A dual cohesin-dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome.

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin-dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.

Validation of a quantitative real-time PCR assay and comparison with fluorescence microscopy and selective agar plate counting for species-specific quantification of an in vitro subgingival biofilm model.

BACKGROUND AND OBJECTIVE: Subgingival biofilms are the prime etiological factor of periodontal disease. Owing to their complex polymicrobial nature, quantification of individual bacterial species within the biofilm for research and diagnostic purposes can be methodologically challenging. The aims of this study were to establish a quantitative real-time PCR (qPCR) assay to quantify the bacteria used in our 10-species in vitro 'subgingival' biofilm model and to compare the quantitative outcome with fluorescence microscopy and colony-forming unit (CFU) counts on selective agar plates. MATERIAL AND METHODS: The 10 species included in the in vitro biofilm were Streptococcus oralis, Streptococcus anginosus, Veillonella dispar, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, Actinomyces oris, Campylobacter rectus, Porphyromonas gingivalis and Prevotella intermedia. The numbers of each species were quantified at two time points using qPCR, microscopy counting following fluorescence in-situ hybridization (FISH) or immunofluorescence staining, and counting of CFUs after growth on selective agar plates. RESULTS: All 10 species were successfully quantified using qPCR and FISH or immunofluorescence, and the eight species culturable on selective agar plates were also quantified by counting the numbers of CFUs after growth on selective agar. In early biofilm cultures, all methods showed a significant correlation, although the absolute numbers differed between methods. In late biofilm cultures, measurements obtained using qPCR and FISH or immunofluorescence, but not by CFU counts, maintained significant correlation. CFU counts yielded lower values than did measurements made using the other two methods. CONCLUSION: Quantitative PCR and epifluorescence microscopy can be easily combined with each other to determine species-specific bacterial numbers within biofilms. However, conventional bacterial cultures cannot be as efficiently combined using these molecular detection methods. This may be crucial in designing and selecting appropriate clinical diagnostic methods for subgingival biofilm samples.

The relationship between turbidity of mouth-rinsed water and oral health status.

OBJECTIVES: The purpose of this study was to examine the relationship between turbidity of mouth rinsed water and oral health status such as dental and periodontal conditions, oral hygiene status, flow rate of saliva and oral bacteria. MATERIALS AND METHODS: Subjects were 165 patients who visited the Dental Hospital, Tokyo Medical and Dental University. Oral health status, including dental and periodontal conditions, oral hygiene status and flow rate of saliva, was clinically examined. The turbidity was measured with a turbidimeter. Quantification of Fusobacterium spp, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and total bacteria levels was performed using real-time PCR. The Pearson correlation and multiple regression analysis were used to explore the associations between the turbidity and oral health parameters. RESULTS: The turbidity showed significant correlations with the number of decayed teeth and deep pockets, the plaque index, extent of tongue coating and Fusobacterium spp, P. gingivalis, T. forsythia, T. denticola and total bacteria levels. In a multiple regression model, the turbidity was negatively associated with the flow rate of saliva and positively associated with the total number of bacteria (p < 0.001). CONCLUSION: Current findings suggested that turbidity of mouth rinsed water could be used as an indicator to evaluate oral health condition and the amount of bacteria in the oral cavity. In addition, the turbiditimeter appeared as a simple and objective device for screening abnormality of oral health condition at chair side as well as community-based research.

Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers.

OBJECTIVE: The purpose of this study was to investigate colonisation by periodontopathic bacteria and the sites of colonisation in elderly upper and lower complete denture wearers. We also investigated the relationship between level of oral hygiene and colonisation by periodontopathic bacteria. MATERIALS AND METHODS: Forty edentulous and 37 dentate volunteers participated in this study. Samples were collected from whole saliva, and levels of Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum were determined by PCR Invader technology. Detection of these species on oral mucosal and denture surfaces was performed by PCR. Fisher's exact test was used for the statistical analysis. Cluster analysis was employed to investigate trends in the periodontopathic bacteria flora in each sampling area. RESULTS: Detection rates of periodontopathic bacteria in whole saliva were lower under edentulous conditions than under dentulous conditions, except for A. actinomycetemcomitans and F. nucleatum (p < 0.01). Detection rate of F. nucleatum was the highest in all areas. A positive correlation was observed between DNA quantification of P. gingivalis and number of Candida species in saliva. Cluster analysis of the test species identified two clusters. Tongue-coating status was associated with the detection rate of all periodontopathic bacteria investigated, and denture plaque status was associated with the detection rate of T. denticola and F. nucleatum. CONCLUSION: Results indicate the presence of periodontopathic bacteria under edentulous conditions and that the status of oral hygiene of the mucosal or denture surfaces affects colonisation by T. denticola and F. nucleatum.

Nutritional influences on the gut microbiota and the consequences for gastrointestinal health.

The human colonic microbiota degrades dietary substrates that are indigestible in the upper GIT (gastrointestinal tract), releasing bacterial metabolites, some of which are important for gut health. Advances in molecular biology techniques have facilitated detailed analyses of the composition of the bacterial community resident in the lower GIT. Such analyses have indicated that more than 500 different bacterial species colonize an individual, and that, although there is much functional consistency in the resident bacterial groups, there is considerable inter-individual variation at the species/strain level. The bacterial community develops during early childhood until it reaches an adult-like composition. Whereas colonization and host factors influence the species composition, dietary factors also have an important impact, with specific bacterial groups changing in response to specific dietary interventions. Since bacterial species have different metabolic activities, specific diets have various consequences for health, dependent on the effect exerted on the bacterial population.

Microbial shifts during dental biofilm re-development in the absence of oral hygiene in periodontal health and disease.

AIM: To monitor microbial shifts during dental biofilm re-development. MATERIALS AND METHODS: Supra- and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects at baseline and immediately after tooth cleaning. Samples were taken again from seven teeth in randomly selected quadrants during 1, 2, 4 and 7 days of no oral hygiene. Samples were analysed using checkerboard DNA-DNA hybridization. Species counts were averaged within subjects at each time point. Significant differences in the counts between healthy and periodontitis subjects were determined using the Mann-Whitney test. RESULTS: The total supra- and subgingival counts were significantly higher in periodontitis on entry and reached or exceeded the baseline values after day 2. Supragingival counts of Veillonella parvula, Fusobacterium nucleatum ss vincentii and Neisseria mucosa increased from 2 to 7 days. Subgingival counts were greater for Actinomyces, green and orange complex species. Significant differences between groups in supragingival counts occurred for 17 of 41 species at entry, 0 at day 7; for subgingival plaque, these values were 39/41 taxa at entry, 17/41 at day 7. CONCLUSIONS: Supragingival plaque re-development was similar in periodontitis and health, but subgingival species recolonization was more marked in periodontitis.

Early bacterial colonization and soft tissue health around zirconia and titanium abutments: an in vivo study in man.

AIM: To compare the early bacterial colonization and soft tissue health of mucosa adjacent to zirconia (ZrO(2)) and titanium (Ti) abutment surfaces in vivo. MATERIALS AND METHODS: Twenty edentulous subjects received two endosseous mandibular implants. The implants were fitted with either a ZrO(2) or a Ti abutment (non-submerged implant placement, within-subject comparison, left-right randomization). Sulcular bacterial sampling and the assessment of probing pocket depth, recession and bleeding on probing were performed at 2 weeks and 3 months post-surgery. Wilcoxon matched-pairs, sign-rank tests were applied to test differences in the counts of seven marker bacteria and the clinical parameters that were associated with the ZrO(2) and Ti abutments, at the two observation time points. RESULTS: ZrO(2) and Ti abutments harboured similar counts of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Peptostreptococcus micros, Fusobacterium nucleatum and Treponema denticola at 2 weeks and 3 months. Healthy clinical conditions were seen around both ZrO(2) and Ti abutments at all times, without significant differences in most clinical parameters of peri-implant soft tissue health. Mean probing depths around Ti abutments were slightly deeper than around ZrO(2) abutments after 3 months (2.2 SD 0.8 mm vs. 1.7 SD 0.7 mm, P=0.03). CONCLUSIONS: No difference in health of the soft tissues adjacent to ZrO(2) and Ti abutment surfaces or in early bacterial colonization could be demonstrated, although somewhat shallower probing depths were observed around ZrO(2) abutments after 3 month.

Effects of new dietary fiber from Japanese Apricot (Prunus mume Sieb. et Zucc.) on gut function and intestinal microflora in adult mice.

Much attention has been focused recently on functional foods. Ume, the Japanese name for the apricot of Prunus mume Sieb. et Zucc., is an example of a Japanese traditional functional food. There are, however, few reports on the effects of fiber from this fruit on bowel function. With this objective, we prepared ume fiber to test the hypothesis that it can change gut function and intestinal flora in mice. Mice were fed an ume fiber (UF) or cellulose (CF) diet (control) for 40 days. The fecal weight, fecal lipids, plasma lipids and cecal composition of the microflora were analyzed. The amount of feces was significantly greater in the UF group than in the CF group (p < 0.01). The fecal lipids content (% DW) of the feces sampled on the final day of the experiment were significantly greater in the UF group than in the CF group (p < 0.01). Plasma non-esterified fatty acids (NEFA) concentrations tended to be lower in the UF compared to the CF group (p = 0.058). Occupation ratios of Bacteroides and Clostridium cluster IV were significantly greater in the cecal flora of the UF group. Our results suggest that ume fiber possesses the fecal lipid excretion effects and feces bulking effects.

The recolonization hypothesis in a full-mouth or multiple-session treatment protocol: a blinded, randomized clinical trial.

AIM: To test recolonization of periodontal lesions after full-mouth scaling and root planing (FM-SRP) or multiple session-SRP (MS-SRP) in a randomized clinical trial and whether FM-SRP and MS-SRP result in different clinical outcomes. MATERIALS AND METHODS: Thirty-nine subjects were randomly assigned to FM-SRP or MS-SRP groups. At baseline and after 3 months, probing pocket depth (PPD), plaque index (PlI) and bleeding on probing (BoP) were recorded. At baseline, immediately after treatment, after 1, 2, 7, 14 and 90 days, paper point samples from a single site from the maxillary right quadrant were collected for microbiological analysis of five putative pathogens by polymerase chain reaction. RESULTS: FM-SRP and MS-SRP resulted in significant reductions in PPD, BoP and PlI and the overall detection frequencies of the five species after 3 months without significant differences between treatments. Compared with MS-SRP, FM-SRP resulted in less recolonization of the five species, significantly for Treponema denticola, in the tested sites. CONCLUSION: FM-SRP and MS-SRP result in overall clinically and microbiologically comparable outcomes where recolonization of periodontal lesions may be better prevented by FM-SRP.

Mechanical non-surgical treatment of peri-implantitis: a single-blinded randomized longitudinal clinical study. II. Microbiological results.

BACKGROUND: Peri-implantitis is common in patients with dental implants. We performed a single-blinded longitudinal randomized study to assess the effects of mechanical debridement on the peri-implant microbiota in peri-implantitis lesions. MATERIALS AND METHODS: An expanded checkerboard DNA-DNA hybridization assay encompassing 79 different microorganisms was used to study bacterial counts before and during 6 months following mechanical treatment of peri-implantitis in 17 cases treated with curettes and 14 cases treated with an ultrasonic device. Statistics included non-parametric tests and GLM multivariate analysis with p<0001 indicating significance and 80% power. RESULTS: At selected implant test sites, the most prevalent bacteria were: Fusobacterium nucleatum sp., Staphylococci sp., Aggregatibacter actinomycetemcomitans, Helicobacter pylori, and Tannerella forsythia. 30 min. after treatment with curettes, A. actinomycetemcomitans (serotype a), Lactobacillus acidophilus, Streptococcus anginosus, and Veillonella parvula were found at lower counts (p<0.001). No such differences were found for implants treated with the ultrasonic device. Inconsistent changes occurred following the first week. No microbiological differences between baseline and 6-month samples were found for any species or between treatment study methods in peri-implantitis. CONCLUSIONS: Both methods failed to eliminate or reduce bacterial counts in peri-implantitis. No group differences were found in the ability to reduce the microbiota in peri-implantitis.

Relation of body mass index, periodontitis and Tannerella forsythia.

OBJECTIVE: To determine if there were differences in periodontal status and the composition of the subgingival microbiota in individuals who exhibited different body mass indices (BMI). MATERIAL AND METHODS: One hundred and twenty-one periodontally healthy/gingivitis and 574 chronic periodontitis subjects had height and weight determined and were measured for probing pocket depth, clinical attachment level, bleeding on probing, gingival redness and presence of visible plaque. Subgingival plaque samples taken from each tooth were individually analysed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. RESULTS: Crude odds ratios (ORs) [95% confidence interval (CI)] of overweight and obese individuals exhibiting periodontitis were 3.1 (1.9-4.8) and 5.3 (2.8-9.5), respectively, when compared with subjects with normal BMI. Logistic regression analysis indicated an OR (95% CI) of 2.3 (1.2-4.5) for an obese subject to exhibit periodontitis after adjusting for age, gender and smoking status. Individuals <46.8 years (median age) were responsible for this association. Only Tannerella forsythia differed significantly in proportions among BMI groups and was significantly higher in obese periodontally healthy/gingivitis individuals. CONCLUSION: The data suggest that an overgrowth of T. forsythia occurs in the subgingival biofilms of periodontally healthy, overweight and obese individuals that might put them at risk for initiation and progression of periodontitis.

Analysis of neutrophil-derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the innate immune response against periodontogenic bacteria.

INTRODUCTION: During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis. METHODS: GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real-time polymerase chain reactions. The amounts of myeloperoxidase and alpha-defensins (HNP1-3) were determined by enzyme-linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL-37) was assayed by Western blot. RESULTS: Myeloperoxidase concentration was not correlated with levels of LL-37 and HNP1-3 in samples from patients, compared to controls. The amount of HNP1-3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL-37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. CONCLUSION: The lack of a correlation between LL-37, HNP1-3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL-37, because the 11-kDa cathelicidin-derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL-37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis.

Site-specific development of periodontal disease is associated with increased levels of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in subgingival plaque.

BACKGROUND: Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia (previously T. forsythensis), which are regarded as the principal periodontopathogenic bacteria, exist as a consortium in subgingival biofilms. We aimed to examine quantitative relationships between P. gingivalis, T. denticola, and T. forsythia in subgingival biofilms and the relationship between the quantity and prevalence of these three bacteria and site-specific periodontal health. METHODS: This study was cross-sectional. The study population consisted of 35 adult subjects who visited the Kyushu Dental College Hospital. Plaque samples were collected from 105 periodontal pocket sites. Quantitative analyses of each of the three periodontopathogenic bacteria were performed using real-time polymerase chain reaction with species-specific primers and hybridization probes. RESULTS: The plaque samples were divided into four groups based on the presence or absence of a periodontal pocket (probing depth [PD] > or =4 mm) and bleeding on probing (BOP), regardless of attachment loss. The proportions of all three target bacteria detected in samples from sites of periodontal disease (with PD and BOP) were markedly higher than those in the other sample groups. Cell numbers of P. gingivalis, T. denticola, and T. forsythia in the subgingival plaque of each sampling site were significantly mutually correlated and were increased in the plaque of sites of periodontal disease with PD > or =4 mm and BOP. CONCLUSION: The symbiotic effects of P. gingivalis, T. denticola, and T. forsythia, which coaggregate and exist concomitantly in subgingival biofilms, may be associated with the local development of periodontitis.

Interleukin-6 polymorphisms are associated with pathogenic bacteria in subjects with periodontitis.

BACKGROUND: Growing evidence suggests that individual genetic susceptibility may influence the host's response to infections. Previously, we showed that a common variation in the interleukin (IL)-6 gene was associated with increased odds of detection of common periodontal pathogens from individuals with aggressive periodontitis. The aim of this study was to investigate the association between IL-6 polymorphisms and periodontopathogenic bacteria in a larger, ethnically mixed population of subjects with periodontitis. METHODS: Genomic DNA was extracted from 107 subjects diagnosed with severe forms of periodontitis to study a cluster of polymorphisms in inflammatory genes, including IL-6. The presence of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and Tannerella forsythia (previously T. forsythensis) in their subgingival biofilm was determined by polymerase chain reaction analysis. Serum IL-6 and C-reactive protein (CRP) concentrations were analyzed by enzyme-linked immunosorbent assay. RESULTS: Multiple logistic regression analysis revealed that the IL-6 -6106 polymorphism was associated with the detection of A. actinomycetemcomitans (P = 0.009; odds ratio [OR] = 3.5; 95% confidence interval [CI]: 1.38 to 9.16) and the concomitant detection of A. actinomycetemcomitans and P. gingivalis (P = 0.015; OR = 3.6; 95% CI: 1.28 to 10.04). The IL-6 -174 polymorphism was associated with increased odds of the concomitant detection of A. actinomycetemcomitans and P. gingivalis (P = 0.042; OR = 2.8; 95% CI: 1.04 to 7.75). Haplotype analysis of all five IL-6 polymorphisms confirmed an association with the detection of A. actinomycetemcomitans (P = 0.046). The IL-6 -6106 polymorphism was also associated with CRP serum levels at multivariate analysis (P = 0.024). CONCLUSIONS: These findings confirm the hypothesis that complex interactions between the microbiota and host genome are at the basis of susceptibility to periodontitis. Periodontal disease may represent a useful model to study the pathways and mechanisms of host-pathogen interactions in inflammatory diseases.

Persistence of extracrevicular bacterial reservoirs after treatment of aggressive periodontitis.

BACKGROUND: The purpose of this study was to test the hypothesis that periodontal pathogens associated with aggressive periodontitis persist in extracrevicular locations following scaling and root planing, systemic antibiotics, and antimicrobial rinses. METHODS: Eighteen patients with aggressive periodontitis received a clinical examination during which samples of subgingival plaque and buccal epithelial cells were obtained. Treatment consisted of full-mouth root planing, systemic antibiotics, and chlorhexidine rinses. Clinical measurements and sampling were repeated at 3 and 6 months. Quantitative polymerase chain reaction determined the number of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), and Treponema denticola in the plaque. Fluorescence in situ hybridization and confocal microscopy determined the extent of intracellular invasion in epithelial cells. RESULTS: Clinical measurements improved significantly following treatment. All bacterial species except P. gingivalis were significantly reduced in plaque between baseline and 3 months. However, all species showed a trend to repopulate between 3 and 6 months. This increase was statistically significant for log T. denticola counts. All species were detected intracellularly. The percentage of cells infected intracellularly was not affected by therapy. CONCLUSIONS: The 6-month increasing trend in the levels of plaque bacteria suggests that subgingival recolonization was occurring. Because the presence of these species within epithelial cells was not altered after treatment, it is plausible that recolonization may occur from the oral mucosa. Systemic antibiotics and topical chlorhexidine did not reduce the percentage of invaded epithelial cells. These data support the hypothesis that extracrevicular reservoirs of bacteria exist, which might contribute to recurrent or refractory disease in some patients.

Adding mucins to an in vitro batch fermentation model of the large intestine induces changes in microbial population isolated from porcine feces depending on the substrate.

Adding mucus to in vitro fermentation models of the large intestine shows that some genera, namely lactobacilli, are dependent on host-microbiota interactions and that they rely on mucosal layers to increase their activity. This study investigated whether this dependence on mucus is substrate dependent and to what extent other genera are impacted by the presence of mucus. Inulin and cellulose were fermented in vitro by a fecal inoculum from pig in the presence or not of mucin beads in order to compare fermentation patterns and bacterial communities. Mucins increased final gas production with inulin and shifted short-chain fatty acid molar ratios (P < 0.001). Quantitative real-time PCR analyses revealed that Lactobacillus spp. and Bifidobacterium spp. decreased with mucins, but Bacteroides spp. increased when inulin was fermented. A more in-depth community analysis indicated that the mucins increased Proteobacteria (0.55 vs 0.25%, P = 0.013), Verrucomicrobia (5.25 vs 0.03%, P = 0.032), Ruminococcaceae, Bacteroidaceae and Akkermansia spp. Proteobacteria (5.67 vs 0.55%, P < 0.001) and Lachnospiraceae (33 vs 10.4%) were promoted in the mucus compared with the broth, while Ruminococcaceae decreased. The introduction of mucins affected many microbial genera and fermentation patterns, but from PCA results, the impact of mucus was independent of the fermentation substrate.

Interaction between H2-producing and non-H2-producing cellulolytic bacteria from the human colon.

The cellulose-degrading species recently isolated from the human colon showed diverse ability to degrade and ferment cellulose. In the present study, the nature of the inter-relation existing between one H(2)-producing cellulolytic isolate (Ruminococcus sp. nov.) and one non-H(2)-producing cellulose-degrading species (Bacteroides sp. nov.) was investigated in vitro. Coculture experiments revealed synergism in cellulose degradation between these two cellulolytic species. An increase in total bacterial population was measured in the coculture, Bacteroides sp. being the predominant organism. As a result, a large decrease in H(2) production from cellulose fermentation was observed. Predominance of Bacteroides sp. might thus contribute to limit gas produced from fibre fermentation in the gut.

Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis are components of a polymicrobial intracellular flora within human buccal cells.

Previously, we used in situ hybridization and confocal microscopy to detect the periodontal pathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis within buccal epithelial cells taken directly from the mouth. This study tested the hypothesis that the intracellular flora of buccal cells is polymicrobial. Mixtures containing a red fluorescent universal probe paired with green fluorescent versions of either A. actinomycetemcomitans-, P. gingivalis-, or T. forsythensis-specific probes were hybridized with buccal cells collected from each of 38 healthy humans. We verified co-localization of probe pairs within cells by generating three-dimensional reconstructions. Intracellular bacteria were detected in every subject. Each cell that was labeled with a species-specific probe also contained bacteria recognized only by the universal probe. Bacteria labeled with specific probes often occupied smaller regions within larger masses of bacteria. Those findings suggest that future studies of invasion by oral bacteria may need to include microbial consortia.

Bread crust melanoidins as potential prebiotic ingredients.

Melanoidins are the final products of the Maillard reaction. They are a heterogeneous mixture of compounds characterized by brown color and high molecular weight. The physiological properties of melanoidins have been widely investigated and there is a general consensus on their poor digestibility and bioavailability. In vitro studies on food melanoidins are in many cases limited by their poor water solubility. This problem was recently overcome for bread melanoidins using an enzymatic digestion procedure. Bread melanoidins are constituted by low-molecular-weight, colored compounds linked to the gluten polymer. In this work, melanoidins from different bread types were investigated for their potential prebiotic activity by a static batch culture. Results showed that anaerobic bacteria, particularly Bifidobacteria strains, are able to use bread melanoidins as carbon source. The bacterial growth is different for the various types of melanoidins samples indicating that starting materials and processing conditions have a strong influence on the prebiotic potential of bread melanoidins. In all cases the bacterial growth obtained using bread melanoidins is lower than that previously observed using melanoidins from other sources, such as coffee silverskin.

The effect of crude extracts of nine African chewing sticks on oral anaerobes.

Chewing sticks are widely used in Nigeria for dental and oral hygiene. In-vitro susceptibility tests were done with crude extracts from nine popular sticks on four species of Bacteroides. Serindeia warneckei chewing stick had the greatest and most consistent inhibitory effect on the four species; extracts from bark and pulp were bactericidal at concentrations of less than or equal to 1%. Extracts of other sticks, when inhibitory, were only so at higher concentrations--in the range 2-30%. All the black-pigmented oral anaerobes were very susceptible to eight of the nine chewing-stick extracts but non-pigmented anaerobes showed variable susceptibilities.