Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling

AIMS: The aim of this study was to use confocal laser scanning microscopy (CLSM) to examine the spatial distribution of both viable and nonviable bacteria within microcosm dental plaques grown in vitro. Previous in vivo studies have reported upon the distribution of viable bacteria only. METHODS AND RESULTS: Oral biofilms were grown on hydroxyapatite (HA) discs in a constant-depth film fermenter (CDFF) from a saliva inoculum. The biofilms were stained with the BacLight LIVE/DEAD system and examined by CLSM. Fluorescence intensity profiles through the depth of the biofilm showed an offset between the maximum viable intensity and the maximum nonviable intensity. Topographical differences between the surface properties of the viable and nonviable biofilm virtual surfaces were also measured. CONCLUSIONS: The profile of fluorescence intensity from viable and nonviable staining suggested that the upper layers of the biofilm contain proportionally more viable bacteria than the lower regions of the biofilm. SIGNIFICANCE AND IMPACT OF STUDY: Viability profiling records the transition from predominantly viable to nonviable bacteria through biofilms suggesting that this technique may be of use for quantifying the effects of antimicrobial compounds upon biofilms. The distribution of viable bacteria was similar to that found in dental plaque in vivo suggesting that the CDFF produces in vitro biofilms which are comparable to their in vivo counterparts in terms of the spatial distribution of viable bacteria.     

Measuring the thickness of an outer layer of viable bacteria in an oral biofilm by viability mapping

Bacterial biofilms have been reported to contain distinct regions of viable and nonviable bacteria. The purpose of this study was to identify such regions in biofilms of oral bacteria and to determine their dimensions. Oral biofilms were grown aerobically in a constant-depth film fermenter (CDFF) and studied using confocal laser scanning microscopy (CLSM) incorporating viability staining with water immersion lenses. A variety of viability distributions were observed, including biofilm "stacks" possessing an outer layer of viable bacteria surrounding an internal core of nonviable bacteria. Using image analysis tools, we measured the thickness of this outer viable region, in the x-y plane, from single confocal optical sections, and determined the mean angle (theta) of these portions of the biofilm stack (10.93 degrees ). x-y plane thickness data in conjunction with the data on the angle of the stack returned the thickness of the outer viable layer perpendicular to the bulk medium flow as 36.62 microm (31.61-42.21 microm accounting for 95% confidence for variation in both the x-y plane thickness and theta). We have shown that CLSM, in conjunction with vital stains and image analysis techniques, can reveal viability patterns in biofilms and where appropriate can be used to measure the dimensions of these structures.     

Effect of Chlorhexidine on Multi-Species Biofilms

With human saliva as an inoculum, microcosm dental plaques were grown on dental amalgam in a constant-depth film fermentor (CDFF) in the presence (suc +ve) and absence (suc −ve) of sucrose. The biofilms were then exposed to 0.2% chlorhexidine gluconate (CHG) for 1, 5, or 60 min and the survivors enumerated. Suc +ve biofilms had higher proportions of streptococci but lower proportions of veillonellae than suc −ve biofilms. Exposure to CHG for 1 min reduced the viable count of suc −ve and suc +ve biofilms by 53% and 89% respectively. In both cases, reductions were mainly attributable to killing of streptococci and actinomyces. After 60 min of exposure, 4% of the bacteria in the suc −ve and 2% in the suc +ve biofilms remained viable. This study has shown that large numbers of bacteria in microcosm dental plaques can survive a 1-min exposure to 0.2% CHG and that even after a 60-min exposure, substantial numbers of bacteria remain viable. Received: 16 May 1997 / Accepted: 7 July 1997     

Application of carbon source utilization patterns to measure the metabolic similarity of complex dental plaque biofilm microcosms

Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H2-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (Streptococcus mutans plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.     

Application of Carbon Source Utilization Patterns To Measure the Metabolic Similarity of Complex Dental Plaque Biofilm Microcosms

Biolog technology was applied to measure the metabolic similarity of plaque biofilm microcosms, which model the complex properties of dental plaque in vivo. The choice of Biolog plate, incubation time, and incubation conditions strongly influenced utilization profiles. For plaque biofilm microcosms, Biolog GP2 plates incubated anaerobically in an H₂-free atmosphere gave the clearest profile. To test the application of the Biolog GP2 assay, plaque microcosms were developed under different nutrient conditions in which the frequency of sucrose application was varied. Cluster analysis of Biolog GP2 data from 10 microcosm biofilms correlated with sucrose frequency. Aciduric bacteria (Streptococcus mutans plus lactobacilli) predominated in the plaques receiving high-frequency sucrose applications. Agreement between the Biolog GP2 groupings with nutrient and compositional changes suggests that Biolog analysis is a valuable technique for analyzing the metabolic similarity of dental plaque biofilm microcosms and other high-nutrient or predominantly anaerobic ecosystems.     

A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms

Dental plaque bacteria form complex and robust cell aggregates which cannot be counted accurately using epifluorescence microscopy. This causes a significant problem for quantifying their viability. The aim of the investigation was to develop a fluorescence assay to quantify the viable biomass of dental plaque biofilms. Using an artificial mouth system, microcosm plaques were grown under a range of fluoride and mineralizing conditions, and were treated with the oral antiseptics chlorhexidine (CHX) and Listerine. Plaques were harvested, made into suspension and stained in microtitre plates with a di-chromatic fluorescent stain (Live/Dead BacLight). The percentage of viable biomass was calculated from the regression data generated from a viability standard. The standard was constructed using different proportions of viable (green fluorescence) and non-viable (red fluorescence) plaque bacteria, and growth conditions for optimizing green fluorescence were investigated. The results from the assay showed that fluoride at 1000 and 3000 ppm promoted plaque viability by at least 15%, from approximately 45 to 60%, and at 5000 ppm to approximately 87% (P<0.05). Plaques treated with Listerine and CHX from d 0 yielded insufficient biomass to be tested for viability, however 14 d post-treatment, viability was comparable to untreated plaques (approximately 55%, P>0.05). Treatment with Listerine and CHX from d 3 reduced biomass but not viability. Development of this assay enabled viability of plaque bacteria which cannot be resolved with epifluorescence microscopy to be evaluated. It offers a rapid alternative to epifluorescence microscopy and could be applied to nonoral bacteria.     

Modeling shifts in microbial populations associated with health or disease

Stable microbial communities associated with health can be disrupted by altered environmental conditions. Periodontal diseases are associated with changes in the resident oral microflora. For example, as gingivitis develops, a key change in the microbial composition of dental plaque is the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. We describe the use of an in vitro model to replicate this population shift, first with a dual-species model (Actinomyces naeslundii and Streptococcus sobrinus) and then using a microcosm model of dental plaque. The population shift was induced by environmental changes associated with gingivitis, first by the addition of artificial gingival crevicular fluid and then by a switch to a microaerophilic atmosphere. In addition to the observed population shifts, confocal laser scanning microscopy also revealed structural changes and differences in the distribution of viable and nonviable bacteria associated with the change in environmental conditions. This model provides an appropriate system for the further understanding of microbial population shifts associated with gingivitis and for the testing of, for example, antimicrobial agents.     

Growth and Activities of Sulfate-Reducing and Methanogenic Bacteria in Human Oral Cavity

Viable counts and activities of sulfate-reducing bacteria (SRB) and methanogenic bacteria were determined in the oral cavities of eight volunteers. Of these, seven harbored viable SRB populations, and six harbored viable methanogenic bacterial populations. Two volunteers classified as type III periodontal patients had both SRB and methanogenic bacteria. Six separate sites were sampled: posterior tongue, anterior tongue, mid-buccal mucosa, vestibular mucosa, supragingival plaque, and subgingival plaque. The SRB was found in all areas in one volunteer, and it was mostly present in posterior tongue, anterior tongue, supragingival, and subgingival plaques in many volunteers. The methanogenic bacteria were mostly found in supragingival and subgingival plaques. The activities of sulfate reduction and methane production were determined in randomly selected isolates. Received: 27 July 2002 / Accepted: 27 August 2002     

Modeling Shifts in Microbial Populations Associated with Health or Disease

Stable microbial communities associated with health can be disrupted by altered environmental conditions. Periodontal diseases are associated with changes in the resident oral microflora. For example, as gingivitis develops, a key change in the microbial composition of dental plaque is the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. We describe the use of an in vitro model to replicate this population shift, first with a dual-species model (Actinomyces naeslundii and Streptococcus sobrinus) and then using a microcosm model of dental plaque. The population shift was induced by environmental changes associated with gingivitis, first by the addition of artificial gingival crevicular fluid and then by a switch to a microaerophilic atmosphere. In addition to the observed population shifts, confocal laser scanning microscopy also revealed structural changes and differences in the distribution of viable and nonviable bacteria associated with the change in environmental conditions. This model provides an appropriate system for the further understanding of microbial population shifts associated with gingivitis and for the testing of, for example, antimicrobial agents.     

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。     

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.     

Detection of dental plaque and its potential pathogenicity using quantitative light‐induced fluorescence

Quantitative light‐induced fluorescence (QLF) technology can detect some dental plaque as red fluorescence. This in vivo study aimed to identify the microbial characteristics of red fluorescent (RF) dental plaque using 16S rRNA gene sequencing and evaluate the correlations between RF plaque and the clinical symptoms of dental diseases. Paired supragingival plaque samples collected from each 10 subjects and consisted of RF and non‐RF dental plaques as observed by QLF technology using a 405 nm blue light source for excitation. The characteristics of the bacterial communities in the RF and non‐RF plaque samples were compared by sequencing analysis. An increase in microbial diversity was observed in RF plaque compared with the non‐RF plaque. There were significant differences in the community compositions between the 2 types of dental plaque. Periodontopathic bacteria were significantly more abundant in the RF plaque than non‐RF plaque. The fluorescence intensity of RF plaque was significantly related to the proportion of the periodontopathic bacterial community and the presence of gingival inflammation. In conclusion, the plaque red fluorescence is associated with changes in the microbial composition and enrichment of periodontopathic pathogens, which suggests that RF plaque detected by QLF technology could be used as a risk indicator for gingival inflammation.      

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.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

Conformational change of the hexagonally packed intermediate layer of Deinococcus radiodurans monitored by atomic force microscopy.

Both surfaces of the hexagonally packed intermediate (HPI) layer of Deinococcus radiodurans were imaged in buffer solution by atomic force microscopy. When adsorbed to freshly cleaved mica, the hydrophilic outer surface of the HPI layer was attached to the substrate and the hydrophobic inner surface was exposed to the stylus. The height of a single HPI layer was 7.0 nm, while overlapping edges of adjacent single layers adsorbed to mica had a height of 14.7 nm. However, double-layered stacks with inner surfaces facing each other exhibited a height of 17.4 nm. These stacks exposed the outer surface to the stylus. The different heights of overlapping layers and stacks are attributed to differences in the interaction between inner and outer surfaces. At high resolution, the inner surface revealed a protruding core with a central pore connected by six emanating arms. The pores exhibited two conformations, one with and the other without a central plug. Individual pores were observed to switch from one state to the other.     

Effect of minocycline on subgingival plaque bacteria

The effects of minocycline on subgingival plaque samples from patients with chronic periodontitis were investigated in vitro. Minocycline concentrations as low as 1.0 microgram/ml inhibited 95.7% of the cultivable bacteria in the samples but 256 micrograms/ml was necessary to inhibit all of the cultivable bacteria in the samples. Although up to 99.9% of bacteria in the plaque samples were killed by a 6 h exposure to 8.0 micrograms/ml of minocycline, large numbers of viable bacteria remained. These results imply that adequate reductions in the numbers of viable subgingival plaque bacteria are unlikely to occur after exposure to minocycline at concentrations attainable in gingival crevicular fluid after systemic administration.     

The effect of chlorhexidine on defined, mixed culture oral biofilms grown in a novel model system

In order to develop an improved method to evaluate antimicrobial agents for use in clinical dentistry, a constant-depth film fermenter (CDFF) has been used to generate biofilms of fixed depth comprising nine species of bacteria commonly found in dental plaque in health and disease. These bacteria were grown together initially in a conventional chemostat which was used to inoculate the CDFF over an 8 h period. Medium was then supplied directly to the CDFF and biofilms allowed to develop. The biofilms were then challenged with eight short pulses of two concentrations of chlorhexidine (0·0125 and 0·125% w/v). The lower concentration had a limited effect on the composition of the biofilms while a differential and substantial inhibition was obtained with a higher concentration. Actinomyces naeslundii was lost from the biofilm, and the viable counts of streptococci, Fusobacterium nucleatum and Porphyromonas gingivalis were inhibited by over three orders of magnitude by 0·125% chlorhexidine, whereas Veillonella dispar was only transiently affected. The findings were consistent with those from clinical studies of dental plaque, suggesting that this model would have a predictive value when evaluating novel antiplaque or antimicrobial inhibitors.     

Effect of minocycline on subgingival plaque bacteria

The effects of minocycline on subgingival plaque samples from patients with chronic periodontitis were investigated in vitro. Minocycline concentrations as low as 1.0 μg/ml inhibited 95.7% of the cultivable bacteria in the samples but 256 μg/ml was necessary to inhibit all of the cultivable bacteria in the samples. Although up to 99.9% of bacteria in the plaque samples were killed by a 6 h exposure to 8.0 μg/ml of minocycline, large numbers of viable bacteria remained. These results imply that adequate reductions in the numbers of viable subgingival plaque bacteria are unlikely to occur after exposure to minocycline at concentrations attainable in gin-gival crevicular fluid after systemic administration.     

Relationship of periodontal clinical parameters with bacterial composition in human dental plaque

More than 600 bacterial species have been identified in the oral cavity, but only a limited number of species show a strong association with periodontitis. The purpose of the present study was to provide a comprehensive outline of the microbiota in dental plaque related to periodontal status. Dental plaque from 90 subjects was sampled, and the subjects were clustered based on bacterial composition using the terminal restriction fragment length polymorphism of 16S rRNA genes. Here, we evaluated (1) periodontal clinical parameters between clusters; (2) the correlation of subgingival bacterial composition with supragingival bacterial composition; and (3) the association between bacterial interspecies in dental plaque using a graphical Gaussian model. Cluster 1 (C1) having high prevalence of pathogenic bacteria in subgingival plaque showed increasing values of the parameters. The values of the parameters in Cluster 2a (C2a) having high prevalence of non-pathogenic bacteria were markedly lower than those in C1. A cluster having low prevalence of non-pathogenic bacteria in supragingival plaque showed increasing values of the parameters. The bacterial patterns between subgingival plaque and supragingival plaque were significantly correlated. Chief pathogens, such as Porphyromonas gingivalis, formed a network with other pathogenic species in C1, whereas a network of non-pathogenic species, such as Rothia sp. and Lautropia sp., tended to compete with a network of pathogenic species in C2a. Periodontal status relates to non-pathogenic species as well as to pathogenic species, suggesting that the bacterial interspecies connection affects dental plaque virulence.