Paraphocaeicola brunensis gen. nov., sp. nov., Carrying Two Variants of nimB Resistance Gene from Bacteroides fragilis, and Caecibacteroides pullorum gen. nov., sp. nov., Two Novel Genera Isolated from Chicken Caeca.

Three difficult-to-cultivate, strictly anaerobic strains, AN20T, AN421T, and AN502, were analyzed within a project studying possible probiotics for newly hatched chickens. Phylogenetic analyses showed that strains AN20T, AN421T, and AN502 formed two well-separated phylogenetic lineages in all phylogenetic and phylogenomic trees comprising members of the family Bacteroidaceae. Comparison to reference genomes of type species Bacteroides fragilis NCTC 9343T, Phocaeicola abscessus CCUG 55929T, and Capsularis zoogleoformans ATCC 33285T showed low relatedness based on the calculated genome-to-genome distance and orthologous average nucleotide identity. Analysis of fatty acid profiles showed iso-C15:0, anteiso-C15:0, C16:0, C18:1ω9c, and iso-C17:0 3OH as the major fatty acids for all three strains and additionally C16:0 3OH for AN421T and AN502. A specific combination of respiratory quinones different from related taxa was found in analyzed strains, MK-5 plus MK-11 in strain AN20T and MK-5 plus MK-10 in strains AN421T and AN502. Strains AN421T and AN502 harbor complete CRISPR loci with CRISPR array, type II-C, accompanied by a set of cas genes (cas9, cas1, and cas2) in close proximity. Interestingly, strain AN20T was found to harbor two copies of nimB gene with >95% similarity to nimB of B. fragilis, suggesting a horizontal gene transfer between these taxa. In summary, three isolates characterized in this study represent two novel species, which we proposed to be classified in two novel genera of the family Bacteroidaceae, for which the names Paraphocaeicola brunensis sp. nov. (AN20T = CCM 9041T = DSM 111154T) and Caecibacteroides pullorum sp. nov. (AN421T= CCM 9040T = DSM 111155T) are proposed. IMPORTANCE This study represents follow-up research on three difficult-to-cultivate anaerobic isolates originally isolated within a project focused on strains that are able to stably colonize newly hatched chickens, thus representing possible probiotics. This project is exceptional in that it successfully isolates several miscellaneous strains that required modified and richly supplemented anaerobic media, as information on many gut-colonizing bacteria is based predominantly on metagenomic studies. Superior colonization of newly hatched chickens by Bacteroides spp., Phocaeicola spp., or related taxa can be considered of importance for development of future probiotics. Although different experiments can also be performed with provisionally characterized isolates, precise taxonomical definition is necessary for subsequent broad communication. The aim of this study is therefore to thoroughly characterize these isolates that represent novel genera and precisely determine their taxonomic position among related taxa to facilitate further research and communication involving these strains.

Serum IgG titers against periodontal pathogens are associated with cerebral hemorrhage growth and 3-month outcome.

To assess the influence of periodontal disease on cerebral hemorrhage and its clinical course, we examined the association of the serum IgG titer of periodontal pathogens with hemorrhage growth and 3-month outcome. We consecutively enrolled 115 patients with acute cerebral hemorrhage (44 females, aged 71.3 ± 13.1 years) and used ELISA to evaluate the serum IgG titers of 9 periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter (A.) actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Fusobacterium (F.) nucleatum, Treponema denticola, Tannerella forsythensis, Campylobacter rectus, and Eikenella corrodens. Significant hematoma growth was defined as an increase in the volume of >33% or an absolute increase in the volume of >12.5 mL. A poor outcome was defined as a 3 or higher on the modified Rankin Scale. We observed hemorrhage growth in 13 patients (11.3%). Multivariate analysis revealed that increased IgG titers of A. actinomycetemcomitans independently predicted the elevated hemorrhage growth (odds ratio 5.26, 95% confidence interval 1.52-18.25, p = 0.01). Notably, augmented IgG titers of F. nucleatum but not A. actinomycetemcomitans led to a poorer 3-month outcome (odds ratio 7.86, 95% confidence interval 1.08-57.08, p = 0.04). Thus, we demonstrate that elevated serum IgG titers of A. actinomycetemcomitans are an independent factor for predicting cerebral hemorrhage growth and that high serum IgG titers of F. nucleatum may predict a poor outcome in patients with this disease. Together, these novel data reveal how systemic periodontal pathogens may affect stroke patients, and, should, therefore, be taken into consideration in the management and treatment of these individuals.

Comparison of microbiomes in ulcerative and normal mucosa of recurrent aphthous stomatitis (RAS)-affected patients.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is the most common form of oral ulcerative disease, whose cause is still unknown. Researchers have found the association of many factors with the occurrence of RAS, and proposed oral bacterial infection could be a cause for this disease. METHODS: To investigate whether the occurrence of RAS is associated with oral bacterial infection, we performed high throughput sequencing analysis of bacterial samples collected from the normal oral mucosa and aphthous ulcers of 24 patients. RESULTS: Firmicutes, Proteobacteria and Bacteriodetes were the most abundant phyla in the microbiomes analysed. The alpha diversities of the oral mucosa and aphthous ulcer microbiomes were similar, suggesting a similar richness and diversity. The NMDS analysis showed the oral mucosa and aphthous ulcer microbiomes are significantly different. This suggestion is further supported by Anosim, MRPP, and Adonis analyses. More detailed comparison of the two groups of microbiomes suggested that the occurrence of RAS is significantly associated with the increase of Escherichia coli and Alloprevotella, as well as the decrease of Streptococcus. CONCLUSIONS: Considering E. coli is a very common intestinal bacterium, we propose that E. coli colonization could be a cause for RAS, and controlling E. coli colonization could help curing RAS.

Enrichment of intracellular sulphur cycle -associated bacteria in intertidal benthic foraminifera revealed by 16S and aprA gene analysis.

Benthic foraminifera are known to play an important role in marine carbon and nitrogen cycles. Here, we report an enrichment of sulphur cycle -associated bacteria inside intertidal benthic foraminifera (Ammonia sp. (T6), Haynesina sp. (S16) and Elphidium sp. (S5)), using a metabarcoding approach targeting the 16S rRNA and aprA -genes. The most abundant intracellular bacterial groups included the genus Sulfurovum and the order Desulfobacterales. The bacterial 16S OTUs are likely to originate from the sediment bacterial communities, as the taxa found inside the foraminifera were also present in the sediment. The fact that 16S rRNA and aprA -gene derived intracellular bacterial OTUs were species-specific and significantly different from the ambient sediment community implies that bacterivory is an unlikely scenario, as benthic foraminifera are known to digest bacteria only randomly. Furthermore, these foraminiferal species are known to prefer other food sources than bacteria. The detection of sulphur-cycle related bacterial genes in this study suggests a putative role for these bacteria in the metabolism of the foraminiferal host. Future investigation into environmental conditions under which transcription of S-cycle genes are activated would enable assessment of their role and the potential foraminiferal/endobiont contribution to the sulphur-cycle.

Establishing a mucosal gut microbial community in vitro using an artificial simulator.

The Twin Simulator of the Human Intestinal Microbial Ecosystem (TWINSHIME®) was initially developed to study the luminal gut microbiota of the ascending (AC), transverse (TC), and descending (DC) colon regions. Given the unique composition and potential importance of the mucosal microbiota for human health, the TWINSHIME was recently adapted to simulate the mucosal microbiota as well as the luminal community. It has been previously demonstrated that the luminal community in the TWINSHIME reaches a steady state within two weeks post inoculation, and is able to differentiate into region specific communities. However, less is known regarding the mucosal community structure and dynamics. During the current study, the luminal and mucosal communities in each region of the TWINSHIME were evaluated over the course of six weeks. Based on 16S rRNA gene sequencing and short chain fatty acid analysis, it was determined that both the luminal and mucosal communities reached stability 10-20 days after inoculation, and remained stable until the end of the experiment. Bioinformatics analysis revealed the formation of unique community structures between the mucosal and luminal phases in all three colon regions, yet these communities were similar to the inoculum. Specific colonizers of the mucus mainly belonged to the Firmicutes phylum and included Lachnospiraceae (AC/TC/DC), Ruminococcaceae and Eubacteriaceae (AC), Lactobacillaceae (AC/TC), Clostridiaceae and Erysipelotrichaceae (TC/DC). In contrast, Bacteroidaceae were enriched in the gut lumen of all three colon regions. The unique profile of short chain fatty acid (SCFA) production further demonstrated system stability, but also proved to be an area of marked differences between the in vitro system and in vivo reports. Results of this study demonstrate that it is possible to replicate the community structure and composition of the gut microbiota in vitro. Through implementation of this system, the human gut microbiota can be studied in a dynamic and continuous fashion.

Cospeciation of gut microbiota with hominids.

The evolutionary origins of the bacterial lineages that populate the human gut are unknown. Here we show that multiple lineages of the predominant bacterial taxa in the gut arose via cospeciation with humans, chimpanzees, bonobos, and gorillas over the past 15 million years. Analyses of strain-level bacterial diversity within hominid gut microbiomes revealed that clades of Bacteroidaceae and Bifidobacteriaceae have been maintained exclusively within host lineages across hundreds of thousands of host generations. Divergence times of these cospeciating gut bacteria are congruent with those of hominids, indicating that nuclear, mitochondrial, and gut bacterial genomes diversified in concert during hominid evolution. This study identifies human gut bacteria descended from ancient symbionts that speciated simultaneously with humans and the African apes.

Molecular identification of black-pigmented bacteria from subgingival samples of cats suffering from periodontal disease.

OBJECTIVES: To characterise the black-pigmented bacterial species found in the subgingival samples of cats with periodontal disease using molecular-based microbiological techniques. METHODS: Sixty-five subgingival samples obtained from 50 cats with periodontal disease were analysed by polymerase chain reaction amplified ribosomal DNA restriction analysis and cloning and sequencing of the 16S rRNA genes. RESULTS: Among the 65 subgingival samples, eight phylogenetic profiles were obtained, of which the most prevalent species were: Porphyromonas gulae (40%), P. gingivalis/P. gulae (36 · 9%), P. gulae/Porphyromonas sp. UQD 406 (9 · 2%), Odoribacter denticanis (6 · 2%), P. gulae/Porphyromonas sp. UQD 348 (1 · 5%) and P. circumdentaria (1 · 5%). When compared with the species resulting from biochemical diagnosis, the identification of P. gulae was congruent in 70% of the cases, while colonies identified as P. intermedia-like corresponded in 80% of cases to P. gulae. CLINICAL SIGNIFICANCE: The use of molecular-based microbiological diagnostic techniques resulted in a predominance of Porphyromonas spp. in the subgingival plaque of cats suffering from periodontal disease. Further characterisation of these bacteria identified P. gulae, O. denticanis and P. circumdentaria. The more frequently detected phylogenetic profiles corresponded to P. gingivalis and P. gulae.

Extensive strain-level copy-number variation across human gut microbiome species.

Within each bacterial species, different strains may vary in the set of genes they encode or in the copy number of these genes. Yet, taxonomic characterization of the human microbiota is often limited to the species level or to previously sequenced strains, and accordingly, the prevalence of intra-species variation, its functional role, and its relation to host health remain unclear. Here, we present a comprehensive large-scale analysis of intra-species copy-number variation in the gut microbiome, introducing a rigorous computational pipeline for detecting such variation directly from shotgun metagenomic data. We uncover a large set of variable genes in numerous species and demonstrate that this variation has significant functional and clinically relevant implications. We additionally infer intra-species compositional profiles, identifying population structure shifts and the presence of yet uncharacterized variants. Our results highlight the complex relationship between microbiome composition and functional capacity, linking metagenome-level compositional shifts to strain-level variation.

Mucosal microbiome in patients with recurrent aphthous stomatitis.

Recurrent aphthous stomatitis (RAS) is the most common disease affecting oral mucosae. Etiology is unknown, but several factors have been implicated, all of which influence the composition of microbiota residing on oral mucosae, which in turn modulates immunity and thereby affects disease progression. Although no individual pathogens have been conclusively shown to be causative agents of RAS, imbalanced composition of the oral microbiota may play a key role. In this study, we sought to determine composition profiles of bacterial microbiota in the oral mucosa associated with RAS. Using high-throughput 16S rRNA gene sequencing, we characterized the most abundant bacterial populations residing on healthy and ulcerated mucosae in patients with RAS (recruited using highly stringent criteria) and no associated medical conditions; we also compared these to the bacterial microbiota of healthy controls (HCs). Phylum-level diversity comparisons revealed decreased Firmicutes and increased Proteobacteria in ulcerated sites, as compared with healthy sites in RAS patients, and no differences between RAS patients with healthy sites and HCs. Genus-level analysis demonstrated higher abundance of total Bacteroidales in RAS patients with healthy sites over HCs. Porphyromonadaceae comprising species associated with periodontal disease and Veillonellaceae predominated in ulcerated sites over HCs, while no quantitative differences of these families were observed between healthy sites in RAS patients and HCs. Streptococcaceae comprising species associated with oral health predominated in HCs over ulcerated sites but not in HCs over healthy sites in RAS patients. This study demonstrates that mucosal microbiome changes in patients with idiopathic RAS--namely, increased Bacteroidales species in mucosae of RAS patients not affected by active ulceration. While these changes suggest a microbial role in initiation of RAS, this study does not provide data on causality. Within this limitation, the study contributes to the understanding of the potential role of mucosal microbiome changes in oral mucosal disease.

Susceptibility of as-yet-uncultivated and difficult-to-culture bacteria to chemomechanical procedures.

INTRODUCTION: A significant portion of the bacteria taking part of the microbiome associated with apical periodontitis still remain to be cultivated and phenotypically characterized. This molecular study evaluated the prevalence of selected as-yet-uncultivated and difficult-to-culture bacterial taxa in infected root canals and their susceptibility to chemomechanical procedures. METHODS: Root canals of single-rooted teeth with apical periodontitis were prepared using rotary nickel-titanium instruments and 2.5% sodium hypochlorite as the irrigant. DNA extracts from samples taken before (S1) and after (S2) chemomechanical preparation were surveyed for the presence of 7 as-yet-uncultivated phylotypes and 1 difficult-to-culture species using end-point polymerase chain reaction. Samples were also subjected to quantitative analysis of total bacteria and levels of the 2 most prevalent taxa. RESULTS: Bacteroidaceae sp. HOT-272 (24%) and Fretibacterium fastidiosum (20%) were the most prevalent taxa in S1. Their mean counts in S1 were 8.25 × 10(3) and 2.13 × 10(3) rRNA gene copies, corresponding to 0.18% and 0.55% of the total bacteria. Chemomechanical debridement promoted a highly statistically significant reduction in total bacterial counts (P < .001), but 64% of the canals were still positive for bacterial presence. Of the target taxa, only Bacteroidaceae sp. HOT-272 and F. fastidiosum were detected in S2 (each one in 1 sample). The reduction in counts of both taxa was also highly significant (P < .001). CONCLUSIONS: Findings confirmed that several as-yet-uncultivated and difficult-to-grow bacterial taxa can participate in the microbiome associated with apical periodontitis. Two of them were found in relatively high prevalence but rarely as a dominant species. Chemomechanical procedures were highly effective in completely eliminating these taxa or at least substantially reducing their numbers.

Falsiporphyromonas endometrii gen. nov., sp. nov., isolated from the post-partum bovine uterus, and emended description of the genus Porphyromonas Shah and Collins 1988.

Two black-pigmented, anaerobic bacterial strains, designated LMM 40(T) and LMM 41, were isolated from the bovine post-partum endometrium of two Holstein cows. The 16S rRNA gene sequences of the two strains were identical and showed the highest similarity to the 16S rRNA gene sequence of the type strain of Porphyromonas crevioricanis (90.2%) but only 85.1% 16S rRNA gene sequence similarity to the type strain of the type species of the genus Porphyromonas, Porphyromonas asaccharolytica. The major fatty acid profiles of the two strains were similar to those of species of the genus Porphyromonas, containing iso-C(15 : 0) as the major component and moderate amounts of anteiso-C(15 : 0), iso-C(13 : 0), C(15 : 0) and C(16 : 0). Hydroxylated fatty acids, such as iso-C(14 : 0) 3-OH, iso-C(16 : 0) 3-OH and iso-C(17 : 0) 3-OH, were also detected. The quinone profiles were dominated by the menaquinones MK-8 and MK-9, while spermidine was the major polyamine. The polar lipid profiles contained major amounts of phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminophospholipid and two unidentified lipids and minor amounts of phosphatidylglycerol, an unidentified aminolipid, a second unidentified aminophospholipid and an unidentified glycolipid. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The genomic DNA G+C contents of LMM 40(T) and LMM 41 were 40.7 and 41.3 mol%, respectively. Based on a polyphasic approach, including phylogenetic analysis, physiological and biochemical tests as well as metabolic fingerprinting, it is proposed that the two strains are members of a novel genus and species, for which the name Falsiporphyromonas endometrii gen. nov., sp. nov. is proposed. The type strain of Falsiporphyromonas endometrii is LMM 40(T) ( = DSM 27210(T) = CCUG 64267(T)). An emended description of the genus Porphyromonas is also presented.

Murine gut microbiota is defined by host genetics and modulates variation of metabolic traits.

The gastrointestinal tract harbors a complex and diverse microbiota that has an important role in host metabolism. Microbial diversity is influenced by a combination of environmental and host genetic factors and is associated with several polygenic diseases. In this study we combined next-generation sequencing, genetic mapping, and a set of physiological traits of the BXD mouse population to explore genetic factors that explain differences in gut microbiota and its impact on metabolic traits. Molecular profiling of the gut microbiota revealed important quantitative differences in microbial composition among BXD strains. These differences in gut microbial composition are influenced by host-genetics, which is complex and involves many loci. Linkage analysis defined Quantitative Trait Loci (QTLs) restricted to a particular taxon, branch or that influenced the variation of taxa across phyla. Gene expression within the gastrointestinal tract and sequence analysis of the parental genomes in the QTL regions uncovered candidate genes with potential to alter gut immunological profiles and impact the balance between gut microbial communities. A QTL region on Chr 4 that overlaps several interferon genes modulates the population of Bacteroides, and potentially Bacteroidetes and Firmicutes-the predominant BXD gut phyla. Irak4, a signaling molecule in the Toll-like receptor pathways is a candidate for the QTL on Chr15 that modulates Rikenellaceae, whereas Tgfb3, a cytokine modulating the barrier function of the intestine and tolerance to commensal bacteria, overlaps a QTL on Chr 12 that influence Prevotellaceae. Relationships between gut microflora, morphological and metabolic traits were uncovered, some potentially a result of common genetic sources of variation.

Inulin and fructo-oligosaccharides have divergent effects on colitis and commensal microbiota in HLA-B27 transgenic rats.

Modulation of intestinal microbiota by non-digestible carbohydrates may reduce inflammation in inflammatory bowel disease (IBD). The aim of the present study was to assess the effects of inulin and fructo-oligosaccharides (FOS) on intestinal microbiota and colitis in HLA-B27 transgenic rats, a well-validated rodent model for IBD. In this study, 4-week-old rats were fed 8 g/kg body weight inulin or FOS for 12 weeks, or not. Faeces were collected at 4 and 16 weeks of age; and caecal samples were collected at necropsy. The effects of inulin and FOS on chronic intestinal inflammation were assessed using a gross gut score, histology score and levels of mucosal IL-1ß. Intestinal microbiota were characterised by quantitative PCR and denaturing gradient gel electrophoresis. Colitis was significantly reduced in all FOS-fed rats compared to the control diet, whereas inulin decreased chronic intestinal inflammation in only half the number of animals. Quantitative analysis of caecal microbiota demonstrated that inulin increased the numbers of total bacteria and the Bacteroides-Prevotella-Porphyromonas group, FOS increased bifidobacteria, and both fructans decreased Clostridium cluster XI. In the faecal samples, both inulin and FOS decreased total bacteria, Bacteroides-Prevotella-Porphyromonas group, and Clostridium clusters XI and XIVa. FOS increased Bifidobacterium spp., and mediated a decrease of gene copies of Enterobacteriaceae and Clostridium difficile toxin B in faeces. SCFA concentrations in the faecal and caecal samples were unaffected by the diets. In conclusion, FOS increased the abundance of Bifidobacterium spp., whereas both fructans reduced Clostridium cluster XI and C. difficile toxin gene expression, correlating with a reduction of chronic intestinal inflammation.

RNA-oligonucleotide quantification technique (ROQT) for the enumeration of uncultivated bacterial species in subgingival biofilms.

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp. OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 10(4) cells. Fusobacterium nucleatum ss. polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.

Detection of selected bacterial species in intraoral sites of patients with chronic periodontitis using multiplex polymerase chain reaction.

OBJECTIVE: The aim of this study was to detect the prevalence of selected bacterial species in intraoral sites of patients with chronic periodontitis (CP) using multiplex polymerase chain reaction (PCR). METHODOLOGY: Samples were collected from the tongue dorsum, buccal mucosa, supragingival and subgingival plaque and saliva of 30 patients with untreated CP. Multiplex PCR was used to determine prevalence rates, which were then compared using a chi-square test. Significance level was set at p<0.05. Mean and standard deviation values were used to evaluate variations in prevalence according to site. RESULTS: The prevalence of S. mutans was 70% in saliva; 60% in samples collected from the tongue dorsum; 50% in samples collected from the buccal mucosa; 56.5% in the supragingival plaque; and 53.5% in the subgingival plaque. The prevalence of E. faecalis ranged from 3.5% to 13.5% in all intraoral microenvironment. The highest prevalence of P. gingivalis was found in subgingival plaque (53.5%), and of P. intermedia in supragingival plaque (33.5%), subgingival plaque (30%) and tongue dorsum (33.5%). The prevalence of bacteria did not vary significantly among the intraoral sites. CONCLUSIONS: All studied bacteria were identified in intraoral sites. S. mutans, P. gingivalis and P. intermedia had high prevalence rates, but the prevalence of E. faecalis was low. Multiplex PCR proved to be an adequate method for epidemiological studies.

Detection of selected bacterial species in intraoral sites of patients with chronic periodontitis using multiplex polymerase chain reaction

OBJECTIVE: The aim of this study was to detect the prevalence of selected bacterial species in intraoral sites of patients with chronic periodontitis (CP) using multiplex polymerase chain reaction (PCR). METHODOLOGY: Samples were collected from the tongue dorsum, buccal mucosa, supragingival and subgingival plaque and saliva of 30 patients with untreated CP. Multiplex PCR was used to determine prevalence rates, which were then compared using a chi-square test. Significance level was set at p<0.05. Mean and standard deviation values were used to evaluate variations in prevalence according to site. RESULTS: The prevalence of S. mutans was 70 percent in saliva; 60 percent in samples collected from the tongue dorsum; 50 percent in samples collected from the buccal mucosa; 56.5 percent in the supragingival plaque; and 53.5 percent in the subgingival plaque. The prevalence of E. faecalis ranged from 3.5 percent to 13.5 percent in all intraoral microenvironment. The highest prevalence of P. gingivalis was found in subgingival plaque (53.5 percent), and of P. intermedia in supragingival plaque (33.5 percent), subgingival plaque (30 percent) and tongue dorsum (33.5 percent). The prevalence of bacteria did not vary significantly among the intraoral sites. CONCLUSIONS: All studied bacteria were identified in intraoral sites. S. mutans, P. gingivalis and P. intermedia had high prevalence rates, but the prevalence of E. faecalis was low. Multiplex PCR proved to be an adequate method for epidemiological studies.

Microbial complexes in supragingival plaque.

BACKGROUND/AIMS: To examine microbial communities in supragingival biofilm samples. METHODS: Supragingival plaque samples were taken from 187 subjects at baseline (n = 4745). Fifty-five subjects provided supragingival plaque samples at 1-7 days after professional tooth cleaning (n = 1456); 93 subjects provided 8044 samples between 3 and 24 months post-therapy. All samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization. Microbial associations among species were sought using cluster analysis and community ordination techniques for the three groups separately. RESULTS: Six complexes were formed for the baseline samples. Similar complexes were formed for the samples taken 3-24 months post-therapy. However, distinct changes were observed in microbial communities in samples taken during the 7 days of plaque redevelopment. The complexes related to clinical parameters of periodontal disease. CONCLUSION: There were specific microbial complexes in supragingival plaque that were similar to those found in subgingival plaque samples with a few minor differences. The relation of previously unclustered taxa to the complexes was also described.

Genus Megamonas should be placed in the lineage of Firmicutes; Clostridia; Clostridiales; 'Acidaminococcaceae'; Megamonas.

Megamonas hypermegale is the sole species of the genus Megamonas included in the List of Prokaryotic Names with Standing in Nomenclature and in the databases of DDBJ, EBI/EMBL and NCBI/GenBank it is placed in the lineage of Bacteroidetes; Bacteroidetes (class); 'Bacteroidales'; Bacteroidaceae; Megamonas. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that this species clustered with species of the family 'Acidaminococcaceae' but not with those of the Bacteroidaceae. The genus Megamonas should be placed in the lineage of Firmicutes; Clostridia; Clostridiales; 'Acidaminococcaceae'; Megamonas.

Endosymbiotic Bacteroidales bacteria of the flagellated protist Pseudotrichonympha grassii in the gut of the termite Coptotermes formosanus.

A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.