Plaque Microcosms Research Articles

Bacteriological effects of a Lactobacillus reuteri probiotic on in vitro oral biofilms

ObjectiveDespite the targeted and incidental exposure of the oral cavity to probiotic bacteria, relatively little information is available concerning their bacteriological effects or ecological fate in this environment. We have investigated the effects of an oral probiotic bacterium, Lactobacillus reuteri on salivary biofilm microcosms. DesignNascent in vitro plaques in hydroxyapatite disc model and mature continuous culture plaques within constant depth film fermenters (CDFFs) were exposed to probiotic strains of L. reuteri. Microbial composition was assessed using differential viable counting and CDFF plaques were additionally characterised using qPCR and PCR-DGGE. ResultsProbiotic dosing of nascent plaques was associated with significant increases in lactobacilli and Gram-negative anaerobes in both biofilm and planktonic phases, and with elevated counts of streptococci in the planktonic phase that were not accompanied by decreases in pH. In mature CDFF plaques, differential culture indicated that whilst significant increases in lactobacillus counts occurred during dosing, numbers of other bacterial groups and pH did not significantly change. Monitoring the probiotic strain in CDFFs using selective culture and qPCR indicated that the exogenous bacterium reached and maintained c. 5log10CFU/mm2 during dosing and 20 days after cessation. The apparent lack of marked bacterial inhibition by L. reuteri in both model systems was supported by the absence of antagonism in binary antagonism assays. ConclusionThe largest compositional changes occurred in nascent plaques where the addition of lactobacilli caused significant increases in streptococci and Gram-negative anaerobes, the latter possibly via lactate syntrophy. Exogenous L. reuteri persisted in mature plaque microcosms after dosing ceased.

The Effects of Fractions from Shiitake Mushroom on Composition and Cariogenicity of Dental Plaque Microcosms in an In Vitro Caries Model

The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) in in vitro caries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential.

An in vitro evaluation of hydrolytic enzymes as dental plaque control agents

The plaque-control potential of commercially available amylase, lipase and protease was evaluated by observing their effects on coaggregation and on bacterial viability within various plaque microcosms. A quantitative coaggregation assay indicated that protease significantly inhibited the extent of coaggregation of Actinomyces naeslundii and Streptococcus oralis (P <0.05) and of Porphyromonas gingivalis and S. oralis. Amylase significantly (P <0.05) increased the coaggregation of A. naeslundii versus Fusobacterium nucleatum and A. naeslundii versus P. gingivalis. Concomitant challenge of constant-depth film fermenter-grown plaques with the enzymes did not result in detectable ecological perturbations (assessed by differential culture and denaturing gradient gel electrophoresis). Similar dosing and analysis of multiple Sorbarod devices did not reveal increases in bacterial dispersion which could result from disaggregation of extant plaques. A short-term hydroxyapatite colonization model was therefore used to investigate possible enzyme effects on early-stage plaque development. Whilst culture did not indicate significant reductions in adhesion or plaque accumulation, a vital visual assay revealed significantly increased aggregation frequency following enzyme exposure. In summary, although hydrolytic enzymes negatively influenced binary coaggregation, they did not cause statistically significant changes in bacterial viability within plaque microcosms. In contrast, enzyme exposure increased aggregation within extant plaques.

Development of an in vitro denture plaque biofilm to model denture malodour

This study aimed to develop an in vitro denture plaque biofilm to model denture malodour. No previous studies have attempted to characterize the malodour associated with dentures and the effect of Candida spp. (main aetiological agent of denture-related stomatitis) on malodour. Pooled denture plaque microcosms and ‘model’ denture plaque biofilms (pooled saliva supplemented with additional microbial species) with and without addition of candida were grown aerobically at 37 °C for up to 13 days in a constant depth film fermenter (CDFF) on denture acrylic discs. Sample discs were removed, rinsed in sterile water and placed in phosphate buffered saline (PBS). The discs were vortex mixed to remove the biofilms, diluted in PBS and plated in duplicate onto general and selective media. The composition and stability of the biofilms over time were assessed. CDFF-grown microcosms and ‘model’ denture plaque biofilms were relatively stable in composition, with streptococci remaining the dominant microbial group. Model denture plaque biofilms were comparable in composition to denture plaque microcosms. This model system has the potential for evaluation of agents that might affect these parameters such as denture cleansers and other oral hygiene treatments.

Microbiota of Plaque Microcosm Biofilms: Effect of Three Times Daily Sucrose Pulses in Different Simulated Oral Environments

Aim: To explore the Ecological Plaque Hypothesis for dental caries. To test modification of the microbiota of dental plaque microcosm biofilms by sucrose pulsing during growth in two different simulated oral fluids, and with a urea-induced plaque pH elevation. Methods: Plaque microcosm biofilms were cultured in an ‘artificial mouth’ with and without 6-min 5% w/v sucrose pulses every 8 h in an environment of continuously supplied saliva-like defined medium with mucin (DMM), or basal medium mucin (BMM, a high-peptone-yeast extract oral fluid analogue), and also in DMM + 10 mmol/l urea, with sucrose pulsing. Forty plaque species were quantified by checkerboard DNA:DNA hybridization analysis. Results: Sucrose pulsing extended rapid plaque growth in DMM and BMM, inducing major microbiota changes in DMM but not in BMM. In DMM, some streptococci and lactobacilli were unaffected while others implicated in caries, together with Candida albicans and Capnocytophaga gingivalis, increased. Aerobic, microaerophilic and major anaerobic species decreased. Elevation of the pH_max from 6.4 to 7.0 had almost no effect on the microbiota. BMM plaques were distinct from DMM plaques with particularly low levels of Candida albicans and Actinomyces. Conclusions: Modest sucrose exposure in a saliva-like environment causes profound changes in the developmental self-organization of plaque microcosms, supporting the Ecological Plaque Hypothesis. Nevertheless, there is significant stability in microbial composition with varying pH near neutrality. Increases in levels of specific bacteria in response to sucrose could be characteristic of organisms particularly important in caries.

Caries‐related plaque microcosm biofilms developed in microplates

In vivo dental plaque biofilms consist of complex communities of oral bacteria that are a challenge to replicate in vitro. The aim of this investigation was to establish human dental plaque microcosms in microplates to reflect conditions that are relevant to dental caries. Microcosm plaque biofilms were initiated from the saliva of two different donors, grown for up to 10 days in 24-welled microplates on Thermanox coverslips in various types of artificial saliva with and without sucrose, which were replaced daily. Microbiota composition of 40 species associated with oral health and dental caries was monitored in the plaques using Checkerboard DNA-DNA hybridization analysis. pH was measured as an indicator of cariogenic potential. The composition of the saliva inocula was different, and yielded plaque microcosms with different composition and growth responses to sucrose. Artificial saliva type and presence of sucrose, and the resulting growth and pH conditions, modified the growth of individual species and hence the ecological profile of the microplate plaques during development. Complex population shifts were observed during development, and older plaques comprised predominantly facultative anaerobic species. Sucrose supplementation limited the decline of Streptococci over time but did not increase the abundance of mutans Streptococci. Sucrose at 0.15% increased levels of caries-associated species including Lactobacillus fermentum, Lactobacillus acidophilus and Actinomyces gerensceriae; these were further increased with sucrose at 0.5%, in addition to Actinomyces israelii, Rothia dentocariosa and Capnocytophaga gingivalis. The microplate plaques demonstrated complex community dynamics that appeared to reflect the maturation of natural plaques, and sucrose induced a cariogenic plaque composition and pH.

Variation in bacterial DGGE patterns from human saliva: over time, between individuals and in corresponding dental plaque microcosms

Eubacterial 16S rDNA fingerprints of human saliva and dental plaque microcosm biofilms grown in the multi-plaque artificial mouth (MAM) were characterised using denaturing gradient gel electrophoresis (DGGE). The stability of the bacterial community in the saliva of one individual collected over 7 years was assessed and compared with bacterial patterns in the saliva of 10 different individuals. DGGE was also used to assess changes in bacterial composition between saliva and mature plaque microcosms developed in the MAM from these 10 individual saliva samples. A relatively stable bacterial community (>87% concordance) was maintained within the individual oral environment of the standard donor over 7 years of monitoring. By comparison, DGGE fingerprint patterns of saliva from 10 different donors displayed greater variability (66% concordance). Variability between individual DGGE profiles increased further in mature plaque microcosms grown from the saliva of the 10 donors (52% concordance) with an increase in detected species diversity and evidence for conserved similarity and hence the maintenance of organisation during community development. These results suggest that stable ecological conditions were maintained long-term within the oral environment of the individual saliva donor but that transient fluctuations also occurred. The ecology and predominating microbiota in different individuals was host-specific and these differences were maintained to a degree during development into mature plaque microcosms. These findings also demonstrate the potential usefulness of applying DGGE to monitor temporal and developmental changes and possibly pathogenic patterns in oral bacterial communities from saliva and plaque.

Effects of triclosan-containing rinse on the dynamics and antimicrobial susceptibility of in vitro plaque ecosystems.

Dental plaque microcosms were established under a feast-famine regimen within constant-depth film fermentors and exposed four times daily postfeeding to a triclosan (TR)-containing rinse (dentifrice) (TRD). This was diluted so that the antimicrobial content was 0.6 mg/ml. Microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene (rDNA). Dominant isolates and PCR amplicons were identified by partial sequencing of 16S rDNA. TRD caused considerable decreases in the counts of both gram-negative organisms and total anaerobic cells, transiently lowered the numbers of streptococci and actinomycetes, and markedly increased the proportion of lactobacilli. DGGE indicated the presence of putatively unculturable bacteria and showed that a Porphyromonas sp. and Selenomonas infelix had been inhibited by TRD. Pure culture studies of 10 oral bacteria (eight genera) showed that Neisseria subflava, Prevotella nigrescens, and Porphyromonas gingivalis were highly susceptible to TR, while the lactobacilli and streptococci were the least susceptible. Clonal expansion of the lactobacilli in the pulsed microcosm could be explained on the basis of TR activity. The mean MICs of TR, chlorhexidine, erythromycin, penicillin V, and vancomycin for the population before and after 5 days of exposure to TRD showed few significant changes. In conclusion, changes in plaque microcosm populations following repeated exposure to TRD showed inhibition of the most susceptible flora and clonal expansion of less susceptible species.

Effects of a chlorhexidine gluconate-containing mouthwash on the vitality and antimicrobial susceptibility of in vitro oral bacterial ecosystems.

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (P < 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in Prevotella sp. and Selenomonas infelix. Pure-culture studies of 10 oral bacteria (eight genera) showed that Actinomyces naeslundii, Veillonella dispar, Prevotella nigrescens, and the streptococci were highly susceptible to CHX, while Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (P < 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.

Growth and molecular characterization of dental plaque microcosms.

(i) To compare the effects of feeding protocols upon the composition and stability of dental plaque microcosms formed in constant-depth film fermenters (CDFF). (ii) To evaluate the utility of denaturing gradient gel electrophoresis (DGGE) and culture methodologies for the investigation of such models. Microcosms were established anaerobically in the CDFFs from freshly collected saliva. These were fed either with artificial saliva alone (famine) or combined with discontinuous feeding (feast-famine). Culture and 16s rDNA sequencing indicated that supplemental feeding gave ca. 2 log increases in Lactobacillus rhamnosus and Prevotella buccae. Feast-famine microcosms were then further characterized by DGGE using primers specific for the V2-V3 region of eubacterial rDNA. These gave single major bands with pure cultures (eight species) and resolved all strains apart from Lact. rhamnosus and Actinomyces naeslundii. Whilst culture with selective media indicated a degree of stability and reproducibility between replicate microcosms, DGGE showed a considerable degree of variability that related to several putatively uncultured bacteria. Feast-famine regimes altered community composition. DGGE analyses identified putatively unculturable species and demonstrated variability between replicate fermenters. This study demonstrates the utility of DGGE for the analysis of dental plaque, especially with respect to unculturable bacteria. Results question the assumptions of reproducibility of plaque microcosms established in non-replicated CDFFs made on the basis of selective media. Feeding regimes, particularly those involving complex nutrients, will dramatically affect population dynamics.

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 comparison of human dental plaque microcosm biofilms grown in an undefined medium and a chemically defined artificial saliva

The growth and pathogenic properties of dental plaque result from interactions between the microbiota and the oral environment and have been studied in laboratory experimental systems ranging from single or a few species (such as in chemostats) to dental plaque microcosms. Microcosm plaque is an in vitro version of natural plaque and has been explored as a microflora model because it is sited a more manipulable and controllable environment. It is obtained as microcosm biofilms in an ‘artificial mouth’ plaque culture system by culturing the bacteria in natural plaque-enriched saliva (i.e. salivary bacteria where a whole-saliva donor has abstained from oral hygiene for 24 h to increase the plaque bacteria in the saliva). The aim here was to examine whether a new, chemically defined analogue of saliva (defined medium mucin, DMM) could substitute for a previously used, chemically undefined medium (basal medium mucin, BMM) as an analogue of saliva for large-scale biofilm culturing. DMM contains various ions, mucin, amino acids, vitamins and growth factors at concentrations generally similar to those in saliva, whereas BMM contains yeast extract, peptones and mucin. To model the nutrient functions of salivary proteins, amino acids equivalent to 5 g/l casein were also included in DMM. In earlier studies, BMM-grown plaques were similar to natural plaques in structure, composition, growth rate and pH response to substrates. Their doubling-time patterns over a 20-day period were similar, except that the DMM-grown plaques showed biphasic growth patterns that were more pronounced than with BMM. Variation in enzyme profiles between BMM- and DMM-grown plaque, measured using the API-ZYM technique, provided evidence of nutritional effects on plaque composition. It was concluded that realistic growth rates and patterns are generated in microcosm plaque biofilms by supplying both DMM and BMM. However, the use of DMM enables specific modifications to be made to nutrient conditions during large-scale culture in our ‘artificial mouth’ biofilm system.