Isolation and description of Selenomonas timonae sp. nov., a novel Selenomonas species detected in a gingivitis patient.

A Gram-stain-negative bacterium, designated strain Marseille-Q3039T, was isolated from subgingival dental plaque of a woman with gingivitis in Marseille, France. Strain Marseille-Q3039T was found to be an anaerobic, motile and spore-forming crescent-shaped bacterium that grew at 25-41.5 °C (optimum, 37 °C), pH 5.5-8.5 (optimum, pH 7.5) and salinity of 5.0 g l-1 NaCl. The results of 16S rRNA gene sequence analysis revealed that strain Marseille-Q3039T was closely related to Selenomonas infelix ATCC 43532T (98.42 % similarity), Selenomonas dianae ATCC 43527T (97.25 %) and Centipedia periodontii DSM 2778T (97.19 %). The orthologous average nucleotide identity and digital DNA-DNA hybridization relatedness between strain Q3039T and its closest phylogenetic neighbours were respectively 84.57 and 28.2 % for S. infelix ATCC 43532T and 83.93 and 27.2 % for C. periodontii DSM 2778T. The major fatty acids were identified as C13 : 0 (27.7 %), C15 : 0 (24.4 %) and specific C13 : 0 3-OH (12.3 %). Genome sequencing revealed a genome size of 2 351 779 bp and a G+C content of 57.2 mol%. On the basis of the results from phenotypic, chemotaxonomic, genomic and phylogenetic analyses and data, we concluded that strain Marseille-Q3039T represents a novel species of the genus Selenomonas, for which the name Selenomonas timonae sp. nov. is proposed (=CSUR Q3039=CECT 30128).

A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia.

The class Clostridia in the phylum Firmicutes (formerly low-G+C Gram-positive bacteria) includes diverse bacteria of medical, environmental and biotechnological importance. The Selenomonas-Megasphaera-Sporomusa branch, which unifies members of the Firmicutes with Gram-negative-type cell envelopes, was recently moved from Clostridia to a separate class Negativicutes. However, draft genome sequences of the spore-forming members of the Negativicutes revealed typically clostridial sets of sporulation genes. To address this and other questions in clostridial phylogeny, we have compared a phylogenetic tree for a concatenated set of 50 widespread ribosomal proteins with the trees for beta subunits of the RNA polymerase (RpoB) and DNA gyrase (GyrB) and with the 16S rRNA-based phylogeny. The results obtained by these methods showed remarkable consistency, suggesting that they reflect the true evolutionary history of these bacteria. These data put the Selenomonas-Megasphaera-Sporomusa group back within the Clostridia. They also support placement of Clostridium difficile and its close relatives within the family Peptostreptococcaceae; we suggest resolving the long-standing naming conundrum by renaming it Peptoclostridium difficile. These data also indicate the existence of a group of cellulolytic clostridia that belong to the family Ruminococcaceae. As a tentative solution to resolve the current taxonomical problems, we propose assigning 78 validly described Clostridium species that clearly fall outside the family Clostridiaceae to six new genera: Peptoclostridium, Lachnoclostridium, Ruminiclostridium, Erysipelatoclostridium, Gottschalkia and Tyzzerella. This work reaffirms that 16S rRNA and ribosomal protein sequences are better indicators of evolutionary proximity than phenotypic traits, even such key ones as the structure of the cell envelope and Gram-staining pattern.

Subgingival microbiome in smokers and non-smokers in periodontitis: an exploratory study using traditional targeted techniques and a next-generation sequencing.

AIM: To compare the results of two targeted techniques to an open-ended technique in periodontitis patients, differentiated on the basis of smoking habit. MATERIALS & METHODS: Thirty periodontitis patients (15 smokers and 15 non-smokers) provided subgingival plaque samples for 16S rRNA gene amplicon sequencing, culturing and quantitative polymerase chain reaction (qPCR). RESULTS: No differences were found in the composition of the subgingival microbiome between smokers and non-smokers with culture and qPCR. With pyrosequencing, operational taxonomic units (OTUs) classified to genera Fusobacterium, Prevotella and Selenomonas were more abundant in smokers, while OTUs belonging to the genera Peptococcus and Capnocytophaga were more abundant in non-smokers. Principal coordinate analysis identified two clusters; one was composed mainly of smokers (80%) and revealed significantly lower taxonomic diversity, higher attachment loss and higher proportion of the genera Fusobacterium, Paludibacter and Desulfobubus. CONCLUSION: In periodontitis, there is a difference in the composition of the subgingival microbiome between smokers and non-smokers, as revealed by pyrosequencing. This difference was not identified by the targeted techniques. Low taxonomic diversity was associated with higher disease severity, especially in smokers. This supports the hypothesis of the ecological microbial-host interaction in the severity of periodontal disease.

Pyrosequencing reveals unique microbial signatures associated with healthy and failing dental implants.

AIM: Although it is established that peri-implantitis is a bacterially induced disease, little is known about the bacterial profile of peri-implant communities in health and disease. The purpose of the present investigation was to examine the microbial signatures of the peri-implant microbiome in health and disease. MATERIALS AND METHODS: Subgingival and submucosal plaque samples were collected from forty subjects with periodontitis, peri-implantitis, periodontal and peri-implant health and analysed using 16S pyrosequencing. RESULTS: Peri-implant biofilms demonstrated significantly lower diversity than subgingival biofilms in both health and disease, however, several species, including previously unsuspected and unknown organisms, were unique to this niche. The predominant species in peri-implant communities belonged to the genera Butyrivibrio, Campylobacter, Eubacterium, Prevotella, Selenomonas, Streptococcus, Actinomyces, Leptotrichia, Propionibacterium, Peptococcus, Lactococcus and Treponema. Peri-implant disease was associated with lower levels of Prevotella and Leptotrichia and higher levels of Actinomyces, Peptococcus, Campylobacter, non-mutans Streptococcus, Butyrivibrio and Streptococcus mutans than healthy implants. These communities also demonstrated lower levels of Prevotella, non-mutans Streptococcus, Lactobacillus, Selenomonas, Leptotrichia, Actinomyces and higher levels of Peptococcus, Mycoplasma, Eubacterium, Campylobacter, Butyrivibrio, S. mutans and Treponema when compared to periodontitis-associated biofilms. CONCLUSION: The peri-implant microbiome differs significantly from the periodontal community in both health and disease. Peri-implantitis is a microbially heterogeneous infection with predominantly gram-negative species, and is less complex than periodontitis.

Microbial profiles in saliva from children with and without caries in mixed dentition.

OBJECTIVE: The purpose of this study was to determine the bacterial profiles in saliva of the isolated children for studying caries etiology. MATERIALS AND METHODS: Samples were collected from isolated children from 6 to 8years old including 20 caries-free (dmfs=0) (healthy) and 30 caries-active individuals (dmfs>8) (patients). 16S rRNA genes were amplified by PCR from bacterial DNA of saliva sample and labeled via incorporation of Cy3-dCTP in second nested PCR. After hybridization of labeled amplicons on HOMIM, the microarray slides were scanned and original data acquired from professional software. RESULTS: Collectively, 94 bacterial species or clusters representing six bacterial phyla and 30 genera were detected. A higher bacterial diversity was observed in patients than in healthy samples. Statistical analyses revealed eight species or clusters were detected more frequently in diseased patients than in healthy samples, while six different species were detected more frequently in healthy as compared to diseased patients. CONCLUSION: The diversity of microbe within saliva derived from isolated population increased in caries-active status, and there are some bacteria in salivary flora can be as candidate biomarkers for caries prognosis in mixed dentition. The imbalances in the resident microflora may be the ultimate mechanism of dental caries.

Molecular epidemiology and spatial distribution of Selenomonas spp. in subgingival biofilms.

The aetiology of periodontal disease has been a field of intensive research in the past decades. Along with a variety of other putative pathogens, different members of the genus Selenomonas have repeatedly been associated with both generalized aggressive periodontitis and chronic periodontitis. For the present study, a specific oligonucleotide probe targeting the majority of all oral Selenomonas spp. was designed. Their prevalence was determined, using dot-blot hybridization, in a total of 742 subgingival samples collected from patients with generalized aggressive (n=62) and chronic periodontitis (n=82), and from periodontitis-resistant subjects (n=19). In addition, fluorescence in situ hybridization (FISH) and electron microscopy were performed to analyze the spatial arrangement of Selenomonas in subgingival biofilms collected from patients with generalized aggressive periodontitis. In the samples from patients, Selenomonas spp. showed a lower prevalence in both diseased groups compared with other putative pathogens, and a relatively high prevalence in the periodontitis-resistant group. Consequently, Selenomonas spp. do not seem to be suitable diagnostic marker organisms for periodontal disease. By contrast, FISH and electron microscopic analysis of periodontal carriers revealed that Selenomonas spp. appeared in large numbers in all parts of the collected biofilms and seemed, if present in a site from patients, to make a relevant contribution to their structural organization.

Assessment of intraradicular bacterial composition by terminal restriction fragment length polymorphism analysis.

BACKGROUND: The aim of the study was to assess the bacterial community structures associated with endodontic infections using terminal restriction fragment length polymorphism (T-RFLP), and to investigate the correlation of whole community profiles with the manifestation of particular clinical features. METHODS: Intraradicular samples were collected from 34 subjects and classified into three study groups based on the observed clinical symptoms: acute (n = 16), sub-acute (n = 8), and asymptomatic (n = 10). Genomic DNA was extracted from each sample, submitted to polymerase chain reaction using a fluorescently labeled 16S ribosomal DNA forward primer, and digested with two tetrameric endonucleases (HhaI and MspI). The terminal restriction fragments (T-RFs) were subsequently discriminated in an automated DNA sequencer, and the results were filtered using a statistics-based criterion. RESULTS: Totals of 138 (HhaI) and 145 (MspI) unique T-RFs were detected (means 13.1 and 11.9) and there was high inter-subject variability in the bacterial assemblages. Odds-ratio analysis unveiled the existence of higher order groups of positively associated T-RFs, restating the concept that intricate ecological relationships may take place in the root canal space. A significantly greater T-RF prevalence was detected in acute cases, suggesting a straight correlation between species richness and spontaneous pain. CONCLUSION: Overall, no T-RFLP profile representing a specific bacterial consortium could be associated with the manifestation of symptoms of endodontic origin.

Comparisons of subgingival microbial profiles of refractory periodontitis, severe periodontitis, and periodontal health using the human oral microbe identification microarray.

BACKGROUND: This study compared the subgingival microbiota of subjects with refractory periodontitis (RP) to those in subjects with treatable periodontitis (GRs = good responders) or periodontal health (PH) using the Human Oral Microbe Identification Microarray (HOMIM). METHODS: At baseline, subgingival plaque samples were taken from 47 subjects with periodontitis and 20 individuals with PH and analyzed for the presence of 300 species by HOMIM. The subjects with periodontitis were classified as having RP (n = 17) based on mean attachment loss (AL) and/or more than three sites with AL >or=2.5 mm after scaling and root planing, surgery, and systemically administered amoxicillin and metronidazole or as GRs (n = 30) based on mean attachment gain and no sites with AL >or=2.5 mm after treatment. Significant differences in taxa among the groups were sought using the Kruskal-Wallis and chi(2) tests. RESULTS: More species were detected in patients with disease (GR or RP) than in those without disease (PH). Subjects with RP were distinguished from GRs or those with PH by a significantly higher frequency of putative periodontal pathogens, such as Parvimonas micra (previously Peptostreptococcus micros or Micromonas micros), Campylobacter gracilis, Eubacterium nodatum, Selenomonas noxia, Tannerella forsythia (previously T. forsythensis), Porphyromonas gingivalis, Prevotella spp., Treponema spp., and Eikenella corrodens, as well as unusual species (Pseudoramibacter alactolyticus, TM7 spp. oral taxon [OT] 346/356, Bacteroidetes sp. OT 272/274, Solobacterium moorei, Desulfobulbus sp. OT 041, Brevundimonas diminuta, Sphaerocytophaga sp. OT 337, Shuttleworthia satelles, Filifactor alocis, Dialister invisus/pneumosintes, Granulicatella adiacens, Mogibacterium timidum, Veillonella atypica, Mycoplasma salivarium, Synergistes sp. cluster II, and Acidaminococcaceae [G-1] sp. OT 132/150/155/148/135) (P <0.05). Species that were more prevalent in subjects with PH than in patients with periodontitis included Actinomyces sp. OT 170, Actinomyces spp. cluster I, Capnocytophaga sputigena, Cardiobacterium hominis, Haemophilus parainfluenzae, Lautropia mirabilis, Propionibacterium propionicum, Rothia dentocariosa/mucilaginosa, and Streptococcus sanguinis (P <0.05). CONCLUSION: As determined by HOMIM, patients with RP presented a distinct microbial profile compared to patients in the GR and PH groups.

The Microbiome in Populations with a Low and High Prevalence of Caries.

The oral microbiota was compared between Romanian adolescents with a high prevalence of caries and no dental care and Swedish caries-active and caries-free adolescents in caries prevention programs and with a low prevalence of caries. Biofilm samples were analyzed by FLX+ pyrosequencing of the V1 to V4 hypervariable regions of the 16S rRNA gene and polymerase chain reaction (PCR)/quantitative PCR (qPCR) for Streptococcus mutans and Streptococcus sobrinus. Sequences obtained blasted to 9 phyla, 66 genera, and 401 human oral taxa (HOT) in the 16S rRNA Human Oral Microbiome Database, of which 295 were represented by ≥20 sequences. The Romanian adolescents had more sequences in Firmicutes and fewer in Actinobacteria phyla and more sequences in the genera Bacteroidetes [G-3], Porphyromonas, Abiotrophia, Filifactor, Peptostreptococcaceae [11][G-4], Pseudoramibacter, Streptococcus, and Neisseria and fewer in Actinomyces, Selenomonas, Veillonella, Campylobacter, and TM7 [G-1] than the Swedish groups. Multivariate modeling employing HOT, S. sobrinus and S. mutans (PCR/qPCR), and sugar snacks separated Romanian from Swedish adolescents. The Romanian adolescents' microbiota was characterized by a panel of streptococci, including S. mutans, S. sobrinus, and Streptococcus australis, and Alloprevotella, Leptotrichia, Neisseria, Porphyromonas, and Prevotella. The Swedish adolescents were characterized by sweet snacks, and those with caries activity were also characterized by Prevotella, Actinomyces, and Capnocytophaga species and those free of caries by Actinomyces, Prevotella, Selenomonas, Streptococcus, and Mycoplasma. Eight species including Streptococcus mitis and Streptococcus species HOT070 were prevalent in Romanian and Swedish caries-active subjects but not caries-free subjects. In conclusion, S. mutans and S. sobrinus correlated with Romanian adolescents with caries and with limited access to dental care, whereas S. mutans and S. sobrinus were detected infrequently in Swedish adolescents in dental care programs. Swedish caries-active adolescents were typically colonized by Actinomyces, Selenomonas, Prevotella, and Capnocytophaga. Hence, the role of mutans streptococci as a primary caries pathogen appears less pronounced in populations with prevention programs compared to populations lacking caries treatment and prevention strategies.

Complete genome sequence of Selenomonas ruminantium subsp. lactilytica will accelerate further understanding of the nature of the class Negativicutes.

Selenomonas ruminantium subsp. lactilytica, a strictly anaerobic ruminal bacterium, possesses typical Gram-negative cell surface structure comprising cytoplasmic membrane, peptidoglycan layer and outer membrane, whereas its 16S rRNA-based taxonomy shows that the bacteria belongs to Gram-positive Firmicutes. Complete genome analysis showed that genes or gene clusters involved in Gram-negative cell structure were scattered in the S. ruminantium genome, and might provide the new insight of phylogenetic relationship between the bacterium and other bacterial species.

Restriction and modification systems of ruminal bacteria.

A high frequency of type II restriction endonuclease activities was detected in Selenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated from S. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected in S. ruminantium strains as well as in several other species belonging to the Sporomusa subbranch of low G + C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).

Molecular basis for the maintenance of envelope integrity in Selenomonas ruminantium: cadaverine biosynthesis and covalent modification into the peptidoglycan play a major role.

Polyamine is a small organic polycation composed of a hydrocarbon backbone with multiple amino groups which ubiquitously exists in all living organisms from bacteria to higher animals. The critical step of polyamine biosynthesis generally includes the amino acid-decarboxylating reaction to produce the primary diamines, such as a synthesis of putrescine (NH(3)(+)·(CH(2))(4)·NH(3)(+)) from ornithine, and cadaverine (NH(3)(+)·(CH(2))(5)·NH(3)(+)) from lysine, which are catalyzed by pyridoxal-5'-phosphate (PLP; vitamin B(6))-dependent decarboxylases. Synthesized polyamines are implicated in a wide variety of cytoplasmic reactions such as DNA replication and protein synthesis, and are essential for proper growth of the organisms. However, in Selenomonas ruminantium, a strictly anaerobic Gram-negative bacterium dominant in sheep rumen, cadaverine displays its function in a quite distinctive scheme compared to the general bacteria reported. It serves as an essential constituent of the peptidoglycan for the maintenance of envelope integrity through an interaction with the periplasm-exposed SLH domain of Mep45, the outer membrane protein of this bacterium. Furthermore, cytoplasmic biosynthesis of cadaverine occurs totally in a eukaryotic-like manner rather than in a conventional way of bacteria. Lysine/ornithine decarboxylase (LDC/ODC), a PLP-dependent enzyme responsible for cadaverine synthesis in this bacterium, displays significant homology to the eukaryotic ODC but not to the general bacterial LDC nor ODC, and its activity is tightly regulated by antizyme-mediated proteolysis, a regulatory process generally found in eukaryotes. These findings represent the biological diversity of this bacterium beyond the preexisting knowledge related to the polyamine-physiology, cell envelope-architecture, and the regulatory system for the enzyme. In this review we will describe (i) the cadaverine-containing peptidoglycan of S. ruminantium: its chemical structure, biosynthesis, and biological function, and (ii) cellular biosynthesis of cadaverine by LDC/ODC and its antizyme-mediated regulation. In addition, we will briefly refer to (iii) the phylogenetic position and characteristics of S. ruminantium and its unique cadaverine-physiology.

The subgingival microbiota of Papillon-Lefèvre syndrome.

BACKGROUND: There is little information about the microbiologic profiles of periodontal lesions in Papillon-Lefèvre syndrome (PLS) and the significance of bacteria in the pathogenesis of periodontitis in these patients. This comprehensive analysis of the subgingival microbiota in patients with PLS used 16S ribosomal RNA (rRNA) clonal analysis and the 16S rRNA-based Human Oral Microbe Identification Microarray (HOMIM). METHODS: Thirteen patients with PLS from seven unrelated families volunteered for this microbiologic study. Subgingival plaque was collected with sterile paper points from multiple sites with ≥5 mm probing depth, and whole genomic DNA was extracted. The 16S rRNA genes were amplified, cloned, and sequenced. The samples were then probed for ≈300 predominant oral bacterial species using the HOMIM. RESULTS: The most commonly detected phylotypes in the clonal analysis were Gemella morbillorum, Gemella haemolysans, Granulicatella adiacens, Lachnospiraceae OT 100 (EI074), Parvimonas micra, Selenomonas noxia, and Veillonella parvula. As a group, streptococci were commonly detected in these individuals. In the HOMIM analysis, a total of 170 bacterial species/phylotypes were detected, with a range of 40 to 80 species per patient with PLS. Of these, 12 bacterial species were detected in medium to high levels in ≥50% of the individuals. The high-frequency strains were clustered into eight groups: Aggregatibacter actinomycetemcomitans, Campylobacter spp., Capnocytophaga granulosa, G. morbillorum, P. micra, Porphyromonas endodontalis, Streptococcus spp., and Tannerella forsythia. CONCLUSIONS: The subgingival microbiota in PLS is diverse. Periodontal pathogens commonly associated with chronic and aggressive periodontitis and opportunistic pathogens may be associated with the development of severe periodontitis in patients with PLS.

Bacterial composting of animal fleshing generated from tannery industries.

Animal fleshing (ANFL) is the major proteinaceous solid waste generated during the manufacture of leather, which requires to be disposed of by environmentally sound manner. This study reports about the treatment of ANFL into an organic compost and its effects on physiological parameters of different crops in a laboratory study. The ANFL was hydrolysed using Selenomonas ruminantium HM000123 and then the hydrolysed ANFL was mixed with cow dung and leaf litter for producing composted organic fertilizer (COF). The COF was characterized for pH, electrical conductivity (EC), total Kjeldhal nitrogen (TKN), and total organic carbon (TOC). The composting resulted in a significant reduction in pH, TOC and C:N ratio and an increase in TKN after 49 days in a compost reactor. Scanning electron microscope and FT-IR were used to analyse the hydrolysis of intra structural ANFL matrix and changes in the functional groups, respectively, in initial and final day COF. Thermogravimetry (TG) analysis was carried out for the raw mixture and COF samples to identify the weight loss under the nitrogen environment. The relative seed germination was found to be 94% in tomato (Lycopersicon esculentum), 92% in green gram (Vigna radiata), 86% in bottle gourd (Lagenaria siceraria (Mol.) Standl.) and 84% in cucumber (Cucumis sativus L.) using the extracts of COF. The results indicate that the combination of both hydrolysis and bacterial composting reduced the overall time required for composting and producing a nutrient-enriched compost product.

Selenomonas bovis sp. nov., isolated from yak rumen contents.

Two strictly anaerobic, crescent-shaped bacterial strains, designated WGT and Ycb08, were isolated from a cellulose-degrading mixed culture enriched from yak rumen contents. The strains were Gram-negative, non-spore-forming and motile, with four to six flagella situated at the centre of the concave side of the cell. The cells were 0.9-1.1x4-6 microm. Growth was observed at 27-46 degrees C (optimum 39 degrees C) and pH 4.2-8.3 (optimum pH 7.0-7.2). Arabinose, glucose, mannose, cellobiose, lactose, sucrose, trehalose, melibiose, raffinose, salicin and aesculin were fermented. The end products of glucose fermentation were acetate, propionate and CO2. The G+C contents of strains WGT and Ycb08 were respectively 63.9+/-0.2 and 62.5+/-0.2 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains were related to the genera Mitsuokella and Selenomonas at similarity levels below 97%; however, they differed from members of the genus Mitsuokella in their flagellar arrangement. On the basis of phenotypic, genotypic and physiological evidence, strains WGT and Ycb08 are identified as members of a novel species of the genus Selenomonas, for which the name Selenomonas bovis sp. nov. is proposed. The type strain is WGT (=CGMCC 1.5073T=JCM 15470T).

Beta-D-xylosidase from Selenomonas ruminantium of glycoside hydrolase family 43.

Beta-D-xylosidase from the ruminal anaerobic bacterium, Selenomonas ruminantium (SXA), catalyzes hydrolysis of beta-1,4-xylooligosacharides and has potential utility in saccharification processes. The enzyme, heterologously produced in Escherichia coli and purified to homogeneity, has an isoelectric point of approx 4.4, an intact N terminus, and a Stokes radius that defines a homotetramer. SXA denatures between pH 4.0 and 4.3 at 25 degrees C and between 50 and 60 degrees C at pH 5.3. Following heat or acid treatment, partially inactivated SXA exhibits lower k(cat) values, but similar K(m) values as untreated SXA. D-glucose and D-xylose protect SXA from inactivation at high temperature and low pH.

Profiling of bacterial flora in crevices around titanium orthodontic anchor plates.

OBJECTIVES: The aims of this study were to characterize the microflora in crevices around titanium orthodontic anchor plates using anaerobic culture and molecular biological techniques for bacterial identification, and to compare the microbial composition between crevices around anchor plates and gingival crevices. MATERIAL AND METHODS: Samples from crevices around titanium anchor plates and healthy gingival crevices of 17 subjects (aged 20-29) were cultured anaerobically, and isolated bacteria were identified by 16S rRNA sequencing. RESULTS: The average logarithm colony-forming units/ml were 6.84, 7.51 and 8.88 in healthy anchor plate crevices, inflamed anchor plate crevices and healthy gingival crevices, respectively, indicating that the bacterial density of anchor plate crevices was lower than that of healthy gingival crevices. Of 184 strains isolated from healthy anchor plate crevices of seven subjects, 108 (59%) were anaerobic bacteria, while 73 (40%) were facultative bacteria. Predominant isolates were Gram-negative rods, such as Campylobacter (12%), Fusobacterium (10%) and Selenomonas (10%), and Gram-positive facultative bacteria, such as Actinomyces (17%) and Streptococcus (8.2%). Of 133 strains isolated from inflamed anchor plate crevices of three subjects, 110 (83%) were anaerobic bacteria, while predominant isolates were Gram-negative rods, such as Prevotella (47%), Fusobacterium (33%) and Campylobacter (16%). On the other hand, of 146 strains isolated from healthy gingival crevices of seven subjects, 98 (67%) were facultative bacteria, while 45 (31%) were anaerobic bacteria. Predominant isolates were Gram-positive facultative bacteria, such as Actinomyces (37%) and Streptococcus (20%). CONCLUSIONS: These results suggest that the environment in crevices around titanium orthodontic anchor plates is anaerobic and supportive of anaerobic growth of bacteria, which may trigger inflammation in the tissue around the plates.

Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets.

INTRODUCTION: Molecular ecological analysis based on 16S rRNA gene sequence analysis is well established for the characterisation of complex bacterial communities. However, 'universal' PCR primers can introduce biases into the analysis of the species composition of clone libraries because of mismatches between the primer and target organism sequences. In this study, three universal primer sets were compared for the analysis of the microflora in subgingival plaque. METHODS: Three subgingival plaque samples were collected from two subjects with localised severe chronic periodontitis. Half of each sample was cultured while DNA was extracted from the remaining half and 16S rDNA amplified with universal primer pairs 27F, 1525R (A); 27F, 1492R (B) and 530F, 1525R (C). Amplified genes were cloned, sequenced and identified by comparison with 16S rRNA databases. RESULTS: 137 taxa were identified among 177 isolates and 417 clones sequenced. Of these, 86 were detected only by the molecular technique whereas 26 were found only by culture. Sequences from 81 taxa did not correspond to those of named species and of these, 38 were not represented in the nucleotide databases. 16S RNA genes for these 38 taxa were sequenced and deposited with GenBank. CONCLUSION: The use of three sets of universal primers allowed the identification of 38 novel bacterial phylotypes. There were marked differences in the composition of the libraries generated by the different primer sets. A combination of molecular and cultural techniques is recommended to maximise the coverage of detection of bacterial taxa in oral samples.

Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing.

It is well recognized that a dynamic biofilm develops upon plant biomass in the herbivore gastrointestinal tract, but this component of the microbiome has not previously been specifically sampled, or directly compared with the biodiversity present in the planktonic fraction of digesta. In this study, the digesta collected from four sheep fed two different diets was separated into three fractions: the planktonic phase, and the microbial populations either weakly or tightly adherent to plant biomass. The community DNA prepared from each fraction was then subjected to both ribosomal intergenic spacer analysis (RISA) and denaturing gradient gel electrophoresis (DGGE). Both types of analysis showed that dietary factors influence community structure, and that the adherent fractions produced more complex profiles. The RIS-clone libraries prepared from the planktonic and adherent populations were then subjected to restriction fragment length polymorphism (RFLP) and DNA sequence analyses, which resulted in a far greater degree of discrimination among the fractions. Although many of the sequenced clones from the adherent populations were assigned to various clusters within the low G+C Gram-positive bacteria, the clone libraries from animals consuming an all-grass diet were largely comprised of novel lineages of Clostridium, while in animals consuming the starch-containing diet, Selenomonas and Ruminococcus spp. were the dominant low G+C Gram-positive bacteria. Additionally, the libraries from hay-fed animals also contained clones most similar to asaccharolytic Clostridia, and other Gram-positive bacteria that specialize in the transformation of plant phenolic compounds and the formation of cinnamic, phenylacetic and phenylpropionic acids. These results reveal, for the first time, the phylogeny of adherent subpopulations that specialize in the transformation of plant lignins and other secondary compounds, which potentiate polysaccharide hydrolysis by other members of the biofilm.

Phylogenetic characterization of Centipeda periodontii, Selenomonas sputigena and Selenomonas species by 16S rRNA gene sequence analysis.

The nearly complete 16S rRNA gene sequences for oral Gram-negative anaerobic motile bacteria, Centipeda periodontii, Selenomonas sputigena and Selenomonas species (formerly S. sputigena type strain), were determined in order to unveil their relationship to other oral motile bacteria. To determine the phylogenetic characterization of these bacteria, their 16S rRNA gene sequences were obtained and compared with those from the ribosomal sequence databases previously reported. The 16S rRNA gene sequences of these bacteria were similar to those of Selenomonas ruminantium and Schwartzia succinivorans isolated from rumens, and to Pectinatus cerevisiiphilus isolated from spoiled beer. Among oral bacteria, the nucleotide sequence analysis of these bacteria revealed high nucleotide similarity to Veillonella species, whereas low similarity to oral motile bacteria such as Campylobacter species. Phylogenetic analysis clearly confirmed that C. periodontii and two Selenomonas species were classified as relatives of a group besides Selenomonas, Schwartzia, and Pectinatus species, and not as close relatives to oral motile bacteria, such as Campylobacter species. These results suggest that such oral Gram-negative anaerobic motile bacteria are close relatives of oral bacteria.