Roseovarius pelagicus sp. nov., a facultatively anaerobic bacterium with potential for degrading polypropylene, isolated from Arctic seawater.

A Gram-stain-negative, facultatively anaerobic, non-motile, rod-shaped bacterial strain, designated HL-MP18T, was isolated from Arctic seawater after a prolonged incubation employing polypropylene as the sole carbon source. Phylogenetic analyses of the 16S rRNA gene sequence revealed that strain HL-MP18T was affiliated to the genus Roseovarius with close relatives Roseovarius carneus LXJ103T (96.8 %) and Roseovarius litorisediminis KCTC 32327T (96.5 %). The complete genome sequence of strain HL-MP18T comprised a circular chromosome of 3.86 Mbp and two circular plasmids of 0.17 and 0.24 Mbp. Genomic comparisons based on average nucleotide identity and digital DNA-DNA hybridization showed that strain HL-MP18T was consistently discriminated from its closely related taxa in the genus Roseovarius. Strain HL-MP18T showed optimal growth at 25 °C, pH 7.0 and 2.5 % (w/v) sea salts. The major cellular fatty acids were C18 : 1 ω6c and/or C18 : 1 ω7c (49.6 %), C19 : 0 cyclo ω8c (13.5 %), and C16 : 0 (12.8 %). The major respiratory quinone was ubiquinone-10. The polar lipids consisted of phosphatidylcholine, phosphatidylglycerol, an unidentified aminolipid and three unidentified lipids. The genomic DNA G+C content of the strain was 59.2 mol%. The phylogenetic, genomic, phenotypic and chemotaxonomic results indicate that strain HL-MP18T is distinguishable from the recognized species of the genus Roseovarius. Therefore, we propose that strain HL-MP18T represents a novel species belonging to the genus Roseovarius, for which the name Roseovarius pelagicus sp. nov. is proposed. The type strain is HL-MP18T (=KCCM 90405T=JCM 35639T).

Antimicrobial susceptibility pattern of oral gram negative anaerobes from Indian subjects.

OBJECTIVES: There is paucity of information on the antimicrobial susceptibility pattern of oral anaerobic bacteria. In this study, an attempt has been made to evaluate the antimicrobial susceptibility/resistance trend of oral Gram negative bacteria from Indian subjects. METHODS: Minimum inhibitory concentrations (MIC) of 304 isolates against twelve different antibiotics were determined using gradient diffusion MIC strips. The organisms were isolated and identified based on phenotypic characteristics and included Porphyromonas gingivalis, Prevotella species, Tannerella forsythia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcoitans, Eickenella corrodens and Capnocytophaga species. For each antimicrobial agent, MIC50 and MIC90 were calculated and expressed. RESULTS: Resistance to azithromycin, clindamycin, and amoxicillin was observed in most of the anaerobic bacterial species studied. High degree of susceptibility was observed to amoxillin-clavulanic acid, doxycycline and moxifloxacin. A single strain of P. melaninogenica was resistant to moxifloxacin. The susceptibility pattern varied with cephalosporins among species. Ceftriaxone showed highest and cefazolin least efficacy among cephalosporins. All anaerobic bacteria tested were susceptible to metronidazole. Strains of T. forsythia were more resistant to several antibiotics than other anaerobic bacteria. All three species of capnophilic bacteria displayed high degree of resistance to metronidazole and significant resistance to amoxicillin, azithromycin, clindamycin, cefazolin and cefuroxime. CONCLUSIONS: Amoxicillin-clavulanic acid, doxycycline, moxifloxacin and metronidazole appeared to be the most effective drugs against gram negative anaerobic bacteria. However, the MIC50 and MIC90 values against metronidazole were on the higher side of the normal indicating a potential for developing resistance.

Multiple factors drive the abundance and diversity of the diazotrophic community in typical farmland soils of China.

Biological nitrogen fixation plays an important role in nitrogen cycling by transferring atmospheric N2 to plant-available N in the soil. However, the diazotrophic activity and distribution in different types of soils remain to be further explored. In this study, 152 upland soils were sampled to examine the diazotrophic abundance, nitrogenase activity, diversity and community composition by quantitative polymerase chain reaction, acetylene reduction assay and the MiSeq sequencing of nifH genes, respectively. The results showed that diazotrophic abundance and nitrogenase activity varied among the three soil types. The diazotrophic community was mainly dominated by Bradyrhizobium, Azospirillum, Myxobacter, Desulfovibrio and Methylobacterium. The symbiotic diazotroph Bradyrhizobium was widely distributed among soils, while the distribution of free-living diazotrophs showed large variation and was greatly affected by multiple factors. Crop type and soil properties directly affected the diazotrophic ɑ-diversity, while soil properties, climatic factors and spatial distance together influenced the diazotrophic community. Network structures were completely different among all three types of soils, with most complex interactions observed in the Red soil. These findings suggest that diazotrophs have various activities and distributions in the three soil types, which played different roles in nitrogen input in agricultural soil in China, being driven by multiple environmental factors.

A Prospective Evaluation of Two Rapid Phenotypical Antimicrobial Susceptibility Technologies for the Diagnostic Stewardship of Sepsis.

Rapid identification of bloodstream pathogens by MALDI-TOF MS and the recently introduced rapid antimicrobial susceptibility testing (rAST) directly from positive blood cultures allow clinicians to promptly achieve a targeted therapy, especially for multidrug resistant microorganisms. In the present study, we propose a comparison between phenotypical rASTs performed in light-scattering technology (Alfred 60AST, Alifax®) and fluorescence in situ hybridization (Pheno™, Accelerate) directly from positive blood cultures, providing results in 4-7 hours. Blood samples from 67 patients admitted to the Azienda Ospedaliero-Universitaria Pisana were analyzed. After the direct MALDI-TOF MS identification, the rAST was performed at the same time both on Alfred 60AST and Pheno. Alfred 60AST provided qualitative results, interpreted in terms of clinical categories (SIR). Pheno provided identification and MIC values for each antibiotic tested. Results were compared to the broth microdilution assay (SensiTitre™, Thermo Fisher Scientific), according to EUCAST rules. Using Alfred 60AST, an agreement was reached, 91.1% for Gram-negative and 95.7% for Gram-positive bacteria, while using Pheno, the agreement was 90.6% for Gram-negative and 100% for Gram-positive bacteria. Both methods provided reliable results; Alfred 60AST combined with MALDI-TOF MS proved itself faster and cheaper. Pheno provided identification and MIC determination in a single test and, although more expensive, may be useful whenever MIC value is necessary and where MALDI-TOF MS is not present.

Abditibacterium utsteinense sp. nov., the first cultivated member of candidate phylum FBP, isolated from ice-free Antarctic soil samples.

Most bacterial lineages are known only by molecular sequence data from environmental surveys and represent the uncultivated majority. One of these lineages, candidate phylum FBP, is widespread in extreme environments on Earth, ranging from polar and desert ecosystems to wastewater and contaminated mine sites. Here we report on the characterization of strain LMG 29911T, the first cultivated representative of the FBP lineage. The strain was isolated from a terrestrial surface sample from Utsteinen, Sør Rondane Mountains, East Antarctica and is a Gram-negative, aerobic, oligotrophic chemoheterotrophic bacterium. It displays growth in a very narrow pH range, use of only a limited number of carbon sources, but also a metabolism optimized for survival in low-nutrient habitats. Remarkably, phenotypic and genome analysis indicated an extreme resistance against antibiotics and toxic compounds. We propose the names Abditibacterium utsteinense for this bacterium and Abditibacteriota for the former candidate phylum FBP. Furthermore, inter- and intra-phylum relationships indicate Armatimonadetes, a neighboring lineage to the Abditibacteriota, to be a superphylum.

Description of a novel pectin-degrading bacterial species Prevotella pectinovora sp. nov., based on its phenotypic and genomic traits.

Five strictly anaerobic Gram-negative bacterial strains, P4-65, P4-76(T), P5-60, P5-119, and P5-125, presumably belonging to the genus Prevotella were isolated from pig fecal samples. Strains were tested for various phenotypic traits and nearcomplete genome sequences were obtained and analyzed. Phylogenetic analysis based on 16S rRNA gene sequences and multilocus sequence analysis based on five conserved genes confirmed that the strains belong to the genus Prevotella, revealing that they represent a novel and discrete lineage distinct from other known species of this genus. The size of the genome of the isolated strains is 3-3.3 Mbp, and the DNA G+C content is 47.5-48.1 mol%. The isolates are strictly anaerobic, rod-shaped with rounded ends, non-motile and non-spore-forming. The main fermentation products are succinate and acetate, with minor concentrations of isovalerate, propionate and isobutyrate. Hydrogen is also produced. Major cellular fatty acids consist of anteiso-C(15:0) and iso-C(15:0), and a number of additional acids are present in lower concentrations. A substantial portion of genes involved in carbohydrate utilization is devoted to pectin degradation and utilization, while those supporting growth on xylan in ruminal Prevotella could not have been revealed. On the basis of the presented results, a novel species, Prevotella pectinovora sp. nov. is proposed. The type strain is P4-76(T) (=DSM 29996(T) =ZIM B1020(T)).

Characterization of Sporohalobacter salinus sp. nov., an anaerobic, halophilic, fermentative bacterium isolated from a hypersaline lake.

Halophilic, obligately anaerobic, Gram-stain-negative bacterial strains were isolated from a sediment sample taken from under the salt crust of El-Jerid hypersaline lake in southern Tunisia by using tryptone or glucose as the substrate. One strain, CEJFT1B(T), was characterized phenotypically and phylogenetically. Cells were non-motile, non-spore-forming, short rods. Strain CEJFT1B(T) was able to grow in the presence of 5-30 % (w/v) NaCl (optimum 20 %) and at 30-60 °C (optimum 45 °C). It grew at pH 5.5-7.8 and the optimum pH for growth was 6.8. The isolate required yeast extract for growth. Substrates utilized by strain CEJFT1B(T) as the sole carbon source included glucose, fructose, sucrose, pyruvate, Casamino acids and starch. Individual amino acids such as glutamate, lysine, methionine, serine, tyrosine, and amino acid mixtures formed by the Stickland reaction such as alanine-glycine, valine-proline, leucine-proline, isoleucine-proline were also utilized. Products of glucose fermentation were acetate (major product), butyrate, H2 and CO2. The genomic DNA G+C content of strain CEJFT1B(T) was 32.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CEJFT1B(T) should be assigned to the genus Sporohalobacter. The sequence similarity between strain CEJFT1B(T) and Sporohalobacter lortetii was 98.5 %, but DNA-DNA hybridization between the two strains revealed a relatedness value of 56.4 %, indicating that they are not related at the species level. The combination of phylogenetic analysis, DNA-DNA hybridization data, and differences in substrate utilization support the view that strain CEJFT1B(T) represents a novel species of the genus Sporohalobacter, for which the name Sporohalobacter salinus sp. nov. is proposed. The type strain is CEJFT1B(T) ( = DSM 26781(T) = JCM 19279(T)).

The subgingival microbiome of clinically healthy current and never smokers.

Dysbiotic oral bacterial communities have a critical role in the etiology and progression of periodontal diseases. The goal of this study was to investigate the extent to which smoking increases risk for disease by influencing the composition of the subgingival microbiome in states of clinical health. Subgingival plaque samples were collected from 200 systemically and periodontally healthy smokers and nonsmokers. 16S pyrotag sequencing was preformed generating 1,623,713 classifiable sequences, which were compared with a curated version of the Greengenes database using the quantitative insights into microbial ecology pipeline. The subgingival microbial profiles of smokers and never-smokers were different at all taxonomic levels, and principal coordinate analysis revealed distinct clustering of the microbial communities based on smoking status. Smokers demonstrated a highly diverse, pathogen-rich, commensal-poor, anaerobic microbiome that is more closely aligned with a disease-associated community in clinically healthy individuals, suggesting that it creates an at-risk-for-harm environment that is primed for a future ecological catastrophe.

Newly identified pathogens associated with periodontitis: a systematic review.

There is substantial evidence supporting the role of certain oral bacteria species in the onset and progression of periodontitis. Nevertheless, results of independent-culture diagnostic methods introduced about a decade ago have pointed to the existence of new periodontal pathogens. However, the data of these studies have not been evaluated together, which may generate some misunderstanding on the actual role of these microorganisms in the etiology of periodontitis. The aim of this systematic review was to determine the current weight of evidence for newly identified periodontal pathogens based on the results of "association" studies. This review was conducted and reported in accordance with the PRISMA statement. The MEDLINE, EMBASE, and Cochrane databases were searched up to September 2013 for studies (1) comparing microbial data of subgingival plaque samples collected from subjects with periodontitis and periodontal health and (2) evaluating at least 1 microorganism other than the already-known periodontal pathogens. From 1,450 papers identified, 41 studies were eligible. The data were extracted and registered in predefined piloted forms. The results suggested that there is moderate evidence in the literature to support the association of 17 species or phylotypes from the phyla Bacteroidetes, Candidatus Saccharibacteria, Firmicutes, Proteobacteria, Spirochaetes, and Synergistetes. The phylum Candidatus Saccharibacteria and the Archaea domain also seem to have an association with disease. These data point out the importance of previously unidentified species in the etiology of periodontitis and might guide future investigations on the actual role of these suspected new pathogens in the onset and progression of this infection.

Utilidad de la espectrometría de masas MALDI-TOF en la identificación de bacterias anaerobias / Utility of MALDI-TOF MS for the identification of anaerobic bacteria

El análisis de espectrometría de masas mediante la metodología hoy conocida como MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry) se ha convertido en un recurso de referencia para la identificación de microorganismos en microbiología clínica. No obstante, los datos relativos a algunos grupos de microorganismos son todavía controvertidos. El objetivo del presente estudio fue determinar la utilidad del MALDI-TOF MS para la identificación de aislamientos clínicos de bacterias anaerobias. Se analizaron 106 aislamientos de bacterias anaerobias mediante MALDI-TOF MS y por pruebas bioquímicas convencionales. En aquellos casos en los que la identificación por metodología convencional no era aplicable o frente a una discordancia de resultados entre las metodologías citadas, se realizó la secuenciación del gen 16S del ARNr. El método convencional y el MALDI-TOF MS coincidieron a nivel de género y especie en un 95,3 % de los casos considerando la totalidad de los aislamientos estudiados. Al considerar solo el conjunto de los bacilos gram negativos, la coincidencia fue del 91,4 %; entre los bacilos gram positivos, fue del 100 %; los 8 aislados de cocos gram positivos estudiados coincidieron y también hubo coincidencia en el único coco gram negativo incluido. Los datos obtenidos en este estudio demuestran que el MALDI-TOF MS ofrece la posibilidad de llegar a una adecuada identificación de bacterias anaerobias

The analysis by MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry) has become a reference method for the identification of microorganisms in Clinical Microbiology. However, data on some groups of microorganisms are still controversial. The aim of this study is to determine the utility of MALDI-TOF MS for the identification of clinical isolates of anaerobic bacteria. One-hundred and six anaerobic bacteria isolates were analyzed by MALDI-TOF MS and by conventional biochemical tests. In those cases where identification by conventional methodology was not applicable or in the face of discordance between sequencing methodologies, 16 S rRNA gene sequence analysis was performed. The conventional method and MALDI-TOF MS agreed at genus and species level by 95.3 %. Concordance in gram-negative bacilli was 91.4% and 100% among gram-positive bacilli; there was also concordance both in the 8 isolates studied in gram-positive cocci and in the single gram-negative cocci included. The data obtained in this study demonstrate that MALDI-TOF MS offers the possibility of adequate identification of anaerobic bacteria

Kosmotoga pacifica sp. nov., a thermophilic chemoorganoheterotrophic bacterium isolated from an East Pacific hydrothermal sediment.

A novel strictly anaerobic thermophilic heterotrophic bacterium, strain SLHLJ1(T), was isolated from a Pacific hydrothermal sediment. Cells were Gram-negative coccobacilli (approximately 1.0 × 0.6 µm) with a toga. It grew at temperatures between 33 and 78 °C (optimum 70 °C). Elemental sulphur and L-cystine stimulated its growth. It contained C16:0, C16:1 ω11c, C18:0 and C18:1 ω9c as major fatty acids (>5%), 3 phospholipids and 2 glycolipids as polar lipids. Its DNA G+C content was 43.7 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLHLJ1(T) within the family Thermotogaceae. The novel isolate was most closely related to Kosmotoga arenicorallina (97.93 % 16S rRNA gene sequence similarity), K. olearia (92.43%) and K. shengliensis (92.17 %). On the basis of phenotypic, chemotaxonomic and phylogenetic comparisons with its closest relatives, we propose its assignment to a novel species of the genus Kosmotoga. The name Kosmotoga pacifica sp. nov. is proposed with strain SLHLJ1(T) (=DSM 26965(T) = JCM 19180(T) = UBOCC 3254(T)) as the type species.

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.

Patient-specific analysis of periodontal and peri-implant microbiomes.

Periodontally involved teeth have been implicated as 'microbial reservoirs' in the etiology of peri-implant diseases. Therefore, the purpose of this investigation was to use a deep-sequencing approach to identify the degree of congruence between adjacent peri-implant and periodontal microbiomes in states of health and disease. Subgingival and peri-implant biofilm samples were collected from 81 partially edentulous individuals with periodontal and peri-implant health and disease. Bacterial DNA was isolated, and the 16S rRNA gene was amplified and sequenced by pyrotag sequencing. Chimera-depleted sequences were compared against a locally hosted curated database for bacterial identification. Statistical significance was determined by paired Student's t tests between tooth-implant pairs. The 1.9 million sequences identified represented 523 species. Sixty percent of individuals shared less than 50% of all species between their periodontal and peri-implant biofilms, and 85% of individuals shared less than 8% of abundant species between tooth and implant. Additionally, the periodontal microbiome demonstrated significantly higher diversity than the implant, and distinct bacterial lineages were associated with health and disease in each ecosystem. Analysis of our data suggests that simple geographic proximity is not a sufficient determinant of colonization of topographically distinct niches, and that the peri-implant and periodontal microbiomes represent microbiologically distinct ecosystems.

Microbial analysis in primary and persistent endodontic infections by using pyrosequencing.

INTRODUCTION: The aim of this study was to investigate the bacterial community profile of intracanal microbiota in primary and persistent endodontic infections associated with asymptomatic chronic apical periodontitis by using GS-FLX Titanium pyrosequencing. The null hypothesis was that there is no difference in diversity of overall bacterial community profiles between primary and persistent infections. METHODS: Pyrosequencing analysis from 10 untreated and 8 root-filled samples was conducted. RESULTS: Analysis from 18 samples yielded total of 124,767 16S rRNA gene sequences (with a mean of 6932 reads per sample) that were taxonomically assigned into 803 operational taxonomic units (3% distinction), 148 genera, and 10 phyla including unclassified. Bacteroidetes was the most abundant phylum in both primary and persistent infections. There were no significant differences in bacterial diversity between the 2 infection groups (P > .05). The bacterial community profile that was based on dendrogram showed that bacterial population in both infections was not significantly different in their structure and composition (P > .05). CONCLUSIONS: The present pyrosequencing study demonstrates that persistent infections have as diverse bacterial community as primary infections.

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.

Synergistetes cluster A in saliva is associated with periodontitis.

BACKGROUND AND OBJECTIVES: Synergistetes is a novel bacterial phylum consisting of gram-negative anaerobes. Increasing lines of evidence demonstrate that this phylum is associated with periodontal diseases. This study aimed to compare the presence and levels of Synergistetes clusters A and B, in saliva of patients with chronic periodontitis (CP), generalized aggressive periodontitis (G-AgP) and non-periodontitis subjects, and investigate their correlation with clinical parameters. MATERIAL AND METHODS: Saliva was collected from patients with CP (n = 20), G-AgP (n = 21) and non-periodontitis subjects (n = 18). Full mouth clinical periodontal measurements were recorded. The numbers of Synergistetes cluster A and cluster B or the associated species Jonquetella anthropi were quantified by fluorescent in situ hybridization and microscopy. RESULTS: Synergistetes cluster A bacteria were detected more frequently, and at higher numbers and proportions in the two periodontitis groups, than the non-periodontitis control group. The prevalence was 27.7% in the control group, 85% in CP and 86% in G-AgP. Compared to the control group, the numbers were significantly higher by 12.5-fold in CP and 26.5-fold in G-AgP, whereas the difference between the two forms of periodontitis was not statistically significant. Within the total bacterial population, the proportion of this cluster was increased in CP and G-AgP compared to the control group, with the difference between the two forms of periodontitis being also significant. There was a positive correlation between the levels of Synergistetes cluster A in saliva and all full mouth clinical periodontal parameters. Nevertheless, Synergistetes cluster B bacteria and J. anthropi species were detected infrequently and at low levels in all the three subject groups. CONCLUSION: Synergistetes cluster A, but not cluster B, bacteria are found at higher prevalence, numbers and proportions in saliva from patients with periodontitis, than non-periodontitis subjects. These findings support the association of this cluster with periodontitis.

Symptomatic and asymptomatic apical periodontitis associated with red complex bacteria: clinical and microbiological evaluation.

In this study, the association of red complex (RC) bacteria that include Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis with acute, exacerbated or chronic apical periodontitis was evaluated. Seventy-one patients with periapical disease were evaluated by clinical examination and microbiological samples obtained from the root canals were analyzed by a polymerase chain reaction assay. Twenty-one (29.6%) samples were positive for RC bacteria, with T. denticola, T. forsythia and P. gingivalis being detected in 14 (19.7%), 10 (14.1%) and 6 (8.5%) samples, respectively. RC bacteria were mainly associated with acute apical periodontitis (29.2%) and phoenix abscess (63.2%), while they were only sporadically detected (7.1%) in patients with chronic apical periodontitis. Generally, RC bacteria were associated with pain and a higher frequency of intracanalar/intrasulcular pus drainage. Involvement of RC bacteria in symptomatic periapical disease should be suspected in the presence of particularly severe clinical pain and pus drainage.

Prevalence and diversity of Synergistetes taxa in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Members of the phylum Synergistetes have previously been identified within periodontitis subgingival plaque and are considered putative periodontopathogens. This study compared the diversity of subginigval Synergistetes in a cohort of subjects with periodontitis (n = 10) vs. periodontitis-free controls (n = 10). MATERIAL AND METHODS: Pooled subgingival plaque samples from all deep periodontal pockets or all sulci were collected from the periodontitis and periodontitis-free subjects, respectively. Bacterial 16S rRNA genes were PCR-amplified from purified subgingival plaque DNA using a Synergistetes 'selective' primer set. PCR products were cloned and sequenced to analyze the prevalence and diversity of Synergistetes operational taxonomic units (OTUs) present in plaque samples of both subject groups. RESULTS: A total of 1030 non-chimeric 16S rRNA clones were obtained, of which 162 corresponded to members of the phylum Synergistetes. A significantly larger number of Synergistetes clones were obtained from periodontitis subgingival plaque than from periodontitis-free controls (25.4% vs. 5.9%, p < 0.001). All Synergistetes clones corresponded to cluster A oral Synergistetes, and fell into 31 OTUs (99% sequence identity cut-off). Twenty-nine Synergistetes OTUs were detected in the periodontitis group while eight were detected in the periodontitis-free group (p < 0.001). Five Synergistetes OTUs; including one OTU corresponding to the recently-characterized species Fretibacterium fastidiosum, were more prevalent in the periodontitis subjects (p < 0.05). CONCLUSION: OTUs belonging to oral Synergistetes cluster A were more readily detectable and were more diverse in subgingival plaque from periodontitis subjects compared with periodontitis-free controls. Specific Synergistetes OTUs appear to be associated with periodontitis.