Alcohol Consumption and the Diversity of the Oral Microbiome in Postmenopausal Women.

BACKGROUND: Alcohol reduces neutrophil function and decreases salivary flow, which could affect the composition of the oral microbiome. OBJECTIVE: We hypothesized that the α- and ß-diversity of the oral microbiome and the relative abundance of bacterial taxa would differ by frequency and type of alcohol consumption. METHODS: We used a food frequency questionnaire to assess the frequency of consumption of beer, wine, and liquor (drinks/week) in a sample of 1179 postmenopausal women in the Osteoporosis and Periodontal Disease Study. Women were categorized as nondrinkers, drinking <1 drink/wk, ≥1 to <7 drinks/wk, or ≥7 drinks/wk for total alcohol consumption and for beer, wine, and liquor consumption. The composition and diversity of the oral microbiome was assessed from subgingival plaque samples using 16S ribosomal RNA amplicon sequencing. Permutational multivariate analysis of variance (PERMANOVA) was used to examine ß-diversity (between-sample diversity) in the microbiome between alcohol consumption categories. Analysis of covariance was used to examine the mean α-diversity (within-sample diversity), assessed by the Shannon index (species evenness), Chao1 index (species richness), and observed operational taxonomic unit (OTU) count and the mean relative abundance of 245 bacterial taxa across alcohol consumption categories. RESULTS: Over half of the participants (67%) consumed alcohol, with 14% reporting ≥1 drink/d. The ß-diversity across categories of total alcohol consumption, but not categories of alcohol type, was statistically significantly different (P for PERMANOVA = 0.016). Mean α-diversity measures were statistically significantly higher (P < 0.05) in the highest category of total alcohol and wine consumption compared to nondrinkers; no significant associations were found for beer or liquor consumption. The relative abundance of 1 OTU, Selenomonassp._oral_taxon_133, was significantly lower in the highest level of total alcohol consumption compared to nondrinkers after adjustment for multiple comparisons. CONCLUSIONS: Alcohol consumption was associated with the diversity and composition of the subgingival microbiome.

Relationship between the subgingival microbiome and menopausal hormone therapy use: The Buffalo OsteoPerio study.

BACKGROUND: This study investigated the association between menopausal hormone therapy (HT) use and the subgingival microbiome, for which published information is limited. METHODS: This cross-sectional study included 1270 postmenopausal women, aged 53-81 years, who completed clinical examinations. Detailed information on HT use (type, delivery mode, duration) was obtained from questionnaires. HT use was categorized into three groups (never, former, current). 16S rRNA sequencing was performed on subgingival plaque samples obtained during dental examinations. Operational taxonomic units were centered log2-ratio (CLR) transformed to account for the compositional data structure. Analysis of variance was used to compare mean microbial relative abundances across HT categories with Benjamini-Hochberg correction. RESULTS: Significantly higher alpha diversity (Shannon Index) and beta diversity (Aitchison distance) was observed in never compared with current HT users (p < 0.05, each). Of the total 245 microbial taxa identified, 18 taxa differed significantly among the three HT groups, 11 of which were higher in current users and seven of which were lower in current users as compared with never users (p < 0.05, each). Differences in relative abundance between never and current HT users were materially unchanged after adjustment for age, body mass index, and oral hygiene. CONCLUSIONS: Relative abundance of several subgingival bacteria differed significantly between never and current HT users in a cohort of postmenopausal women. Additional studies are needed to determine the extent that these relationships might account for the previously reported inverse association between HT use and periodontal disease in older women.

Dietary carbohydrate intake is associated with the subgingival plaque oral microbiome abundance and diversity in a cohort of postmenopausal women.

Limited research exists on carbohydrate intake and oral microbiome diversity and composition assessed with next-generation sequencing. We aimed to better understand the association between habitual carbohydrate intake and the oral microbiome, as the oral microbiome has been associated with caries, periodontal disease, and systemic diseases. We investigated if total carbohydrates, starch, monosaccharides, disaccharides, fiber, or glycemic load (GL) were associated with the diversity and composition of oral bacteria in subgingival plaque samples of 1204 post-menopausal women. Carbohydrate intake and GL were assessed from a food frequency questionnaire, and adjusted for energy intake. The V3-V4 region of the 16S rRNA gene from subgingival plaque samples were sequenced to identify the relative abundance of microbiome compositional data expressed as operational taxonomic units (OTUs). The abundance of OTUs were centered log(2)-ratio transformed to account for the compositional data structure. Associations between carbohydrate/GL intake and microbiome alpha-diversity measures were examined using linear regression. PERMANOVA analyses were conducted to examine microbiome beta-diversity measures across quartiles of carbohydrate/GL intake. Associations between intake of carbohydrates and GL and the abundance of the 245 identified OTUs were examined by using linear regression. Total carbohydrates, GL, starch, lactose, and sucrose intake were inversely associated with alpha-diversity measures. Beta-diversity across quartiles of total carbohydrates, fiber, GL, sucrose, and galactose, were all statistically significant (p for PERMANOVA p < 0.05). Positive associations were observed between total carbohydrates, GL, sucrose and Streptococcus mutans; GL and both Sphingomonas HOT 006 and Scardovia wiggsiae; and sucrose and Streptococcus lactarius. A negative association was observed between lactose and Aggregatibacter segnis, and between sucrose and both TM7_[G-1] HOT 346 and Leptotrichia HOT 223. Intake of total carbohydrate, GL, and sucrose were inversely associated with subgingival bacteria alpha-diversity, the microbial beta-diversity varied by their intake, and they were associated with the relative abundance of specific OTUs. Higher intake of sucrose, or high GL foods, may influence poor oral health outcomes (and perhaps systemic health outcomes) in older women via their influence on the oral microbiome.

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei.

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole-cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

Unconjugated p-cresol activates macrophage macropinocytosis leading to increased LDL uptake.

Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated that a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first-pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, not only did we measure the effect of altering the intestinal microbiota on lipid accumulation in coronary arteries, but we also examined macrophage lipid uptake and handling pathways in response to uPC. We found that atherosclerosis-prone mice fed a high-fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, and hepatic and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated that uPC exposure increased apoptosis whereas PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate LDL uptake and only uPC induced endocytosis- and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate that uPC itself had a distinct effect on macrophage biology that might have contributed to increased cardiovascular risk in patients with CKD.

Subgingival microbiome is associated with alveolar bone loss measured 5 years later in postmenopausal women.

BACKGROUND: The aim of this study was to quantify the association between subgingival microbiota and periodontal disease progression in older women, for which limited published data exist. METHODS: A total of 1016 postmenopausal women, aged 53 to 81 years, completed baseline (1997 to 2001) and 5-year (2002 to 2006) dental exams that included probing depth, clinical attachment level, gingival bleeding, and radiographic alveolar crestal height (ACH). Baseline microbiota were measured in subgingival plaque using 16S rRNA sequencing. Associations between 52 microbiota we previously found statistically significantly associated with clinical periodontal disease at baseline, were examined with disease progression. The traditional Socransky microbiota complexes also were evaluated. Side-by-side radiograph comparisons were used to define progression as ≥2 teeth with ≥1 mm ACH loss or ≥1 new tooth loss to periodontitis. The association between baseline centered log(2) ratio transformed microbial relative abundances and 5-year periodontal disease progression was measured with generalized linear models. RESULTS: Of 36 microbiota we previously showed were elevated in moderate/severe disease at baseline, 24 had statistically significantly higher baseline mean relative abundance in progressing compared with non-progressing women (P < .05, all); which included all Socransky red bacteria (P. gingivalis, T. forsythia, T. denticola). Of 16 microbiota elevated in none/mild disease at baseline, five had statistically significantly lower baseline abundance in non-progressing compared with progressing women (P < 0.05, all), including one Socransky yellow bacteria (S. oralis). When adjusted for baseline age, socioeconomic status, and self-rated general health status, odds ratios for 5-year progression ranged from 1.18 to 1.51 (per 1-standard deviation increment in relative abundance) for microbiota statistically significantly (P < 0.05) positively associated with progression, and from 0.77 to 0.82 for those statistically significantly (P < 0.05) inversely associated with progression. These associations were similar when stratified on baseline levels of pocket depth, gingival bleeding, ACH, and smoking status. CONCLUSIONS: These prospective results affirm clearly that subgingival microbiota are measurably elevated several years prior to progression of alveolar bone loss, and include antecedent elevations in previously undocumented taxa additional to known Socransky pathogenic complexes.

Composition and diversity of the subgingival microbiome and its relationship with age in postmenopausal women: an epidemiologic investigation.

BACKGROUND: The extent to which the composition and diversity of the oral microbiome varies with age is not clearly understood. METHODS: The 16S rRNA gene of subgingival plaque in 1219 women, aged 53-81 years, was sequenced and its taxonomy annotated against the Human Oral Microbiome Database (v.14.5). Composition of the subgingival microbiome was described in terms of centered log(2)-ratio (CLR) transformed OTU values, relative abundance, and prevalence. Correlations between microbiota abundance and age were evelauted using Pearson Product Moment correlations. P-values were corrected for multiple testing using the Bonferroni method. RESULTS: Of the 267 species identified overall, Veillonella dispar was the most abundant bacteria when described by CLR OTU (mean 8.3) or relative abundance (mean 8.9%); whereas Streptococcus oralis, Veillonella dispar and Veillonella parvula were most prevalent (100%, all) when described as being present at any amount. Linear correlations between age and several CLR OTUs (Pearson r = - 0.18 to 0.18), of which 82 (31%) achieved statistical significance (P < 0.05). The correlations lost significance following Bonferroni correction. Twelve species that differed across age groups (each corrected P < 0.05); 5 (42%) were higher in women ages 50-59 compared to ≥70 (corrected P < 0.05), and 7 (48%) were higher in women 70 years and older. CONCLUSIONS: We identified associations between several bacterial species and age across the age range of postmenopausal women studied. Understanding the functions of these bacteria could identify intervention targets to enhance oral health in later life.

Is the Oral Microbiome Associated with Blood Pressure in Older Women?

INTRODUCTION: A possible role of the oral microbiome, specifically oral nitrate reducing flora, in blood pressure (BP) homeostasis, if proven etiologic in nature, could lead to novel mechanism-based therapy to improve hypertension prevention and control. AIM: This cross-sectional study characterized and compared the oral microbiome between four study groups based on BP status among 446 postmenopausal women aged 53-82 years. METHODS: Three study groups were not taking hypertension medication and were separated based on BP, as follows: normal BP (systolic < 120 and diastolic < 80; N = 179), elevated BP/Stage I hypertension (systolic 120-139 or diastolic 80-90; N = 106), Stage II hypertension (systolic > 140 or diastolic > 90; N = 42). The forth group consisted of anyone taking hypertension medications, regardless of BP (N = 119). Subgingival microbiome composition was determined using 16S rRNA sequencing with the Illumina MiSeq platform. Kruskal-Wallis tests were used to compare species-level relative abundance of bacterial operational taxonomic units across the four groups. RESULTS: Sixty-five bacterial species demonstrated significant differences in relative abundance in women with elevated BP or using hypertension medication as compared to those with normal BP. After correction for multiple testing, two species, Prevotella oral (species 317) and Streptococcus oralis, remained significant and were lower in abundance among women taking antihypertension medications compared to those with normal BP (corrected P < 0.05). CONCLUSIONS: These data provide novel description of oral subgingival bacteria grouped according to BP status. Additional larger studies including functional analysis and prospective designs will help further assess the potential role of the oral microbiome in BP regulation and hypertension.

Fecal microbiota analysis of polycystic kidney disease patients according to renal function: A pilot study.

IMPACT STATEMENT: The heterogeneity of the renal disease, therapeutic interventions, and the original cause of the renal failure, all directly affect the microbiota. We delineate in this report the direct effect of decreased renal function on the bacterial composition following stringent criteria to eliminate the possibilities of other confounding factors and dissect the direct effects of the uremic milieu. We analyzed the microbiome following three different approaches to further evaluate the effects of mild, moderate and advanced renal insufficiency on the microbiome. We also present here a detailed functional analysis of the projected altered pathways secondary to changes in the microbiome composition.

Cohort profile: the Buffalo OsteoPerio microbiome prospective cohort study.

PURPOSE: The Buffalo Osteoporosis and Periodontal Disease (OsteoPerio) study is a prospective cohort study focused on the relationship between the microbiome and oral and systemic health outcomes in postmenopausal women. The cohort was established to examine how the oral microbiome is affected by (and how it affects) periodontal disease presence, severity and progression and to characterise the relationship between the microbiome, lifestyle habits and systemic disease outcomes. PARTICIPANTS: Participants (n=1342) were postmenopausal women who were participating in the Women's Health Initiative observational study at the Buffalo, New York clinical centre. There were 1026 participants at the 5-year follow-up visit and 518 at the 15-year visit. FINDINGS TO DATE: Data collected include questionnaires, anthropometric measures, serum blood and saliva samples. At each clinic visit, participants completed a comprehensive oral examination to measure oral health and the oral microbiome. Preliminary findings have contributed to our understanding of risk factors for periodontal disease and the relationship between the oral microbiome and periodontal disease. FUTURE PLANS: The novel microbiome data collected on a large sample of participants at three time points will be used to answer a variety of research questions focused on temporal changes in the microbiome and the relationship between the oral microbiome and oral and systemic disease outcomes. Little is currently known about the relationship between the oral microbiome and health outcomes in older adults; data from the OsteoPerio cohort will fill this gap. Microbiome samples are currently being analysed using next-generation sequencing technology with an anticipated completion date of late 2018.

Substantial Differences in the Subgingival Microbiome Measured by 16S Metagenomics According to Periodontitis Status in Older Women.

Aging invokes physiological changes, such as immunosenescence and inflammation, that could increase host susceptibility to oral microbiome shifts that enable periodontitis progression in later life. At present, there is a dearth of studies specifically evaluating the oral microbiome and periodontitis in older adults. We used high-throughput untargeted sequencing methods and functional metagenomic analyses to assess and compare the subgingival biofilm of postmenopausal women (mean age 71 years) according to periodontitis status. Subgingival plaque samples were obtained from 15 postmenopausal women with no periodontitis, and from 15 women with severe periodontitis, determined by probing measures. The 16S rRNA gene (V1⁻V3 region) was sequenced on the 454 FLX platform. The PICRUSt technique was used to provide information on what the potential functional characteristics of microbiota might be in healthy, compared with diseased, periodontium. The subgingival microbiome associated with periodontitis showed clear differences to that associated with health. Of the 464 species identified, 22.8% had elevated abundance in disease, while only 6.3% had elevated abundance in health. Among the 12 most prevalent organisms in periodontitis, one-half have previously been recognized as periodontal pathogens by other investigators. The subgingival microbiome in periodontitis contained genes that could code for specific activities, including microbial mobility, synthesis of endotoxin, and proteolytic degradation. The healthy microbiome included genes that could code for sustaining microbial life, including encoding for transporters, glycolysis, gluconeogenesis, the Krebs cycle, and protein kinases. In the present study on postmenopausal women, aged 60 and older, the subgingival microbiome differed in composition and potential function between those with and without periodontitis. Studies of functional gene expression, such as transcriptomics, are needed to definitively identify the molecules carrying out functions associated with pathogenic subgingival complexes. This, in turn, could lead to identification of targets for enhanced management of periodontitis and, possibly, other diseases, in later life.

Chronic kidney disease, uremic milieu, and its effects on gut bacterial microbiota dysbiosis.

Several lines of evidence suggest that gut bacterial microbiota is altered in patients with chronic kidney disease (CKD), though the mechanism of which this dysbiosis takes place is not well understood. Recent studies delineated changes in gut microbiota in both CKD patients and experimental animal models using microarray chips. We present 16S ribosomal RNA gene sequencing of both stool pellets and small bowel contents of C57BL/6J mice that underwent a remnant kidney model and establish that changes in microbiota take place in the early gastrointestinal tract. Increased intestinal urea concentration has been hypothesized as a leading contributor to dysbiotic changes in CKD. We show that urea transporters (UT)-A and UT-B mRNA are both expressed throughout the whole gastrointestinal tract. The noted increase in intestinal urea concentration appears to be independent of UTs' expression. Urea supplementation in drinking water resulted in alteration in bacterial gut microbiota that is quite different than that seen in CKD. This indicates that increased intestinal urea concentration might not fully explain the CKD- associated dysbiosis.