the manifestation of the genome is the cell. Here we focus on cell-cell interactions and relate them to the process of building multi-species biofilm communities. We propose that dental plaque communities originate as a result of intimate interactions between cells (genomes) of different species and not through clonal growth of genetically identical cells. Although DNA exchange might occur between cells within these communities, we limit our opinions to discussions of the spatiotemporal and metabolic relationships that exist here. We believe the multi-species interactions occurring during the early stages of biofilm formation determine the species composition and nature of the mature biofilm. The human oral cavity provides easy access to natural biofilms on a retrievable enamel chip, which is an excellent model for the study of genome-genome interactions.     

牙菌斑生物膜的形成与控制

牙菌斑生物膜是附着于牙釉质表面,由复杂的微生物群落构成的一种聚集体。牙菌斑生物膜的形成与生长对口腔健康有着直接或间接的影响,许多研究证实口腔疾病如龋齿和牙周病都与细菌的积累及牙菌斑的形成有关。在牙菌斑生物膜形态建成过程中,牙齿表面最初的定殖菌对生物膜的微生物组成和结构至关重要,这些初级定殖菌决定了后续与之结合形成共生体的微生物种类和数量。不同的微生物组成可能在与生物膜形成相关的口腔病理状况中发挥不同的作用。因此,本文就牙菌斑生物膜的生长及控制进行综述,介绍其微生物的早期定殖和成熟过程、以及通过物理和化学方法对牙菌斑生物膜的控制,以期为了解牙菌斑生物膜的形成机制及相关口腔疾病的预防和治疗提供有价值的参考。     

Study on the relationship between Helicobacter pylori in the dental plaque and the occurrence of dental caries or oral hygiene index

Background: The aims of our study were to determine the presence of Helicobacter pylori DNA in the dental plaque of Chinese children aged 3–6 years by nested polymerase chain reaction (PCR) and to investigate the relationship between this infection and the occurrence of dental caries or oral hygiene index.
Methods: Two hundred and fourteen children from a kindergarten in Guangzhou City of China were evaluated. The children's plaques were assessed by plaque indices of Quigley–Hein. Dental plaque was analyzed using nested PCR for two sets of primers directed to the 860-bp fragment of H. pylori genomic DNA, which have been reported to be highly sensitive and specific by other researchers.
Results: H. pylori was detected in dental plaque samples from 126 children, and 70 children with dental caries carried H. pylori in dental plaque. Of these children without infection, only 36 of 88 suffered dental caries. Besides, the average dental plaque index of 126 H. pylori -positive children was higher than that of 88 children without infection. In the present study, there was a significant correlation between H. pylori infection and dental caries or dental hygiene.
Conclusion: The oral cavity may be a reservoir for H. pylori infection in children. H. pylori in dental plaque may play a role in the occurrence of dental caries, and poor oral hygiene may represent a risk factor for H. pylori in the oral cavity.     

Preferential attachment of barnacle larvae to natural multi-species biofilms: Does surface wettability matter?

We tested the hypothesis that surface wettability does not alter the positive effect of natural biofilms on larval attachment in the barnacle Balanus amphitrite. We also answered the question: Does substratum-biofilm interaction affect the larval choice in barnacle? We developed natural multi-species biofilms of different ages on both high (glass) and low (polystyrene) wettability surfaces at mid-intertidal height (native habitat of B. amphitrite). Attachment choice of both young (0-d-old) and old (6-d-old) larvae to biofilms was determined using still water choice bioassay. Irrespective of larval age, cyprid preferred to attach to un-filmed glass than to un-filmed polystyrene. In contrast to aged larvae, young larvae preferred old (6-d-old) biofilms on polystyrene to young (3-d-old) biofilms on glass. In this study, we were also able to examine the interaction between surface wettability, biofilms and larval attachment, by characterizing bacterial community composition in biofilms. Bacterial community composition showed significant differences between biofilms of different ages. Old biofilms positively influenced larval attachment, irrespective of the type of substrata, thereby supporting the hypothesis that surface wettability does not alter the positive effect of natural biofilms on larval attachment.     

The importance of fungal pathogens and antifungal coatings in medical device infections

In recent years, increasing evidence has been collated on the contributions of fungal species, particularly Candida, to medical device infections. Fungal species can form biofilms by themselves or by participating in polymicrobial biofilms with bacteria. Thus, there is a clear need for effective preventative measures, such as thin coatings that can be applied onto medical devices to stop the attachment, proliferation, and formation of device-associated biofilms.However, fungi being eukaryotes, the challenge is greater than for bacterial infections because antifungal agents are often toxic towards eukaryotic host cells. Whilst there is extensive literature on antibacterial coatings, a far lesser body of literature exists on surfaces or coatings that prevent attachment and biofilm formation on medical devices by fungal pathogens. Here we review strategies for the design and fabrication of medical devices with antifungal surfaces. We also survey the microbiology literature on fundamental mechanisms by which fungi attach and spread on natural and synthetic surfaces. Research in this field requires close collaboration between biomaterials scientists, microbiologists and clinicians; we consider progress in the molecular understanding of fungal recognition of, and attachment to, suitable surfaces, and of ensuing metabolic changes, to be essential for designing rational approaches towards effective antifungal coatings, rather than empirical trial of coatings.     

Composition of in vitro denture plaque biofilms and susceptibility to antifungals

The aim of this study was to investigate the composition of microcosm denture plaque biofilms and the susceptibility of Candida spp. within these biofilms to antifungal agents. An in vitro model was employed to grow oral biofilms derived from denture associated stomatitis (DAS) patient samples to assess fungal growth in the presence and absence of antifungal agents. The compositions of genera present in vitro were found to be similar to those exhibited on the mucosa and denture fitting surfaces of DAS samples. Exposure to single agents, e.g., miconazole, fluconazole or chlorhexidine did not inhibit growth of Candida spp. when used in clinically relevant doses. Combinations of miconazole and chlorhexidine, pulsed into the system to mimic patient use, did reduce bacterial and candidal growth for several days. Hence, the use of dual-therapy appeared to be useful in reducing the number of viable organisms within denture plaque grown in vitro although resistance to these agents was also evident.     

Influence of topical fluoridation of glass ionomer cements on inhibitory activity on cariogenic bacteria

Glass ionomer cements are important options in restorative and preventive dentistry due to their adhesion to the tooth surface and fluoride release, which can decrease the risk of recurrent caries. The aim of this study was to define, in vivo, the influence of the topical use of fluoride gel on dental plaque bacteria growing on the glass ionomer cement. Fifteen patients were included into this study. Thirty five class V restorations from the glass ionomer cement (Ketac Molar Aplicap, ESPE Germany) were placed in the patient's one half of the lower jaw. The sound enamel of other side of the lower jaw was treated as a control. After 6 month 72 old dental plaque was collected from the surfaces of restorations and the surfaces of the sound enamel. Total amount of 30 dental plaque samples were investigated according to the previously described method (17). In dental plaque samples the amount of Streptococcus mutans was calculated at the Department of Microbiology, Medical University of Lód?. Next the topical application of fluoride gel (Fluormex) was performed on the surfaces of glass ionomer (Ketac Molar) fillings and the sound enamel. The patients were asked not to clean the teeth for 72 h. After this time the dental plaque was again collected from the surfaces of restorations and sound enamel. Statistical analysis of collected data was accomplished and showed no statistically significant differences in the amount of Streptococcus mutans both on Ketac Molar and the enamel before and after the topical use of fluoride gel. It was concluded that the topical fluoridation of glass ionomer cement did not affect Streptococcus mutans growing in dental plaque.     

Fungal Biofilms in the Clinical Lab Setting

Device-related infections are often associated with biofilms (microbial communities encased within polysaccharide-rich extracellular matrix) formed by pathogens on surfaces of these devices. Candida species are the most common fungi isolated from infections associated with catheters and dentures, and both Candida and Fusarium are commonly isolated from contact lens–related infections such as fungal keratitis. These biofilms exhibit decreased susceptibility to most antimicrobial agents, which contributes to the persistence of infection. Drug resistance in fungal biofilms is multifactorial and phase-dependent; for example, efflux pumps mediate resistance in biofilms during early phase, whereas altered membrane sterol composition contributes to resistance in mature phase. Both substrate type and surface coatings play an important role in the pathogenesis of device-related fungal biofilms. Host immune cells influence the ability of Candida to form biofilms in vitro. This review summarizes recent advances in research on fungal biofilms and discusses their clinical relevance.     

A continuous culture biofilm model of cariogenic responses

AIMS: To validate an in vitro model for the analysis of physiological and ecological responses to sugar challenge in bacterial populations, and subsequent changes in enamel mineralization. METHODS AND RESULTS: A seven-organism bacterial consortium was grown in a biofilm mode on enamel and hydroxyapatite (HA) surfaces in a continuous culture system and exposed to repeated sucrose challenges. This produced 'pH-cycling' conditions within the system. Populations on HA surfaces were enumerated. Changes in relative proportions of the different populations, and in the total viable count, were observed, between different treatments. Microradiography of the enamel sections showed increasing demineralization with increasing sucrose concentration. The lesions formed were similar to 'white-spot' lesions found in vivo. Differences in the quality of biofilms formed were also observed using Confocal Laser Scanning Microscopy. CONCLUSION: An in vitro model has been validated for the analysis of both physiological and ecological responses to sucrose challenges in bacterial populations, and subsequent changes in enamel mineralization. SIGNIFICANCE AND IMPACT OF THE STUDY: This model should facilitate the study of changes in bacterial populations in response to application of putative anticaries agents and concomitant changes in enamel mineralization.     

Dispersal and inhibition of biofilms associated with infections

As bacteria aggregate and form biofilms on surfaces in the human body such as tissues, indwelling medical devices, dressings and implants, they can cause a significant health risk. Bacterial biofilms possess altered phenotypes: physical features that facilitate antibiotic resistance and evasion of the host immune response. Since metabolic and physical factors contribute to biofilm maturation and persistence, an objective in antibiofilm therapy is to target these factors to deliver innovative approaches for solving these important health problems. Currently, there is little research on the direct immunological effects resulting from the introduction of foreign components to the body pertaining to biofilm inhibition methods. Detailed research involving animal models is necessary to better understand the biological side effects of synthetic peptides, genetically modified bacteriophages and isolated proteins and any resistance that may develop from these approaches.     

Polymerase chain reaction detection of potentially pathogenic free-living amoebae in dental units

Several genera of amoebae can be found in water from dental units and on the inner surface of waterlines. The presence of bacterial biofilms on these surfaces is thought to favor the proliferation of amoebae. Potentially pathogenic Acanthamoeba and Naegleria spp. may be an infection risk for patients through contact with open surgical sites or aerosolization. A polymerase chain reaction of DNA extracted from pelleted samples showed that Acanthamoeba spp. and Naegleria spp. were present in water from dental units, suction lines, and suction filters at the dental clinic of the Université de Montréal. Acanthamoeba spp. were detected in 24.2% of 66 samples and Naegleria spp. in 3.0%. We discuss the infection risk associated with these results.     

Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England

AIMS: To investigate treated water distribution systems in England as a source of Helicobacter pylori. METHODS AND RESULTS: Water and biofilms were obtained from 11 domestic and seven educational properties and from hydrants, reservoirs and water meters supplied by three water utilities. Samples were cultured on nonselective and antibiotic containing media combined with immunomagnetic separation concentration. Viable helicobacters were not detected in any of the 151 samples but Helicobacter-specific PCR assays detected DNA in 26% of samples from domestic properties, schools and hydrants with the highest frequency in biofilms (42%). Direct sequencing of six selected amplicons confirmed >95% sequence homology to H. pylori. CONCLUSIONS: While viable helicobacters were not isolated, evidence was obtained for the presence of Helicobacter DNA, including that of H. pylori. Biofilms on surfaces within water distribution systems may act either as sites for the passive accumulation of helicobacters or as potentially important reservoirs of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings strengthen evidence that H. pylori may be transmitted through drinking water. However, there is currently no evidence that viable cells can survive the disinfection levels used in UK mains supplies and the health risk from this source remains unclear.     

Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces

Candida albicans and mutans streptococci are frequently detected in dental plaque biofilms from toddlers afflicted with early childhood caries. Glucosyltransferases (Gtfs) secreted by Streptococcus mutans bind to saliva-coated apatite (sHA) and to bacterial surfaces, synthesizing exopolymers in situ, which promote cell clustering and adherence to tooth enamel. We investigated the potential role Gtfs may play in mediating the interactions between C. albicans SC5314 and S. mutans UA159, both with each other and with the sHA surface. GtfB adhered effectively to the C. albicans yeast cell surface in an enzymatically active form, as determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the yeast cell surface were more susceptible to dextranase than those synthesized in solution or on sHA and bacterial cell surfaces (P < 0.05), indicating an elevated α-1,6-linked glucose content. Fluorescence imaging revealed that larger numbers of S. mutans cells bound to C. albicans cells with glucans present on their surface than to yeast cells without surface glucans (uncoated). The glucans formed in situ also enhanced C. albicans interactions with sHA, as determined by a novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast cells significantly increased the accumulation of S. mutans on the sHA surface (versus S. mutans incubated alone or mixed with uncoated C. albicans; P < 0.05). These data reveal a novel cross-kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast cell surface. Surface-bound GtfB promotes the formation of a glucan-rich matrix in situ and may enhance the accumulation of S. mutans on the tooth enamel surface, thereby modulating the development of virulent biofilms.     

Microbial biofilms in the human gastrointestinal tract

The human gastrointestinal tract contains rich and diverse microbiotas along its length. However, while extensive studies have been made on lumenal bacterial communities in the gut, less work has been carried out on organisms growing in biofilms, where individual groups of bacteria exist in a multiplicity of different microhabitats and metabolic niches associated with the mucosa, the mucus layer and particulate surfaces in the gut lumen. Bacteria and yeasts also occur in biofilms attached to artificial surfaces and devices implanted in the host, such as in patients being fed via enteral tubes. Although we are just beginning to investigate the composition and metabolic activities of these structures, increasing evidence suggests that they are important to the host in both health and disease. There is mounting interest in mucosal biofilms in the colon, especially with respect to their role in inflammatory bowel disease. Because bacteria growing in biofilms are more resistant to antibiotics than unattached organisms, it is often difficult to modify the structure and composition of these communities, or to eradicate them from the body. However, recent work has shown that there is considerable potential to alter the species composition of mucosal biofilms in a beneficial way using synbiotics.     

A modified chemostat system to study the ecology of oral biofilms

Previously, we developed a chemostat system to study the behaviour and properties of a community of up to 10 species of oral bacteria. The present study describes modification of this system to incorporate removable and replaceable hydroxyapatite (the major mineral in human dental enamel) disks on which biofilms could develop. Hydroxyapatite disks were immersed in the chemostat for known time periods, and the bacterial content of biofilms determined by viable counting. Initial deposition rates were rapid, with all 10 species detected after 1 h, and the numbers of bacteria in biofilms continued to increase for 21 d. The species composition of biofilms reflected that of the surrounding fluid phase, and showed only limited signs of the type of 'species succession' which is observed in developing dental plaque in vivo , although anaerobic species increased in proportion in older biofilms. Four-day biofilms showed the least variability and were chosen as the 'standard biofilm' for more detailed study. Variability in the bacterial composition of 4-d biofilms was comparable both within a single chemostat run and between independent chemostat runs. Glucose pulsing in the absence of pH control resulted in the selection of cariogenic species; the disruption of the biofilm community was less marked than that of the equivalent planktonic culture. The model system has considerable potential in studying the effects of a variety of factors on biofilm development, as well as in comparing the efficacy of antimicrobial systems against biofilms.     

Molecular characterization of subject-specific oral microflora during initial colonization of enamel

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.     

Adherence, accumulation, and cell division of a natural adherent bacterial population.

Developing dental bacterial plaques formed in vivo on enamel surfaces were examined in specimens from 18 adult volunteers during the first day of plaque formation. An intraoral model placing enamel pieces onto teeth was used to study bacterial plaque populations developing naturally to various cell densities per square millimeter of surface area of the enamel (W. F. Liljemark, C. G. Bloomquist, C. L. Bandt, B. L. Philstrom, J. E. Hinrichs, and L. F. Wolff, Oral Microbiol. Immunol. 8:5-15, 1993). Radiolabeled nucleoside incorporation was used to measure DNA synthesis concurrent with the taking of standard viable cell counts of the plaque samples. Results showed that in vivo plaque formation began with the rapid adherence of bacteria until ca. 12 to 32% of the enamel's salivary pellicle was saturated (ca. 2.5 x 10(5) to 6.3 x 10(5) cells per mm2). The pioneer adherent species were predominantly those of the "sanguis streptococci." At the above-noted density, the bacteria present on the salivary pellicle incorporated low levels of radiolabeled nucleoside per viable cell. As bacterial numbers reached densities between 8.0 x 10(5) and 2.0 x 10(6) cells per mm2, there was a small increase in the incorporation of radiolabeled nucleosides per cell. At 2.5 x 10(6) to 4.0 x 10(6) cells per mm2 of enamel surface, there was a marked increase in the incorporation of radiolabeled nucleosides per cell which appeared to be cell-density dependent. The predominant species group in developing dental plaque films during density-dependent growth was the sanguis streptococci; however, most other species present showed similar patterns of increased DNA synthesis as the density noted above approached 2.5 x 10(6) to 4.0 x 10(6) cells per mm2.