Associations of sex, oral hygiene and smoking with oral species in distinct habitats at age 32 years.

The oral microbiome is ecologically diverse, complex, dynamic, and little understood. We describe the microbiota of four oral habitats in a birth cohort at age 32 and examine differences by sex, oral hygiene, and current smoking status, dental caries, and periodontal health. Oral biofilm samples collected from anterior labial supragingival, posterior lingual supragingival, subgingival, and tongue sites of 841 Dunedin Multidisciplinary Health and Development Study members were analysed using checkerboard DNA-DNA hybridization; focusing on 30 ecologically important bacterial species. The four habitats exhibited distinct microbial profiles that differed by sex. Streptococcus gordonii was more dominant in supragingival and tongue biofilms of males; Porphyromonas gingivalis exhibited higher relative abundance in subgingival biofilm of females. Males had higher scores than females for periodontal pathogens at supragingival sites. The relative abundance of several putative caries and periodontal pathogens differed in smokers and non-smokers. With poor oral hygiene significantly higher proportions of Gram-negative facultative anaerobes were present in subgingival biofilm and there were higher scores for the principal components characterised by putative cariogenic and periodontal pathogens at each site. Distinctive microenvironments shape oral biofilms and systematic differences exist by sex, oral hygiene, and smoking status.

Use of denaturing gradient gel electrophoresis for the identification of mixed oral yeasts in human saliva.

A PCR-denaturing gradient gel electrophoresis (DGGE) method was established for the simultaneous presumptive identification of multiple yeast species commonly present in the oral cavity. Published primer sets targeting different regions of the Saccharomyces cerevisiae 26-28S rRNA gene (denoted primer sets N and U) and the 18S rRNA gene (primer set E) were evaluated with ten Candida and four non-Candida yeast species, and twenty Candida albicans isolates. Optimized PCR-DGGE conditions using primer set N were applied to presumptively identify, by band matching, yeasts in the saliva of 25 individuals. Identities were confirmed by DNA sequencing and compared with those using CHROMagar Candida culture. All primer sets yielded detectable DGGE bands for all species tested. Primer set N yielded mainly single bands and could distinguish all species examined, including differentiating Candida dubliniensis from C. albicans. Primer set U was less discriminatory among species but yielded multiple bands that distinguished subspecies groups within C. albicans. Primer set E gave poor yeast discrimination. DGGE analysis identified yeasts in 17 of the 25 saliva samples. Six saliva samples contained two yeast species: three contained C. albicans and three C. dubliniensis. C. dubliniensis was present alone in one saliva sample (total prevalence 16 %). CHROMagar culture detected yeasts in 16 of the yeast-containing saliva samples and did not enable identification of 7 yeast species identified by DGGE. In conclusion, DGGE identification of oral yeast species with primer set N is a relatively fast and reliable method for the simultaneous presumptive identification of mixed yeasts in oral saliva samples.

Plaques from different individuals yield different microbiota responses to oral-antiseptic treatment.

Dental caries is a polymicrobial disease and complicated to treat. Understanding the microbiota responses to treatment from different individuals is a key factor in developing effective treatments. The aim of this study was to investigate the 24-h posttreatment effect of two oral antiseptics (chlorhexidine and Listerine) on species composition of microplate plaque biofilms that had been initiated from the saliva of five different donors and grown in both 0.15% and 0.5% sucrose. Plaque composition was analyzed using checkerboard DNA : DNA hybridization analysis, which comprised of a panel of 40 species associated with oral health and disease. The supernatant pH of the plaques grown in 0.15% sucrose ranged from 4.3 to 6 and in 0.5% sucrose, it ranged from 3.8 to 4. Plaque biomass was largely unaffected by either antiseptic. Each donor had a different salivary microbial profile, differentiating according to the prevalence of either caries or periodontal/anaerobic pathogens. Despite similar plaque microbiota compositions being elicited through the sucrose growth conditions, microbiota responses to chlorhexidine and Listerine differentiated according to the donor. These findings indicate that efficacious caries treatments would depend on the responses of an individual's microbiota, which may differ from person to person.

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.

Checkerboard DNA-DNA hybridization technology using digoxigenin detection.

Checkerboard DNA-DNA hybridization (CKB) is a technique that provides a simultaneous quantitative analysis of 40 microbial species against up to 28 mixed microbiota samples on a single membrane; using digoxigenin (DIG)-labeled, whole-genome DNA probes. Developed initially to study the predominantly gram-negative dental plaque microorganisms involved in periodontitis, we modified the probe species composition to focus on putative pathogens involved in the development of dental caries. CKB analysis is applicable to species from other biodiverse ecosystems and to a large number of samples. The major limitations are that high-quality DNA is required for the preparation of DIG-labeled probes and standards, and that probe specificity requires careful evaluation. Overall, CKB analysis provides a powerful ecological fingerprint of highly biodiverse microbiota based on key cultivable bacteria.

Human dental plaque microcosm biofilms: effect of nutrient variation on calcium phosphate deposition and growth.

UNLABELLED: Plaque mineralisation is a multi-factorial process involving plaque pH, nucleation, inhibitors and promotors. It is poorly understood because of its complexity. OBJECTIVE: To establish the effects of amino acids and peptones in the simulated oral fluid BMM, a saliva analogue DMM and modifications of these on mineral deposition into dental plaque biofilm microcosms. METHODS: Microcosms were cultured for up to 35 days in an Artificial Mouth pulsed with sucrose, followed by 10 days periodic treatment with a pH 5.0 calcium-phosphate-monofluorophosphate-urea solution (CPMU). RESULTS: Initial biofilm doubling times were 3-7h, which then slowed and varied under the different nutrient conditions although their pH behaviour was similar. In BMM, mineral deposition was 20% that of DMM, but removal of BMM peptones increased deposition 12-fold. Substitution of the amino acids in DMM by casein did not affect deposition levels, but their removal leaving mucin the sole macronutrient, increased mineral deposition three-fold, reaching 40 mmol Ca/g protein. CONCLUSIONS: These substantial increases in mineral deposition when the macronutrient concentration is reduced indicates probable changes in the nucleating, inhibitory and Ca-binding properties of the simulated oral fluids themselves and/or changes in the plaque microbiota and their crystal nucleators and inhibitors.