dClostridium difficile infection (CDI) causes nearly half a million cases of diarrhea and colitis in the United States each year. Although the importance of the gut microbiota in C. difficile pathogenesis is well recognized, components of the human gut flora critical for colonization resistance are not known. Culture-independent high-density Roche 454 pyrosequencing was used to survey the distal gut microbiota for 39 individuals with CDI, 36 subjects with C. difficile-negative nosocomial diarrhea (CDN), and 40 healthy control subjects. A total of 526,071 partial 16S rRNA sequence reads of the V1 to V3 regions were aligned with 16S databases, identifying 3,531 bacterial phylotypes from 115 fecal samples. Genomic analysis revealed significant alterations of organism lineages in both the CDI and CDN groups, which were accompanied by marked decreases in microbial diversity and species richness driven primarily by a paucity of phylotypes within the Firmicutes phylum. Normally abundant gut commensal organisms, including the Ruminococcaceae and Lachnospiraceae families and butyrate-producing C2 to C4 anaerobic fermenters, were significantly depleted in the CDI and CDN groups. These data demonstrate associations between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota and nosocomial diarrhea, including C. difficile infection. Mechanistic studies focusing on the functional roles of these organisms in diarrheal diseases and resistance against C. difficile colonization are warranted.

Chronic periodontitis is an inflammatory disease of the periodontium affecting nearly 65 million adults in the United States. Changes in subgingival microbiota have long been associated with chronic periodontitis. Recent culture-independent molecular studies have revealed the immense richness and complexity of oral microbial communities. However, data sets across studies have not been directly compared, and whether the observed microbial variations are consistent across different studies is not known. Here, we used 16S rRNA sequencing to survey the subgingival microbiota in 25 subjects with chronic periodontal disease and 25 healthy controls and compared our data sets with those of three previously reported microbiome studies. Consistent with data from previous studies, our results demonstrate a significantly altered microbial community structure with decreased heterogeneity in periodontal disease. Comparison with data from three previously reported studies revealed that subgingival microbiota clustered by study. However, differences between periodontal health and disease were larger than the technical variations across studies. Using a prediction score and applying five different distance metrics, we observed two predominant clusters. One cluster was driven by Fusobacterium and Porphyromonas and was associated with clinically apparent periodontitis, and the second cluster was dominated by Rothia and Streptococcus in the majority of healthy sites. The predicted functional capabilities of the periodontitis microbiome were significantly altered. Genes involved in bacterial motility, energy metabolism, and lipopolysaccharide biosynthesis were overrepresented in periodontal disease, whereas genes associated with transporters, the phosphotransferase system, transcription factors, amino acid biosynthesis, and glycolysis/gluconeogenesis were enriched in healthy controls. These results demonstrate significant alterations in microbial composition and function in periodontitis and suggest genes and metabolic pathways associated with periodontal disease. P eriodontal disease is a common disease affecting many adults in the United States. If left untreated, chronic periodontitis (CP) can lead to serious problems such as tooth loss. Indeed, periodontal disease is the number one cause of tooth loss in the United States, accounting for half of all tooth loss in U.S. adults (1). There is considerable evidence that the clinical impact of periodontal disease extends beyond the oral cavity (2). Strong associations have been found between periodontitis and a number of systemic conditions, including cardiovascular disease (3, 4), preterm delivery and low-birth-weight babies (5), diabetes mellitus (6-8), and rheumatoid arthritis (9-11).The activity of periodontal disease is determined by a complex interplay between the immune system and periodontal pathogens (12, 13). Alterations in subgingival microbiota have long been associated with the development and progression of periodontitis (14). In susceptible individuals, perturbations in...

Clostridium difficile infection (CDI) is characterized by dysbiosis of the intestinal microbiota and a profound derangement in the fecal metabolome. However, the contribution of specific gut microbes to fecal metabolites in C. difficile-associated gut microbiome remains poorly understood. Using gas-chromatography mass spectrometry (GC-MS) and 16S rRNA deep sequencing, we analyzed the metabolome and microbiome of fecal samples obtained longitudinally from subjects with Clostridium difficile infection (n = 7) and healthy controls (n = 6). From 155 fecal metabolites, we identified two sterol metabolites at >95% match to cholesterol and coprostanol that significantly discriminated C. difficile-associated gut microbiome from healthy microbiota. By correlating the levels of cholesterol and coprostanol in fecal extracts with 2,395 bacterial operational taxonomic units (OTUs) determined by 16S rRNA sequencing, we identified 63 OTUs associated with high levels of coprostanol and 2 OTUs correlated with low coprostanol levels. Using indicator species analysis (ISA), 31 of the 63 coprostanol-associated bacteria correlated with health, and two Veillonella species were associated with low coprostanol levels that correlated strongly with CDI. These 65 bacterial taxa could be clustered into 12 sub-communities, with each community containing a consortium of organisms that co-occurred with one another. Our studies identified 63 human gut microbes associated with cholesterol-reducing activities. Given the importance of gut bacteria in reducing and eliminating cholesterol from the GI tract, these results support the recent finding that gut microbiome may play an important role in host lipid metabolism.

No abstract

Leveraging data from a large case-control study and accounting for multiple factors known to influence oral health status, results from this study failed to identify a subgingival microbial fingerprint that could reliably discriminate RA from OA patients.

The subgingival microbiome is one of the most stable microbial ecosystems in the human body. Alterations in the subgingival microbiome have been associated with periodontal disease, but their variations over time and between different subgingival sites in periodontally healthy individuals have not been well described. We performed extensive, longitudinal sampling of the subgingival microbiome from five periodontally healthy individuals to define baseline spatial and temporal variations. A total of 251 subgingival samples from 5 subjects were collected over 6–12 months and deep sequenced. The overall microbial diversity and composition differed significantly between individuals. Within each individual, we observed considerable differences in microbiome composition between different subgingival sites. However, for a given site, the microbiome was remarkably stable over time, and this stability was associated with increased microbial diversity but was inversely correlated with the enrichment of putative periodontal pathogens. In contrast to microbiome composition, the predicted functional metagenome was similar across space and time, suggesting that periodontal health is associated with shared gene functions encoded by different microbiome consortia that are individualized. To our knowledge, this is one of the most detailed longitudinal analysis of the healthy subgingival microbiome to date that examined the longitudinal variability of different subgingival sites within individuals. These results suggest that a single measurement of the healthy subgingival microbiome at a given site can provide long term information of the microbial composition and functional potential, but sampling of each site is necessary to define the composition and community structure at individual subgingival sites.

Background Subgingival microbiome in disease-associated subgingival sites is known to be dysbiotic and significantly altered. In patients with rheumatoid arthritis (RA), the extent of dysbiosis in disease- and health-associated subgingival sites is not clear. Methods 8 RA and 10 non-RA subjects were recruited for this pilot study. All subjects received full oral examination and underwent collection of subgingival plaque samples from both shallow (periodontal health-associated, probing depth ≤ 3mm) and deep subgingival sites (periodontal disease-associated, probing depth ≥ 4 mm). RA subjects also had rheumatological evaluation. Plaque community profiles were analyzed using 16 S rRNA sequencing. Results The phylogenetic diversity of microbial communities in both RA and non-RA controls was significantly higher in deep subgingival sites compared to shallow sites (p = 0.022), and the overall subgingival microbiome clustered primarily according to probing depth (i.e. shallow versus deep sites), and not separated by RA status. While a large number of differentially abundant taxa and gene functions was observed between deep and shallow sites as expected in non-RA controls, we found very few differentially abundant taxa and gene functions between deep and shallow sites in RA subjects. In addition, compared to non-RA controls, the UniFrac distances between deep and shallow sites in RA subjects were smaller, suggesting increased similarity between deep and shallow subgingival microbiome in RA. Streptococcus parasanguinis and Actinomyces meyeri were overabundant in RA subjects, while Gemella morbillorum, Kingella denitrificans, Prevotella melaninogenica and Leptotrichia spp. were more abundant in non-RA subjects. Conclusions The aggregate subgingival microbiome was not significantly different between individuals with and without rheumatoid arthritis. Although the differences in the overall subgingival microbiome was driven primarily by probing depth, in contrast to the substantial microbiome differences typically seen between deep and shallow sites in non-RA patients, the microbiome of deep and shallow sites in RA patients were more similar to each other. These results suggest that factors associated with RA may modulate the ecology of subgingival microbiome and its relationship to periodontal disease, the basis of which remains unknown but warrants further investigation.

Viral variants with decreased susceptibility to HCV protease inhibitors (PIs) occur naturally and preexist at low levels within HCV populations. In patients failing PI monotherapy, single and double mutants conferring intermediate to high-level resistance to PIs have been selected in vivo. The abundance, temporal dynamics and linkage of naturally occurring resistance-associated variants (RAVs), however, have not been characterized in detail. Here, using high-density pyrosequencing, we analyzed HCV NS3 gene segments from 20 subjects with chronic HCV infection, including 12 subjects before and after liver transplantation. Bioinformatics analysis revealed that Q80 substitution was a dominant variant in 40% of the subjects, whereas other RAVs circulate at low levels within quasispecies populations. Low frequency mutation linkage was detectable by Illumina paired-end sequencing in as low as 0.5% of the mock populations constructed from in vitro RNA transcripts but were uncommon in vivo. We show that naturally occurring RAVs are common and can persist long term following liver transplant at low levels not readily detectable by conventional sequencing. Our results indicate that mutation linkage at low levels could be identified using the Illumina paired-end approach. The methods described here should facilitate the analysis of low frequency HCV drug resistance, mutation linkage and evolution, which may inform future therapeutic strategies in patients undergoing direct acting antiviral therapies.