SMDI: An Index for Measuring Subgingival Microbial Dysbiosis.

An intuitive, clinically relevant index of microbial dysbiosis as a summary statistic of subgingival microbiome profiles is needed. Here, we describe a subgingival microbial dysbiosis index (SMDI) based on machine learning analysis of published periodontitis/health 16S microbiome data. The raw sequencing data, split into training and test sets, were quality filtered, taxonomically assigned to the species level, and centered log-ratio transformed. The training data set was subject to random forest analysis to identify discriminating species (DS) between periodontitis and health. DS lists, compiled by various "Gini" importance score cutoffs, were used to compute the SMDI for samples in the training and test data sets as the mean centered log-ratio abundance of periodontitis-associated species subtracted by that of health-associated ones. Diagnostic accuracy was assessed with receiver operating characteristic analysis. An SMDI based on 49 DS provided the highest accuracy with areas under the curve of 0.96 and 0.92 in the training and test data sets, respectively, and ranged from -6 (most normobiotic) to 5 (most dysbiotic) with a value around zero discriminating most of the periodontitis and healthy samples. The top periodontitis-associated DS were Treponema denticola, Mogibacterium timidum, Fretibacterium spp., and Tannerella forsythia, while Actinomyces naeslundii and Streptococcus sanguinis were the top health-associated DS. The index was highly reproducible by hypervariable region. Applying the index to additional test data sets in which nitrate had been used to modulate the microbiome demonstrated that nitrate has dysbiosis-lowering properties in vitro and in vivo. Finally, 3 genera (Treponema, Fretibacterium, and Actinomyces) were identified that could be used for calculation of a simplified SMDI with comparable accuracy. In conclusion, we have developed a nonbiased, reproducible, and easy-to-interpret index that can be used to identify patients/sites at risk of periodontitis, to assess the microbial response to treatment, and, importantly, as a quantitative tool in microbiome modulation studies.

Modeling Normal and Dysbiotic Subgingival Microbiomes: Effect of Nutrients.

Screening for microbiome modulators requires availability of a high-throughput in vitro model that replicates subgingival dysbiosis and normobiosis, with a tool to measure microbial dysbiosis. Here, we tested various formulations to grow health- and periodontitis-associated subgingival microbiomes in parallel, and we describe a new subgingival dysbiosis index. Subgingival plaque samples pooled from 5 healthy subjects and, separately, 5 subjects with periodontitis were used to inoculate a Calgary Biofilm Device containing saliva-conditioned, hydroxyapatite-coated pegs. Microbiomes were grown for 7 d on either nutrient-rich media-including a modification of SHI medium, brain-heart infusion (BHI) supplemented with hemin and vitamin K, and a blend of SHI and BHI, each at 3 sucrose concentrations (0%, 0.05% and 0.1%)-or nutrient-limited media (saliva with 5%, 10%, or 20% inactivated human serum). The microbiomes were assessed for biomass, viability, and 16S rRNA profiles. In addition to richness and diversity, a dysbiosis index was calculated as the ratio of the sum of relative abundances of disease-associated species to that of health-associated species. The supplemented BHI and blend of SHI and BHI resulted in the highest biomass, whereas saliva-serum maximized viability. Distinct groups of bacteria were enriched in the different media. Regardless of medium type, the periodontitis-derived microbiomes showed higher species richness and alpha diversity and clustered with their inoculum separate from the health-derived microbiomes. Microbiomes grown in saliva-serum showed the highest species richness and the highest similarity to the clinical inocula in both health and disease. However, inclusion of serum reduced alpha diversity and increased dysbiosis in healthy microbiomes in a dose-dependent manner, mainly due to overenrichment of Porphyromonas species. The modification of SHI stood second in terms of species richness and diversity but resulted in low biomass and viability and significantly worsened dysbiosis in the periodontitis-derived microbiomes. Overall, saliva with 5% human serum was optimal for replicating subgingival microbiomes from health and disease.

Independent determinants of periodontitis in Yemeni adults: A case-control study.

OBJECTIVES: The relative importance of risk factors of periodontitis varies from one population to another. In this study, we sought to identify independent risk factors of periodontitis in a Yemeni population. METHODS: One hundred and fifty periodontitis cases and 150 healthy controls, all Yemeni adults 30-60 years old, were recruited. Sociodemographic data and history of oral hygiene practices and oral habits were obtained. Plaque index (PI) was measured on index teeth. Periodontal health status was assessed using Community Periodontal Index (CPI) and Clinical Attachment Loss (CAL) according to WHO. Periodontitis was defined as having one or more sextants with a CPI score ≥ 3. Multiple logistic regression modelling was employed to identify distal, intermediate and proximal determinants of periodontitis, while ordinal regression was used to identify those of CAL scores. RESULTS: In logistic regression, PI score was associated with the highest odds of periodontitis (OR = 82.9) followed by cigarette smoking (OR = 12.8), water pipe smoking (OR = 10.2), male gender (OR = 3.4) and age (OR = 1.19); on the other hand, regular visits to the dentist (OR = 0.05), higher level of education (OR = 0.37) and daily dental flossing (OR = 0.95) were associated with lower odds. Somewhat similar associations were seen for CAL scores (ordinal regression); however, qat chewing was identified as an additional determinant (OR = 4.69). CONCLUSION: Water pipe smoking is identified as a risk factor of periodontitis in this cohort in addition to globally known risk factors. Adjusted effect of qat chewing is limited to CAL scores, suggestive of association with recession.

Inflammatory Bacteriome and Oral Squamous Cell Carcinoma.

Results from microbiome studies on oral cancer have been inconsistent, probably because they focused on compositional analysis, which does not account for functional redundancy among oral bacteria. Based on functional prediction, a recent study revealed enrichment of inflammatory bacterial attributes in oral squamous cell carcinoma (OSCC). Given the high relevance of this finding to carcinogenesis, we aimed here to corroborate them in a case-control study involving 25 OSCC cases and 27 fibroepithelial polyp (FEP) controls from Sri Lanka. DNA extracted from fresh biopsies was sequenced for the V1 to V3 region with Illumina's 2 × 300-bp chemistry. High-quality nonchimeric merged reads were classified to the species level with a prioritized BLASTN-based algorithm. Downstream compositional analysis was performed with QIIME (Quantitative Insights into Microbial Ecology) and linear discriminant analysis effect size, while PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was utilized for bacteriome functional prediction. The OSCC tissues tended to have lower species richness and diversity. Genera Capnocytophaga, Pseudomonas, and Atopobium were overrepresented in OSCC, while Lautropia, Staphylococcus, and Propionibacterium were the most abundant in FEP. At the species level, Campylobacter concisus, Prevotella salivae, Prevotella loeschii, and Fusobacterium oral taxon 204 were enriched in OSCC, while Streptococcus mitis, Streptococcus oral taxon 070, Lautropia mirabilis, and Rothia dentocariosa among others were more abundant in FEP. Functionally, proinflammatory bacterial attributes, including lipopolysaccharide biosynthesis and peptidases, were enriched in the OSCC tissues. Thus, while the results in terms of species composition significantly differed from the original study, they were consistent at the functional level, substantiating evidence for the inflammatory nature of the bacteriome associated with OSCC.

Quantitative analysis of classical and new putative periodontal pathogens in subgingival biofilm: a case-control study.

BACKGROUND AND OBJECTIVES: A number of species/phylotypes have been newly implicated as putative periopathogens. The objective of this study was to explore associations among classical and new pathogens in subgingival biofilm and to assess their relative importance to chronic periodontitis. MATERIAL AND METHODS: Pooled subgingival biofilm samples were obtained from 40 patients with chronic periodontitis and 40 healthy controls. Taqman q-PCR assays were used to determine the absolute and relative counts of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Parvimonas micra, Filifactor alocis, oral Synergistetes and oral TM7s. Microbial associations were assessed using cluster analysis. Different statistical models were used to explore associations between microbial parameters and periodontitis. RESULTS: The median log and relative counts were lowest for TM7s (4.4 and 0.0016%, respectively) and highest for oral Synergistetes (7.2 and 1.4%, respectively). Oral Synergistetes clustered strongly with the red complex, particularly T. forsythia (100% rescaled similarity). All species/phylotypes except TM7s were significantly associated with periodontitis (Mann-Whitney test; p ≤ 0.005). However, P. gingivalis and F. alocis lost association after adjusting for confounders (ordinal regression). In receiving operator characteristic curve analysis, the log counts of oral Synergistetes were the best markers of periodontitis (82.5% sensitivity and specificity), followed by those of T. forsythia, P. micra and T. denticola. In prediction analysis, however, P. micra was the only microbial predictor of periodontal parameters. CONCLUSIONS: Oral Synergistetes are presented here as new members of the red complex, with relative importance to periodontitis exceeding that of the classical members. P. micra is shown as an important periodontal pathogen warranting more attention.

Oral health status and treatment needs among school children in Sana'a City, Yemen.

UNLABELLED: Data on the oral health status and treatment needs among Yemeni children are lacking. OBJECTIVES: To assess caries prevalence, treatment needs and gingival health status among school children in Sana'a City and to examine how these are affected by age, gender and khat chewing. METHODS: 1489 children (6- to 14-year old) were randomly selected from 27 schools representing all nine districts of Sana'a City. Dental caries and treatment needs were evaluated using standard WHO oral survey methods. The plaque index (PI), calculus index (CI) and the gingival index (GI), recorded at the six Ramfjord's teeth, were used to assess gingival health status. RESULTS: 4.1% of the study subjects were caries-free. Prevalence of these was significantly higher among the males. Overall, mean dmfs, dmft, DMFS and DMFT scores were 8.45, 4.16, 3.59 and 2.25 respectively. The decayed component accounted for >85% of the scores. The highest dmfs/dmft means were found among the 6-8 years age group, while the highest DMFS/DMFT means were scored by the 12-14 years age group. The need for restorative treatment and extractions was high; the former was significantly higher among the females. All subjects had gingivitis; the mean PI, CI and GI were 1.25, 0.3 and 1.36 respectively. Khat chewing did not affect caries experience; however, it was significantly associated with higher PI, CI and GI scores. CONCLUSIONS: The prevalence of caries, gingivitis and treatment needs among children in Sana'a city is high. More surveys in other Yemeni cities to generate comprehensive data are required.

Prevalence of subgingival bacteria resistant to aminopenicillins and metronidazole in dental patients from Yemen and Norway.

The purpose of this study was to assess the prevalence of resistance to aminopenicillins and metronidazole among selected subgingival species in dental patients from Yemen and Norway. Three subgingival samples were collected by paper points from each of 34 Yemeni and 21 Norwegian adult volunteers and then pooled. Each of the 55 pooled samples was plated on fastidious anaerobic blood agar containing 2 microg/mL ampicillin or metronidazole, or no antimicrobial. Species identification of growth was done using DNA-DNA checkerboard hybridisation. The overall proportion of ampicillin resistance among the 18 identified species was 28.9% and 7.9% in the Yemeni and Norwegian samples, respectively, whereas for metronidazole it was 60.3% and 11.3%. The number of species resistant to ampicillin and metronidazole was significantly higher (P < 0.016 and P = 0.0000, respectively) in the Yemeni than in the Norwegian samples.

Effect of khat chewing on 14 selected periodontal bacteria in sub- and supragingival plaque of a young male population.

BACKGROUND/AIMS: The habit of chewing khat (Catha edulis) for its amphetamine-like effects is highly prevalent in Yemen and east Africa, and has expanded to Western countries. The purpose of this study was to estimate and compare the prevalence and levels of 14 periodontal bacteria in gingival plaque of khat chewers and khat nonchewers, as well as of khat chewing sides and khat nonchewing sides. METHODS: A total of 408 sub- and supragingival plaque samples were collected from 51 young males (29 khat chewers and 22 khat nonchewers; age range 19-28 years) and analyzed using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical parameters were recorded for all teeth at six sites per tooth. RESULTS: Streptococcus intermedius and Veillonella parvula were significantly more prevalent in the subgingival plaque of chewers, which also showed significantly higher levels of V. parvula and Eikenella corrodens. Similar results were found for the subgingival plaque of the chewing sides compared to the nonchewing sides. However, there was a significantly higher prevalence and higher levels of Tannerella forsythia in the subgingival plaque of the nonchewing sides. No significant differences were observed for the supragingival plaque between the two study groups. There was a significantly lower prevalence of Capnocytophaga gingivalis and Fusobacterium nucleatum in the khat chewing sides, and higher levels of V. parvula and Actinomyces israelii. CONCLUSION: The data suggest that khat chewing induces a microbial profile that is not incompatible with gingival health.