Prevotella Oralis Research Articles

Efficacy of virginiamycin for the control of periodontal disease in calves

ABSTRACT: Periodontal diseases are multifactorial infectious processes caused by complexes of microorganisms, with damage to health, production, and animal welfare. The aim of the present study was to evaluate the efficacy of virginiamycin in the prevention and control of two early forms of periodontal disease: gingivitis and necrotizing gingivitis. Ten weaned calves, aged four to six months, were permanently kept in a single lot under the same rotational grazing regime in a newly reformed area of Panicum maximum. Five of the calves were orally administered 340mg of virginiamycin (Virginiamycin Group) daily for a period of 18 weeks, while the remaining five calves (Control Group) remained under the same food management but did not receive virginiamycin. During this period, animals underwent 18 weekly evaluations regarding periodontal health, with monitoring and recording of clinical parameters of the eight deciduous incisor teeth on the labial and lingual faces. At approximately two-week intervals, nine collections of subgingival sulcus material from five sites of the four right incisor teeth of each animal were performed and subjected to microbiological evaluation using polymerase chain reaction with primers of 25 microorganisms considered potentially pathogenic. After 1440 periodontal clinical evaluations of incisor teeth of the 10 calves, a total of 395 episodes of gingivitis were recorded, of which 267 occurred in the Control Group and 128 in the Virginiamycin Group. Similarly, 89 episodes of necrotizing gingivitis were recorded; 58 in the Control Group and 31 in the Virginiamycin Group. Comparison of between-group means found significant differences for teeth with gingivitis and necrotizing gingivitis (t test; p<0.05). The total number of teeth with gingivitis (p<0.01) and necrotizing gingivitis (p<0.01) in Control Group was significantly higher than that of gingivitis (p<0.01) and necrotizing gingivitis (p<0.05) in the Virginiamycin Group. There was a positive correlation between total occurrence of gingivitis and necrotizing gingivitis in the Virginiamycin Group by Pearson’s test. Virginiamycin had a protective effect on treated animals compared with the Control Group (OR = 0.36: CI (95%) = 0.27-0.43). In the Control Group, Actinomyces israelli (4.74%), domain Archaea (1.58%), Eikenella corrodens (1.05%), Fusobacterium nucleatum (27.37%), class Mollicutes (5.26%); Porphyromonas endodontalis(5.26%); Porphyromonas gulae(0.53%), Prevotella buccae (6.32%), Prevotella loescheii (3.68%), Prevotella nigrescens (8.42%), Prevotella oralis (1.58%), Tannerella forsythia (0.53%), and Treponema denticola (4.21%) were detected at healthy sites, and gingivitis or necrotizing gingivitis samples. In the Virginiamycin Group, A. israelli (3.41%), domain Archaea (0.98%), F. nucleatum (9.27%), class Mollicutes(4.39%), P. endodontalis (4.39%), P. gulae (0.49%), P. buccae (8.29%), P. loescheii (6.83%), P. nigrescens (15.61%), P. oralis (1.46%), Selenomonas sputigena (0.49%), T. forsythia (0.49%), and T. denticola (2.44%) were detected. In conclusion, virginiamycin administered at a dosage of 340mg/animal/day significantly reduced the occurrence of gingivitis and necrotizing gingivitis in cattle maintained on reformed pastures, and was revealed to have action against periodontal bacterial microbiota considered to be potentially pathogenic.

Gut Microbiota and Body Weight in School-Aged Children: The KOALA Birth Cohort Study.

ObjectiveThis study aimed to examine the intestinal microbiota composition of school‐aged children in association with (over)weight.MethodsThe fecal microbiota composition of 295 children was analyzed using the Human Intestinal Tract Chip. Anthropometric outcomes (overweight [BMI ≥ 85th percentile], age‐ and sex‐standardized BMI and weight z scores) were measured at 6 to 7 years of age, and elastic net was used to select genus‐like bacterial groups related to all anthropometric outcomes. Subsequently, multiple linear and logistic regression models were used to model associations between selected bacterial groups and anthropometric measures while controlling for confounders.Results Prevotella melaninogenica, Prevotella oralis, Dialister, and uncultured Clostridiales II (UCII) accounted for 26.1% of the variation in microbiota composition. Several bacterial groups were inversely associated with the anthropometric outcomes: Sutterella wadsworthensis, Marvinbryantia formatexigens, Prevotella melanogenica, P oralis, Burkholderia, uncultured Clostridiales II, and Akkermansia, while Streptococcus bovis was positively associated with overweight. Microbial diversity and richness, and Bacteroidetes to Firmicutes ratio, were not significantly associated with any of the outcomes.ConclusionsIn the largest population‐based study on childhood gut microbiota and body weight so far, both new and previously identified bacterial groups were found to be associated with overweight. Further research should elucidate their role in energy metabolism.

Wrażliwość bakterii beztlenowych na australijski olejek mirtowy (Backhousia citriodora F. Muell.)

Introduction. Herbs have been known and utilize since ancient Times in China, Europe and America. The ancient Egiptians used the myrtle to treat fever and infections. The plant have been mentioned in the Bible six times. Dioscorides recommended myrtle oil on patients with blader and lung infections. The herb and essential oil were used in Ayuveric treating infections respiratory tract and neuralgic. Backhousia citriodora F. Muell. belonging to the genus Backhousia and family Myrtaceae. It grown to 20-30 m high, on plantations in Australia, in subtropical rainforests of central and south-eastern Queensland. The leaves of the tree Backhousia citriodora produces an essential oil. It is obtained via hydro distillation. Lemon myrtle oil is known for its characteristic lemon flavor, related with presence of citral. Research indicated, that constituents of oil showed antibacterial activity against aerobic bacteria. There are no information about its activity towards anaerobic bacteria. Aim. The aim of the study was to determine activity of myrtle oil against anaerobic bacteria isolated from patients. Material and methods. The anaerobic bacteria were isolated from various infections of oral cavity. A total 135 strains isolated from patients and 7 reference strains were tested. The suscibility anaerobes to myrtle oil was carried out using plate dilution technique in Brucella agar supplemented with 5% defibrynated sheep blood, menadione and hemin. Inoculum containing 106 in CFU per spot was seeded with Steers replicator upon the surface of agar with myrtle oil and without oil (strains growth control). Incubation the plates was performed in anaerobic jars (10% C02 , 10% H2 , 80% N2 , palladic catalyst and anaerobic indicator) at 37°C for 48 hrs. The MIC was defined as the lowest concentrations of the myrtle oil that completely inhibited growth the tested anaerobic bacteria. Results. The results indicated that the myrtle oil was active against the 44 (50%) from all the tested strains of anaerobes to the concentrations from ranges < 0.12-1.0 mg/ml. The rods from genus of Prevotella oralis and Prevotella oris were sensitive on concentrations < 0.12 mg/ml. The oil was less active against the Gram-negative anaerobes from genus of Prevotella buccalis, Prevotella levii, Prevotella loescheii, Bacteroides uniformis and Parabacteroides distasonis (MIC > 4.0 mg/ml). The Clostridial strains were very sensitive. The growth of this strains was inhibited by concentrations of < 0.12 mg/ml. The volatile oil did appeared to be equally effective against both Gram-negative and Gram-positive anaerobic rods. The growth of 50 and 47% of this bacteria was inhibited in ranges from < 0.12 to 1.0 mg/ml. The tested Gram-negative cocci from genus Veillonella parvula were less sensitive (MIC 1.0-> 4.0 mg/ml). The strains of Gram-positive cocci were the least sensitive. Its growth was inhibited by concentrations 2.0-> 4.0 mg/ml. Conclusions. The myrtle oil was more active against anaerobic bacteria from genus Prevotella oralis, Prevotella oris and strains from genera of Clostridium. The strains from genus Prevotella buccalis, Prevotella levii, Prevotella loescheii, Bacteroides uniformis and Parabacteroides distasonis were the lowest sensitive. The oil was equally effective against Gram-negative and Gram-positive anaerobic bacteria.

Investigation of microbial community associated with mediastinitis by 16S ribosomal DNA sequencing

Here, we document the case of mediastinitis in an adult individual, developed upon contiguous spread of oesophageal infection. Our findings are noteworthy, because the presence of Prevotella oris , P. veroralis , Dialister pneumosintes , Klebsiella pneumoniae in tissue sample from mediastinum cannot be identified by standard culturing and Gram-staining. Importantly, however, 16S rDNA sequencing did lead to an unambiguous diagnosis and guided successful antimicrobial therapy.

Analysis of Oral Microbiota Revealed High Abundance of Prevotella Intermedia in Gout Patients

Background/Aims: Microbes reside in a number of body sites, including the oral cavity, and are associated with the progression of many systemic diseases. In this study, we aimed to investigate the effects of gout and hyperuricemia (HUA) on the composition of oral microbiomes. Methods: Analysis of the oral microbiota from 12 gout patients, 11 HUA patients, and 19 healthy control subjects was performed using a deep sequencing approach, and validation of significant changes in Prevotella intermedia and Serratia marcescens in new patient cohorts was performed using quantitative PCR (qPCR). Results: Our analysis indicated that both gout and HUA significantly altered the composition of the oral microbiome in patients. Patients with gout or HUA had significantly greater levels of salivary Prevotella intermedia but significantly lower levels of Serratia marcescens than healthy control subjects. Conclusion: We demonstrated the association between the oral microbiome and gout and HUA for the first time. In particular, 16S sequencing and qPCR analysis revealed significantly higher levels of oral Prevotella intermedia in gout/HUA patients, which suggests that these patients might be at risk for the development of periodontitis.

Biomarkers and Bacteria Around Implants and Natural Teeth in the Same Individuals

This cross-sectional study assesses cytokine levels in peri-implant crevicular fluid (PICF)/gingival crevicular fluid (GCF) and a selection of subgingival/submucosal plaque bacteria from clinically healthy or diseased sites in the same individuals. Samples from 97 implants/teeth (58 implants [19 healthy, 20 mucositis, 19 peri-implantitis] and 39 natural teeth [19 healthy, 12 gingivitis, eight periodontitis] in 15 systemically healthy patients were investigated by immunoassay and real-time polymerase chain reaction. Samples were obtained first, with probing depth, clinical attachment level, bleeding on probing, plaque index scores, and keratinized tissue width then recorded. Data were analyzed by Wilcoxon, Mann-Whitney U, and permutation tests on dependent, independent, and mixed dependent and independent samples and Spearman correlation. Interleukin (IL)-1β levels were significantly higher in PICF samples of healthy implants than in GCF samples of healthy teeth (P = 0.003), and soluble receptor activator of nuclear factor-κB ligand (sRANKL) concentrations were significantly higher in the gingivitis than the mucositis group (P = 0.004). Biomarker levels were similar in peri-implantitis and periodontitis groups (P >0.05). Actinomyces naeslundi and Streptococcus oralis levels were significantly higher in the healthy implant group than in healthy teeth (P <0.05). Prevotella intermedia and Treponema denticola (Td) levels were lower in the mucositis group than the gingivitis group (P <0.05). Prevotella oralis and S. oralis levels were significantly higher in the periodontitis group (P <0.05), and Td levels were significantly higher in the peri-implantitis group (P <0.05). There were many similarities but, crucially, some differences in biomarker levels (IL-1β and sRANKL) and bacterial species between peri-implant and periodontal sites in the same individuals, suggesting similar pathogenic mechanisms.

Performance of MALDI-TOF MS for identification of oral Prevotella species

During the past decade, the clinically relevant genus Prevotella has expanded considerably. Prevotella species can be isolated from nearly all types of oral infections but also from various non-oral infections. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced in clinical microbiology laboratories as a convenient method for identifying bacterial isolates from clinical specimens. Here we tested the diagnostic accuracy of a total of 123 oral Prevotella isolates, selected based on their biochemical profile, by Bruker MALDI-TOF MS. Partial 16S rRNA sequencing was used as a reference method. The performance of MALDI-TOF MS to identify the isolates to the genus level was excellent with 100.0% accuracy, while a good identification rate of 88.6% was achieved to the species level with a log score of ≥2.0. The isolates representing P. aurantiaca and P. jejuni, which are currently missing from the MALDI BioTyper database, were identified correctly to the genus level. Of the 123 isolates, one P. pallens isolate (0.8%) was identified with a score variation of 1.7–1.999. Overall, biochemical testing produced a high proportion (70.7%) of incorrect identifications within different species. MALDI-TOF MS offers a reliable and rapid method for the identification of Prevotella species included in the database.

Black-pigmented anaerobic bacteria associated with ovine periodontitis

Periodontitis is a polymicrobial infectious disease that causes occlusion change, tooth loss, difficulty in rumination, and premature culling of animals. This study aimed to detect species of the genera Porphyromonas and Prevotella present in the periodontal pocket of sheep with lesions deeper than 5mm (n=14) and in the gingival sulcus of animals considered periodontally healthy (n=20). The presence of microorganisms was evaluated by polymerase chain reaction (PCR) using specific primers for Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gulae, Prevotella buccae, Prevotella intermedia, Prevotella loescheii, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, and Prevotella tannerae. Prevalence and risk analysis were performed using Student’s t-test and Spearman’s correlation. Among the Prevotella and Porphyromonas species detected in the periodontal lesions of sheep, P. melaninogenica (85.7%), P. buccae (64.3%), P. gingivalis (50%), and P. endodontalis (50%) were most prevalent. P. gingivalis (15%) and P. oralis (10%) prevailed in the gingival sulcus. P. gulae and P. tannerae were not detected in the 34 samples studied. Data evaluation by t-test verified that occurrence of P. asaccharolytica, P. endodontalis, P. gingivalis, P. buccae, P. intermedia, P. melalinogenica, and P. nigrescens correlated with sheep periodontitis. The findings of this study will be an important contribution to research on pathogenesis of sheep periodontitis and development of its control measures.

Comparative pan genome analysis of oral Prevotella species implicated in periodontitis.

Prevotella is part of the oral bacterial community implicated in periodontitis. Pan genome analyses of eight oral Prevotella species, P. dentalis, P. enoeca, P. fusca, P. melaninogenica, P. denticola, P. intermedia 17, P. intermedia 17-2 and P. sp. oral taxon 299 are presented in this study. Analysis of the Prevotella pan genome revealed features such as secretion systems, resistance to oxidative stress and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems that enable the bacteria to adapt to the oral environment. We identified the presence of type VI secretion system (T6SS) in P. fusca and P. intermedia strains. For some VgrG and Hcp proteins which were not part of the core T6SS loci, we used gene neighborhood analysis and identified putative effector proteins and putative polyimmunity loci in P. fusca and polymorphic toxin systems in P. intermedia strains. Earlier studies have identified the presence of Por secretion system (PorSS) in P. gingivalis, P. melaninogenica and P. intermedia. We noted the presence of their homologs in six other oral Prevotella studied here. We suggest that in Prevotella, PorSS is used to secrete cysteine proteases such as interpain and C-terminal domain containing proteins with a "Por_secre_tail" domain. We identified subtype I-B CRISPR-Cas system in P. enoeca. Putative CRISPR-Cas system subtypes for 37 oral Prevotella and 30 non-oral Prevotella species were also predicted. Further, we performed a BLASTp search of the Prevotella proteins which are also conserved in the red-complex pathogens, against the human proteome to identify potential broad-spectrum drug targets. In summary, the use of a pan genome approach enabled identification of secretion systems and defense mechanisms in Prevotella that confer adaptation to the oral cavity.

Plain x-ray films in soft tissue infections.

A 39-year-old male with a history of cocaine abuse presents to the emergency room with pain in the right leg and fever (39oC) 6 days after injecting himself in the thigh with midazolam pills diluted in tap water to control symptoms of withdrawal. Physical examination revealed rubor, swelling and tenderness of the thigh without crepitation, suggestive of cellulitis. Laboratory findings consisted of a WBC of 10.700x106/L (86% Neutrophiles) and CRP of 170 mg/dL. An X-ray of the leg was performed, revealing a feathering pattern of gas within the soft tissue of the thigh indicative of gas gangrene. Intraoperatively, we found wide necrosis of the muscles and fatty tissue of the posterior thigh without substantial quantities of pus. An extensive debridement was performed and penicillin and clindamycin were administered for two weeks. Cultural exams revealed the presence of aerobic (Streptococcus mitis and Streptococcus oralis) and anaerobic bacteria (Clostridium clostridioforme and Prevotella oralis). The infection was controlled after further daily debridement and the wound was closed with the assistance of a negative-pressure wound therapy device. The patient was discharged from the hospital to a physical rehabilitation facility and regained most of his functional capacity. Gas gangrene is a life-threatening condition, misdiagnosis and failure of recognition in its early stages can prove fatal. Plain X-ray films are readily available in most hospitals and can provide significant information in patients with soft tissue infections where 'time is tissue'.

Investigation of microbial community associated with mediastinitis by 16S ribosomal DNA sequencing

Here, we document the case of mediastinitis in an adult individual, developed upon contiguous spread of oesophageal infection. Our findings are noteworthy, because the presence of Prevotella oris , P. veroralis , Dialister pneumosintes , Klebsiella pneumoniae in tissue sample from mediastinum cannot be identified by standard culturing and Gram-staining. Importantly, however, 16S rDNA sequencing did lead to an unambiguous diagnosis and guided successful antimicrobial therapy.

Noncontiguous finished genome sequence and description of Prevotella phocaeensis sp. nov., a new anaerobic species isolated from human gut infected by Clostridium difficile.

Prevotella phocaeensis sp. nov. strain SN19T (= DSM 103364) is a new species isolated from the gut microbiota of patient with colitis due to Clostridium difficile. Strain SN19T is Gram-negative rod-shaped bacteria, strictly anaerobic, nonmotile and non–endospore forming. The predominance fatty acid is hexadecanoic acid. Its 16S rRNA showed a 97.70% sequence identity with its phylogenetically closest species, Prevotella oralis. The genome is 2 922 117 bp long and contains 2486 predicted genes including 56 RNA genes.

Microbial Ecosystem Analysis in Root Canal Infections Refractory to Endodontic Treatment

IntroductionThe purpose of this study was to combine multiple displacement amplification and checkerboard DNA-DNA hybridization to qualitatively and quantitatively evaluate the microbiota present in infections refractory to endodontic treatment. MethodsThe subjects of this study were 40 patients presenting with periapical lesions refractory to endodontic treatment. Samples were taken by scraping or filing root canal walls with a #10 K-type hand file. Sample DNA was amplified by multiple displacement amplification, and the levels of 107 bacterial taxa were analyzed by checkerboard DNA-DNA hybridization. The taxa were divided into 3 distinct microbial populations depending on their mean proportion in samples (% DNA probe counts ± standard error of the mean) as follows: dominant (≥3.0%), subdominant (>1.6%–3.0%), and residual (≤1.6%) populations. The significance of differences was determined using the Mann-Whitney test. ResultsThe taxa present with the highest mean proportions (constituting the dominant population) were Corynebacterium diphtheriae (8.03 ± 0.98), Porphyromonas gingivalis (5.42 ± 2.09), Streptococcus sobrinus (5.33 ± 0.69), and Stenotrophomonas maltophilia (4.72 ± 1.73). Among the subdominant population were Eubacterium saphenum (3.85 ± 1.06), Helicobacter pylori (3.16 ± 0.62), Dialister pneumosintes (3.12 ± 1.1), Clostridium difficile (2.74 ± 0.41), Enterobacter agglomerans (2.64 ± 0.54), Salmonella enterica (2.51 ± 0.52), Mobiluncus mulieris (2.44 ± 0.6), and Klebsiella oxytoca (2.32 ± 0.66). In the population of bacteria present at the lowest mean proportions (the residual population), Bacteroides ureolyticus (0.04 ± 0.01), Haemophilus influenzae (0.04 ± 0.02), and Prevotella oris (0.01 ± 0.01) were found at the lowest mean proportions. Enterococcus faecalis was detected in the residual population (0.52 ± 0.26). ConclusionsThe microbial climax community in teeth refractory to endodontic treatment not only harbors medically important species but also contains distinct microbial consortia present with different population levels.

Atypical Lemierre's syndrome caused by Prevotella oris

Lemierre's syndrome is a rare form of severe sepsis secondary to oropharyngeal infection characterized by septic thrombophlebitis of the internal jugular vein with disseminated infections, with the lungs being the most frequent target. We report the case of a previously healthy 15-year-old admitted to the intensive care unit because of septic shock, brain and lung abscesses, and a positive blood culture for Prevotella oris with a clinical presentation compatible with the diagnosis of Lemierre's syndrome. This is an atypical presentation of Lemierre's syndrome because of the isolated microorganism.

Non-alcoholic fatty liver disease (NAFLD) is associated with a distinct fecal microbiome signature – a case control study

Background: Nonalcoholic fatty liver disease is now being recognized as the most common liver disorder worldwide. The majority of NAFLD patients are characterized by mere liver steatosis but up to one third progresses to non-alcoholic steatohepatitis (NASH). The underlying pathophysiology is poorly understood but changes in the gut microbiome have been suggested to be involved. Design: Using a case control design we recruited 84 subjects with liver steatosis and 83 controls from the population-based Study of Health in Pommerania. Subjects with diabetes mellitus, BMI > 25 kg/m2, immoderate alcohol intake or gallstone disease were excluded. Liver fat content was quantitated by confounder corrected chemical shift encoded MRI sequence at 1.5T. Cases with steatosis were defined as subjects with a mean liver fat content of 24.9% and controls with 2.2%. NAFLD and NASH were distinguished by using the FIB-4 score (Cut-off 1.3). Phylogenetic profiling of fecal samples was performed using the Human intestinal tract Chip (HITChip). For phenotypic correlation of the gut microbiome signature up to 224 host variables, including diet, were available and 38 reached significance. Results: By study design the extent of steatosis on liver MRI differed significantly between cases and controls (p < 10 – 6). Hierarchical clustering showed a clustering tendency. Random Forrest analysis revealed 69%± 14% 95CI classification accuracy on 130 genus-level taxa. Diet did not affect the classification accuracy. Reduced Shannon diversity (p = 0.046) and richness (p = 0.007) in cases were detected. PCA cluster analysis identified 4 out of 130 taxa discriminating between cases and controls (Prevotella oralis and P. melaninogenica, Sutterella wadsworthia, Uncultured Clostridiales) all of those with bimodal distribution. NASH cases showed a significantly increased abundancy of Gram-positive taxa as well as several Bacteroides spp. that could be used as a classifier. Conclusion: In the absence of metabolic syndrome NAFLD is associated with a distinct gut microbiome signature, which is unaffected by diet. Decreased abundancy of taxa, previously defined as tipping elements, points to a pathophysiological relevance. Progression to NASH is correlated with additional distinct changes in the microbiome.

Bacteremia due to anaerobic bacteria: epidemiology in a northern Bari Hospital, Italy

&lt;em&gt;Background&lt;/em&gt;. Anaerobic bacteria are part of the commensal bacterial flora of skin and mucosae. Iatrogenic and pathological conditions altering this commensal relationship cause life-threatening diseases. &lt;br /&gt;&lt;em&gt;Materials&lt;/em&gt; &lt;em&gt;and&lt;/em&gt; &lt;em&gt;Methods&lt;/em&gt;. We analysed the blood cultures sent to the microbiology of our hospital between 2008 and the first quarter of 2013 to measure the frequency of bacteraemia caused by anaerobia. We examined 3138 vials of blood cultures for anaerobia, inoculated following in-house standard procedures. The colonies grown in absence of air were subjected to biochemical analysis. The MICs of metronidazole for 23 of the 26 organisms was tested. &lt;br /&gt;&lt;em&gt;Results&lt;/em&gt;. Twelve bacteria of the Bacteroides genus were identified, 9 &lt;em&gt;Propionibacterium&lt;/em&gt; &lt;em&gt;acnes&lt;/em&gt;, 1 &lt;em&gt;Peptosctreptococcus&lt;/em&gt; &lt;em&gt;micros&lt;/em&gt;, 1 &lt;em&gt;Lactobacillus&lt;/em&gt; &lt;em&gt;acidophilus&lt;/em&gt;, 1 &lt;em&gt;Clostridium&lt;/em&gt; &lt;em&gt;perfringens&lt;/em&gt;, 1 &lt;em&gt;Prevotella&lt;/em&gt; &lt;em&gt;oralis&lt;/em&gt;, 1 &lt;em&gt;Eubacterium&lt;/em&gt; &lt;em&gt;lentum&lt;/em&gt;. &lt;br /&gt;&lt;em&gt;Conclusions&lt;/em&gt;. The analysis of the results suggests that the incidence of cultures positive to anaerobia was constant across the years. We note that advanced age, altered mucocutaneous tropism, alterations to the oral and intestinal bacterial flora intensify the risk of anaerobial pathogenicity. The analysis of the metronidazole-determined MIC suggests that the intestinal anaerobic flora responds well to therapy and prophylaxis with Metronidazole, while the anaerobic bacteria residing on skin and other mucosae are resistant. It is however hard to determine the clinical impact of anaerobic bacteremiae and their effect on the outcome of the patient, due to the scarcity of available clinical data.

PTU-200 Why do crohn’s and idiopathic anal fistulae persist?

<h3>Introduction</h3> An interaction between genetic, microbiological and immunological factors drives Crohn’s disease. Bacterial infection probably initiates idiopathic anal fistulae but does not maintain them. Dendritic cells (DC) express Toll-Like receptors (TLR), which are pattern recognition receptors that are activated by bacterial ligands. We compared the expression of TLRs and microbiota profiles of CD and idiopathic fistulae. <h3>Method</h3> Biopsies were taken from 35 Crohn’s and 25 idiopathic fistulae at surgery. DC were identified as HLA-DR-positive and lineage-negative, and characterised by flow cytometry using fresh samples. The expression of TLR2 and 4 was determined. Immunohistochemical techniques determined the expression of TLR2, TLR4 and TLR9 on paraffin-imbedded biopsies. DNA was extracted from frozen samples and bacterial 16S rRNA genes were sequenced using a MiSeq sequencer. <h3>Results</h3> TLR2 and 4 were expressed on DC from both Crohn’s and idiopathic perianal fistulae using flow cytomtery. There was no significant difference in TLR2 (p = 0.27) or TLR4 (p = 0.45) expression on CD and idiopathic fistulae. Immunohistochemistry showed equal expression of TLR2 (p = 0.42) and TLR4 (p = 0.11) on lymphocytes. TLR9 expression was significantly higher in CD fistulae (p = 0.01). Microbiota were classified as common and abundant, infrequent, and rare. The total number of operational taxonomic units (OTUs) observed and the number of species identified in Crohn’s fistulae was significantly higher than idiopathic (p = 0.02). The most abundant species in the Crohn’s group were Bradyrhizobium pachyrhizi followed by Pseudomonas azotoformans and Prevotella oris. <h3>Conclusion</h3> Bacterial products and the local immune response to them are present in perianal fistula tracts. Fistula persistence may be driven by bacterial products rather than live bacteria. This could provide therapeutic treatment targets for Crohn’s anal fistulae. <h3>Disclosure of interest</h3> None Declared.

Development and pyrosequencing analysis of an in-vitro oral biofilm model.

BackgroundDental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers.ResultsPyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms.ConclusionsDiverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0364-1) contains supplementary material, which is available to authorized users.

Polymicrobial Purulent Pericarditis Probably caused by a Broncho-Lymph Node-Pericardial Fistula in a Patient with Tuberculous Lymphadenitis.

Purulent pericarditis is a rare condition with a high mortality rate. We report a case of purulent pericarditis subsequently caused by Candida parapsilosis, Peptostreptococcus asaccharolyticus, Streptococcus anginosus, Staphylococcus aureus, Prevotella oralis, and Mycobacterium tuberculosis in a previously healthy 17-year-old boy with mediastinal tuberculous lymphadenitis. The probable route of infection was a bronchomediastinal lymph node-pericardial fistula. The patient improved with antibiotic, antifungal, and antituberculous medication in addition to pericardiectomy.

Robust species taxonomy assignment algorithm for 16S rRNA NGS reads: application to oral carcinoma samples

BackgroundUsefulness of next-generation sequencing (NGS) in assessing bacteria associated with oral squamous cell carcinoma (OSCC) has been undermined by inability to classify reads to the species level.ObjectiveThe purpose of this study was to develop a robust algorithm for species-level classification of NGS reads from oral samples and to pilot test it for profiling bacteria within OSCC tissues.MethodsBacterial 16S V1-V3 libraries were prepared from three OSCC DNA samples and sequenced using 454's FLX chemistry. High-quality, well-aligned, and non-chimeric reads ≥350 bp were classified using a novel, multi-stage algorithm that involves matching reads to reference sequences in revised versions of the Human Oral Microbiome Database (HOMD), HOMD extended (HOMDEXT), and Greengene Gold (GGG) at alignment coverage and percentage identity ≥98%, followed by assignment to species level based on top hit reference sequences. Priority was given to hits in HOMD, then HOMDEXT and finally GGG. Unmatched reads were subject to operational taxonomic unit analysis.ResultsNearly, 92.8% of the reads were matched to updated-HOMD 13.2, 1.83% to trusted-HOMDEXT, and 1.36% to modified-GGG. Of all matched reads, 99.6% were classified to species level. A total of 228 species-level taxa were identified, representing 11 phyla; the most abundant were Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria. Thirty-five species-level taxa were detected in all samples. On average, Prevotella oris, Neisseria flava, Neisseria flavescens/subflava, Fusobacterium nucleatum ss polymorphum, Aggregatibacter segnis, Streptococcus mitis, and Fusobacterium periodontium were the most abundant. Bacteroides fragilis, a species rarely isolated from the oral cavity, was detected in two samples.ConclusionThis multi-stage algorithm maximizes the fraction of reads classified to the species level while ensuring reliable classification by giving priority to the human, oral reference set. Applying the algorithm to OSCC samples revealed high diversity. In addition to oral taxa, a number of human, non-oral taxa were also identified, some of which are rarely detected in the oral cavity.