Differences in the subgingival microbiome according to stage of periodontitis: A comparison of two geographic regions.

No microbiological criteria were included in the 2018 EFP-AAP classification of periodontal diseases that could be used to differentiate between stages and grades. Furthermore, differences in the subgingival microbiome depending on stage and grade have not been established. Sixty subgingival biofilm samples were collected in Spain (n = 30) and Colombia (n = 30) from three distinct patient categories: those with periodontal health/gingivitis (n = 20), those with stage I-II periodontitis (n = 20), and those with stage III-IV periodontitis (n = 20). Patients were evaluated by 16S rRNA gene amplification sequencing. Amplicon sequence variants were used to assign taxonomic categories compared to the Human Oral Microbiome Database (threshold ≥97% identity). Alpha diversity was established by Shannon and Simpson indices, and principal coordinate analysis, ANOSIM, and PERMANOVA of the UNIFRAC distances were performed using QIIME2. Although differences in the alpha diversity were observed between samples according to country, Filifactor alocis, Peptostreptococcaceae [XI][G-4] bacterium HMT 369, Fretibacterium fastidiosum, Lachnospiraceae [G-8] bacterium HMT 500, Peptostreptococcaceae [XI][G-5] [Eubacterium] saphenum, Peptostreptococcus stomatis, and Tannerella forsythia were associated with periodontitis sites in all stages. However, only F. alocis, Peptostreptococcaceae [XI][G-4] bacterium HMT 369, Peptostreptococcaceae [XI][G-9] [Eubacterium] brachy, Peptostreptococcaceae [XI][G-5] [Eubacterium] saphenum, and Desulfobulbus sp. HMT 041 were consistent in stage III-IV periodontitis in both countries. Porphyromonas gingivalis and Tannerella forsythia were differentially expressed in severe lesions in the countries studied. Although some non-cultivable microorganisms showed differential patterns between the different stages of periodontitis, they were not the same in the two countries evaluated. Further studies using larger samples with advanced next-generation techniques for high-throughput sequencing of phyla and non-cultivable bacteria within the subgingival microbiome could provide more insight into the differences between stages of periodontitis.

Knowing the enemy: homoacetogens in hydrogen production reactors.

One of the bottlenecks of the hydrogen production by dark fermentation is the low yields obtained because of the homoacetogenesis persistence, a metabolic pathway where H2 and CO2 are consumed to produce acetate. The central reactions of H2 production and homoacetogenesis are catalyzed by enzyme hydrogenase and the formyltetrahydrofolate synthetase, respectively. In this work, genes encoding for the formyltetrahydrofolate synthetase (fthfs) and hydrogenase (hydA) were used to investigate the diversity of homoacetogens as well as their phylogenetic relationships through quantitative PCR (qPCR) and next-generation amplicon sequencing. A total of 70 samples from 19 different H2-producing bioreactors with different configurations and operating conditions were analyzed. Quantification through qPCR showed that the abundance of fthfs and hydA was strongly associated with the type of substrate, organic loading rate, and H2 production performance. In particular, fthfs sequencing revealed that homoacetogens diversity was low with one or two dominant homoacetogens in each sample. Clostridium carboxivorans was detected in the reactors fed with agave hydrolisates; Acetobacterium woodii dominated in systems fed with glucose; Blautia coccoides and unclassified Sporoanaerobacter species were present in reactors fed with cheese whey; finally, Eubacterium limosum and Selenomonas sp. were co-dominant in reactors fed with glycerol. Altogether, quantification and sequencing analysis revealed that the occurrence of homoacetogenesis could take place due to (1) metabolic changes of H2-producing bacteria towards homoacetogenesis or (2) the displacement of H2-producing bacteria by homoacetogens. Overall, it was demonstrated that the fthfs gene was a suitable marker to investigate homoacetogens in H2-producing reactors. KEY POINTS: • qPCR and sequencing analysis revealed two homoacetogenesis phenomena. • fthfs gene was a suitable marker to investigate homoacetogens in H2 reactors.

Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows.

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

Breastfeeding may have a long-term effect on oral microbiota: results from the Fin-HIT cohort.

BACKGROUND: Breastfeeding contributes to gastrointestinal microbiota colonization in early life, but its long-term impact is inconclusive. We aimed to evaluate whether the type of feeding during the first six months of life was associated with oral microbiota in adolescence. METHODS: This is a cross-sectional sub-study using baseline information of 423 adolescents from the Finnish Health in Teens (Fin-HIT) cohort. Type of feeding was recalled by parents and dichotomized as (i) No infant formula; (ii) Infant formula (breastmilk + formula or only formula). Saliva microbiota was analysed using 16S rRNA (V3-V4) sequencing. Alpha diversity and beta diversity were compared between feeding type groups using ANCOVA and PERMANOVA, respectively. Differential bacteria abundance was tested using appropriate general linear models. RESULTS: Mean age and body mass index were 11.7 years and 18.0 kg/m2, respectively. The No formula group contained 41% of the participants. Firmicutes (51.0%), Bacteroidetes (19.1%), and Proteobacteria (16.3%) were the most abundant phyla among all participants. Alpha and beta diversity indices did not differ between the two feeding groups. Three Operational Taxonomic Units (OTUs) belonging to Eubacteria and Veillonella genera (phylum Firmicutes) were more abundant in the No formula than in the Infant formula group (log2fold changes/ p - values - 0.920/ < 0.001, - 0.328/ 0.001, - 0.577/ 0.004). CONCLUSION: Differences exist in abundances of some OTUs in adolescence according to feeding type during the first six months of life, but our findings do not support diversity and overall oral microbiota composition in adolescents being affected by early feeding type.

Subgingival bacterial recolonization after scaling and root planing in smokers with chronic periodontitis.

BACKGROUND: The aim of this study was to compare subgingival bacterial recolonization patterns after scaling and root planing in current smokers and non-smokers. METHODS: 15 smokers and 15 non-smokers with chronic periodontitis received scaling and root planing in six visits lasting one hour each, over a period of 21 days. Clinical monitoring was performed at baseline and 180 days, and microbiological monitoring was performed at baseline, immediately after scaling and root planing (Day 0) and at 42, 63 and 180 days post-therapy. Subgingival plaque samples were analysed by checkerboard DNA-DNA hybridization. RESULTS: An improvement in clinical condition was observed for smokers and non-smokers; however, non-smokers showed a greater reduction in mean clinical attachment level in intermediate sites in comparison with smokers (p < 0.05). At Day 0, there was a significant reduction in the mean counts of the three pathogens from the red complex, Eubacterium nodatum and Parvimonas micra only in non-smokers (p < 0.05). There was a significant increase in the proportion of host-compatible species in non-smokers and smokers from baseline to 180 days post-therapy (p < 0.05). However, a significant decrease in the pathogenic species was observed only in non-smokers. CONCLUSIONS: Smokers were more susceptible to the re-establishment of a pathogenic subgingival biofilm than non-smokers.

Microbial diversity in persistent root canal infections investigated by checkerboard DNA-DNA hybridization.

INTRODUCTION: The aim of the present study was to investigate the composition of the root canal microbiota in endodontic failures in order to identify and quantify these microorganisms. METHODS: Microbiological samples were taken from 36 root canals with persistent endodontic infection. The presence, levels, and proportions of 79 bacterial species were determined by checkerboard DNA-DNA hybridization. The Pearson correlation coefficient was used to investigate the relations between bacterial counts and clinical conditions (P ≤ .05). RESULTS: Enterococcus faecium (36%), Streptococcus epidermidis (36%), Eubacterium saburreum (28%), Parvimonas micra (28%), Streptococcus sanguis (28%), Capnocytophaga sputigena (28%), Leptotrichia buccalis (28%), Enterococcus faecalis (28%), and Staphylococcus warneri (28%) were the most prevalent species; and there was a low prevalence of Treponema socranskii (3%), Fusobacterium periodonticum (3%), Capnocytophaga gingivalis (3%), and Spiroplasma ixodetis (3%). The highest mean levels were found for the following species: E. faecium, Dialister pneumosintes, Staphylococcus epidermidis and Helicobacter pylori. There was a statistically significant difference between the levels of gram-negative species and gram-positive species (13.5 × 10(5) vs 6.5 × 10(5), respectively). A positive correlation was found between the area of the periapical lesion and the levels of gram-negative and rod species (P < .05). CONCLUSIONS: The microbiota from teeth with persistent apical periodontitis presents a mixed and complex profile, hosting E. faecium and S. epidermidis as the most highly prevalent species. No correlation was found between any of the species tested and clinical findings; however, periapical lesions with the largest areas presented higher counts of gram-negative and rod species.

Metronidazole and amoxicillin as adjuncts to scaling and root planing for the treatment of type 2 diabetic subjects with periodontitis: 1-year outcomes of a randomized placebo-controlled clinical trial.

AIM: To evaluate the clinical and microbiological effects of the use of metronidazole (MTZ) + amoxicillin (AMX) as adjuncts to scaling and root planing (SRP) for the treatment of chronic periodontitis (ChP) in type 2 diabetic subjects. MATERIAL AND METHODS: Fifty-eight type 2 diabetic subjects (n = 29/group) with generalized ChP were randomly assigned to receive SRP alone or with MTZ [400 mg/thrice a day (TID)]+AMX (500 mg/TID) for 14 days. Subgingival biofilm samples were analyzed by qPCR for the presence of seven periodontal pathogens. Subjects were monitored at baseline, 3, 6 and 12 months post-therapies. RESULTS: The group receiving SRP+MTZ+AMX presented greater mean probing depth (PD) reduction and clinical attachment gain, a lower number of sites with PD ≥5 mm (primary outcome variable) and a reduced number of subjects with ≥9 of these residual pockets than the control group at 1-year post-therapy (p < 0.05). The antibiotic-treated group also presented reduced levels and greater decreases of the three red complex species, Eubacterium nodatum and Prevotella intermedia, compared to the control group at 1 year (p < 0.05). CONCLUSIONS: The adjunctive use of MTZ+AMX significantly improved the clinical and microbiological outcomes of SRP in the treatment of type 2 diabetic subjects with ChP.

Subgingival biodiversity in subjects with uncontrolled type-2 diabetes and chronic periodontitis.

BACKGROUND AND OBJECTIVE: There is a bidirectional relationship between periodontal disease and type-2 diabetes mellitus (DM). Inflammatory mediators may negatively affect glycemic control, and increased glucose levels and resultant glycation end-products may alter the host response against bacterial infection. However, no agreement has been reached regarding the effect of DM on periodontal subgingival microbiota. Therefore, the purpose of the present study was to compare the subgingival biodiversity in deep periodontal pockets of subjects with chronic periodontitis and either uncontrolled type-2 diabetes or no diabetes using 16S rRNA gene cloning and sequencing. MATERIAL AND METHODS: Twelve subjects with uncontrolled type-2 diabetes (glycated hemoglobin > 8%) and eleven nondiabetic subjects presenting severe and generalized chronic periodontitis were selected. Subgingival biofilm from periodontal pockets > 5 mm were assessed using the 16S rRNA gene cloning and sequencing technique. RESULTS: Significant differences were observed in subgingival microbiota between diabetic and nondiabetic subjects. Diabetic subjects presented higher percentages of total clones of TM7, Aggregatibacter, Neisseria, Gemella, Eikenella, Selenomonas, Actinomyces, Capnocytophaga, Fusobacterium, Veillonella and Streptococcus genera, and lower percentages of Porphyromonas, Filifactor, Eubacterium, Synergistetes, Tannerella and Treponema genera than nondiabetic individuals (p < 0.05). Moreover, some phylotypes, such as Fusobacterium nucleatum, Veillonella parvula, V. dispar and Eikenella corrodens were detected significantly more often in diabetic subjects than in nondiabetic subjects (p < 0.05). CONCLUSION: Subjects with uncontrolled type-2 diabetes and chronic periodontitis presented significant dissimilarities in subgingival biodiversity compared with nondiabetic subjects.

Detection of Mogibacterium timidum in subgingival biofilm of aggressive and non-diabetic and diabetic chronic periodontitis patients

The aim of the present study was to evaluate the frequency of detection of Mogibacterium timidum in subgingival samples of subjects with generalized aggressive periodontitis (GAgP) and uncontrolled diabetic and non-diabetic subjects with generalized chronic periodontitis (GChP). 48 patients with GAgP, 50 non-diabetic and 39 uncontrolled (glycated hemoglobin >7%) type 2 diabetic subjects with GChP were enrolled in this study. Subgingival biofilm were collected from deep pockets (probing depth > 7 mm). After DNA extraction, M. timidum was detected by Nested Polymerase Chain Reaction and chi-square test was used to data analysis (p>0.05). There were no differences in the frequency of detection of M. timidum between subjects with GAgP (35%) and non-diabetic subjects with GChP (40%) (p>0.05). The frequency of detection of M. timidum was significantly higher in deep pockets of diabetic subjects with GChP (56%) when compared to GAgP (p<0.05), but similar to non-diabetic subjects with GChP (p>0.05). The frequency of detection of M. timidum was higher in subjects GChP presenting uncontrolled type 2 diabetes mellitus, when compared to GAgP subjects.

Adjunctive azithromycin in the treatment of aggressive periodontitis: microbiological findings of a 12-month randomized clinical trial.

OBJECTIVES: To compare the subgingival microbiological outcomes of azithromycin or placebo as adjuncts to scaling and root planing (SRP) in the treatment of aggressive periodontitis (AgP), and to secondarily evaluate the microbiological effect of supragingival scaling in AgP patients. METHODS: Twenty-four AgP subjects 13-26 years of age received a 15-day programme of supragingival scaling (SC) and were then randomly assigned to SRP with systemic azithromycin or placebo. Subgingival samples were taken with sterile paper points at baseline, 15 days after SC, and at 3, 6 and 12 months following SRP. Microbiological analysis was performed by the checkerboard DNA-DNA hybridization. RESULTS: Changes in bacterial levels from baseline to 15 days after SC were similar in the 2 groups. When subjects were analysed as a single group, significant reductions after SC were observed for Actinomyces gerencseriae, Capnocytophaga ochracea, and Treponema denticola. During the 12-month follow-up, levels of most of the bacteria decreased in both groups in a similar pattern. For instance, Actinomyces israelli, Veillonella parvula, Streptococcus gordonii, C. ochracea, Eikenella corrodens, Eubacterium nodatum, Fusobacterium periodonticum and Fusobacterium nucleatum ssp. polymorphum decreased significantly within the groups. CONCLUSIONS: Azithromycin was ineffective in lowering the subgingival levels of important putative periodontal pathogens in young AgP subjects compared to placebo. CLINICAL SIGNIFICANCE: Scaling and root planing with adjunctive systemic azithromycin provides little additional benefit compared to placebo in reductions of major subgingival periodontal pathogens.

Effect of non-surgical treatment on chronic and aggressive periodontitis: clinical, immunologic, and microbiologic findings.

BACKGROUND: Our goal was to examine differences in clinical, microbiologic, and immunologic responses to non-surgical mechanical therapy in patients with generalized chronic periodontitis (GCP) and generalized aggressive periodontitis (GAgP). METHODS: Twenty patients with GCP and 14 patients with GAgP were evaluated. Clinical data, gingival crevicular fluid (GCF), and subgingival plaque samples were collected at baseline and 3 months after non-surgical periodontal treatment. Levels of 40 subgingival species were measured using checkerboard DNA-DNA hybridization. GCF interleukin (IL)-1ß, -4, and -8 and interferon-γ (IFN-γ) were analyzed using a multiplexed bead immunoassay, and elastase activity was measured using an enzymatic assay. The significance of changes with time was examined using the Wilcoxon rank sum test. Changes in clinical, microbiologic, and immunologic parameters after therapy were compared between groups using the Mann-Whitney U test. RESULTS: After periodontal therapy, we found significant improvements for all clinical parameters in both groups. We also observed significant reductions in elastase activity in shallow and deep sites from the GAgP group and in deep sites from the GCP group. Microbiologic data showed significant reductions in proportions of orange and red complexes and an increase in proportions of Actinomyces species in both clinical groups. When the clinical, microbiologic, and immunologic responses after therapy were compared between groups, only minor differences were found. CONCLUSION: This study fails to show any significant differences between severe forms of GCP and GAgP in response to non-surgical periodontal treatment.

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.

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.

Frequency, microbial interactions, and antimicrobial susceptibility of Fusobacterium nucleatum and Fusobacterium necrophorum isolated from primary endodontic infections.

This study assessed the prevalence and microbial interactions of Fusobacterium nucleatum and Fusobacterium necrophorum in primary endodontic infections from a Brazilian population and their antimicrobial susceptibility to some antibiotics by the E-test. One hundred ten samples from infected teeth with periapical pathologies were analyzed by culture methods. Five hundred eighty individual strains were isolated; 81.4% were strict anaerobes. F. nucleatum was found in 38 root canals and was associated with Porphyromonas gingivalis, Prevotella spp., and Eubacterium spp. F. necrophorum was found in 20 root canals and was associated with Peptostreptococcus prevotii. The simultaneous presence of F. nucleatum and F. necrophorum was not related to endodontic symptoms (p > 0.05). They were 100% susceptible to amoxicillin, amoxicillin/clavulanate, and cephaclor. Fusobacterium spp. is frequently isolated from primary-infected root canals of teeth with periapical pathologies. Amoxicillin is a useful antibiotic against F. nucleatum and F. necrophorum in endodontic infections and has been prescribed as the first choice in Brazil.

Comparative analysis of endodontic pathogens using checkerboard hybridization in relation to culture.

BACKGROUND/AIM: The purpose of this study was to detect bacterial species and to quantify the total number of bacteria from samples of infected root canals before (S1) and after chemo-mechanical preparation using 2% chlorhexidine (CHX) gel as auxiliary chemical substance (S2) and after 7 days of intracanal dressing (S3) to compare microbial changes. METHOD: Twenty-four teeth were selected for this study. Chemo-mechanical preparation was performed using 2% CHX gel, then three different intracanal medicaments [M1: Ca(OH)(2) paste; M2: 2% CHX gel; and M3: Ca(OH)(2) paste plus 2% CHX gel] were used for 7 days. Checkerboard DNA-DNA hybridization was performed to detect 40 bacterial species. Aerobic and anaerobic culture techniques were used to determine the bacterial community by counting the colony-forming units (CFU). RESULTS: The species most frequently identified by checkerboard in S1 were: Fusobacterium nucleatum ssp. polymorphum, Treponema socranskii ssp. socranskii, Parvimonas micra and Enterococcus faecalis. In S2 and S3 a total of eight different species were identified; and only one of them was gram-positive (E. faecalis). Microorganisms were not identified after use of M2 for 7 days. The quantification obtained on agar plates ranged from 4 x 10(5) to 2.6 x 10(6) CFU/ml in S1, mean CFU was reduced by 99.96% in S2, and there was no statistical difference between the CFU in S2 and S3. CONCLUSION: The antibacterial effect of the mechanical preparation supplemented by the use of an antibacterial auxiliary substance greatly reduced the microorganisms in the main root canal.

Influence of local tetracycline on the microbiota of alveolar osteitis in rats.

The aim of the present study was to evaluate the effects of local tetracycline on the occurrence of alveolar osteitis in rats, and on the microbiota associated to this infection. Forty Wistar rats were randomly assigned to 4 groups (n=10): I - the rats had the maxillary right incisor extracted and the alveolar wound did not receive any treatment; II - adrenaline and Ringer-PRAS were introduced into the alveolar wound; III - the alveolar wound was irrigated with sterile saline; and IV - the alveolar wound was irrigated with an aqueous solution of tetracycline. Microbial samples from the alveolar wounds were collected 2 days after surgery and inoculated on blood agar (with and without 8 microg/mL of tetracycline) and other selective media, and were incubated in either aerobiosis or anaerobiosis at 37 degrees C, for 2 to 14 days. It was verified that tetracycline reduced the occurrence of alveolar osteitis in the rats and caused significant changes in the microbiota of the surgical sites, decreasing the number of anaerobes and increasing the participation of tetracycline-resistant and multi-resistant microorganisms.

Influence of local tetracycline on the microbiota of alveolar osteitis in rats

The aim of the present study was to evaluate the effects of local tetracycline on the occurrence of alveolar osteitis in rats, and on the microbiota associated to this infection. Forty Wistar rats were randomly assigned to 4 groups (n=10): I - the rats had the maxillary right incisor extracted and the alveolar wound did not receive any treatment; II - adrenaline and Ringer-PRAS were introduced into the alveolar wound; III - the alveolar wound was irrigated with sterile saline; and IV - the alveolar wound was irrigated with an aqueous solution of tetracycline. Microbial samples from the alveolar wounds were collected 2 days after surgery and inoculated on blood agar (with and without 8 µg/mL of tetracycline) and other selective media, and were incubated in either aerobiosis or anaerobiosis at 37ºC, for 2 to 14 days. It was verified that tetracycline reduced the occurrence of alveolar osteitis in the rats and caused significant changes in the microbiota of the surgical sites, decreasing the number of anaerobes and increasing the participation of tetracycline-resistant and multi-resistant microorganisms.

O objetivo do presente estudo foi avaliar os efeitos do uso tópico de tetraciclina sobre a ocorrência de alveolite em ratos e sobre a microbiota a ela associada. Quarenta ratos foram divididos, ao acaso, em 4 grupos (n=10): grupo I, realizou-se somente a extração do incisivo superior direito e a ferida alveolar não recebeu nenhum tratamento; grupo II, além da extração dental, soluções de adrenalina e Ringer-PRAS foram introduzidas no interior do alvéolo; grupo III, a ferida alveolar foi irrigada com solução salina estéril; grupo IV, a ferida alveolar foi irrigada com solução aquosa de cloridrato de tetraciclina a 10 por cento. As amostras dos alvéolos para processamento microbiológico foram coletadas dois dias após a realização das cirurgias e foram inoculadas em ágar sangue com ou sem 8 µg/mL de tetraciclina e em outros meios de cultura seletivos, incubadas em aerobiose ou anaerobiose, a 37ºC, de 2 a 14 dias. Verificou-se que a tetraciclina reduziu a ocorrência de alveolite e provocou uma modificação significativa na microbiota do sítio cirúrgico, levando a uma redução nas proporções ocupadas pelos microrganismos anaeróbios e uma elevação da participação de microrganismos resistentes à tetraciclina e outros antimicrobianos.

Evaluation of the microbiota of primary endodontic infections using checkerboard DNA-DNA hybridization.

BACKGROUND/AIMS: The aim of this study was to evaluate the composition of the microbiota of primary endodontic infections in 111 selected cases of single-rooted teeth with necrotic pulp. METHODS: Samples were collected from the root canals using #15 Hedströen-type files and two sterile paper points, which were introduced 1 mm short of the apical foramen. The presence, levels, and proportions of 40 different bacterial species in each sample were determined using DNA probes and checkerboard DNA-DNA hybridization techniques. RESULTS: The mean number of species per sample was 22. Enterococcus faecalis (89.3%), Campylobacter gracilis (89.3%), Leptotrichia buccalis (89.3%), Neisseria mucosa (87.5%), Prevotella melaninogenica (86.6%), Fusobacterium nucleatum ssp. vincentii (85.7%), Eubacterium saburreum (75.9%), Streptococcus anginosus (75%), and Veillonella parvula (74.1%) were the most prevalent species. The species found in highest mean counts (over 10(5)) were F. nucleatum ssp. vincentii (13.14 x 10(5)), E. saburreum (5.67 x 10(5)), E. faecalis (5.38 x 10(5)), N. mucosa (4.19 x 10(5)), V. parvula (3.63 x 10(5)), C. gracilis (3.46 x 10(5)), Treponema socranskii (3.34 x 10(5)), Porphyromonas endodontalis (2.96 x 10(5)), Porphyromonas gingivalis (2.85 x 10(5)), Micromonas micros (2.81 x 10(5)), Prevotella nigrescens (2.68 x 10(5)) and Fusobacterium nucleatum ssp. nucleatum (2.64 x 10(5)). Most of these species were also found in high proportions. CONCLUSIONS: Our results suggest that several bacterial species considered to be oral pathogens seem to be implicated in the etiology of primary endodontic infections.

Bacterial examination of endodontic infections by clonal analysis in concert with denaturing high-performance liquid chromatography.

BACKGROUND/AIMS: The aim of this study was to examine the diversity of bacterial species in the infected root canals of teeth associated with endodontic abscesses by cloning and sequencing techniques in concert with denaturing high-performance liquid chromatography. METHODS: Samples collected from five infected root canals were subjected to polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. Products of these PCRs were cloned and sequenced. Denaturing high-performance liquid chromatography (DHPLC) was used as a screening method to reduce the number of clones necessary for DNA sequencing. RESULTS: All samples were positive for the presence of bacteria and a range of 7-13 different bacteria were found per root canal sample. In total, 48 different oral clones were detected among the five root canal samples. Olsenella profusa was the only species present in all samples. Porphyromonas gingivalis, Dialister pneumosintes, Dialister invisus, Lachnospiraceae oral clone, Staphylococcus aureus, Pseudoramibacter alactolyticus, Peptostreptococcus micros and Enterococcus faecalis were found in two of the five samples. The majority of the taxa were present in only one sample, for example Tannerella forsythia, Shuttleworthia satelles and Filifactor alocis. Some facultative anaerobes that are frequently isolated from endodontic infections such as E. faecalis, Streptococcus anginosus and Lactobacillus spp. were also found in this study. CONCLUSION: Clonal analysis of the microflora associated with endodontic infections revealed a wide diversity of oral species.

The function of lexical motifs in the organization of the Actinomycetes 5S rRNAs

This work shows results obtained by employing the linguistic method to identify biologically meaningful sites in Actinomycetes 5S rRNAs. The approach adopted identifies triplet-words, along the base sequence of 5S rRNA, located mainly at the alpha and beta domains of the 5S secondary structure. There are triplet-words representing universal protein binding sites that include important prokaryote signatures, and sites strategically located in critical regions related to the formation of the 5S ribonucleoproteins (RNP) complex. In those sites, where the GC pressure promoted substitutions, the analysis demonstrates that alterations did not affect their biological significance. Sites formed by GGY (or more rarely GGR), continued to play an important role as ribosomal proteins rpL18 and rpL5 protein receptors. The data suggest that instead of increasing the molecular variability, expected for the diversity in species and habitats occupied for the group, GC pressure functioned as a reducer mechanism for the inter-specific diversity.

Neste trabalho são apresentados resultados obtidos empleando o método linguístico para identificar sítios no 5S rRNAs de actinomicetes com significado biológico. A abordagem identificou palavras-tripletes, junto com a sequência de bases do 5S rRNAs, localizados principalmente nos domínios alfa e beta da estrutura secundária. Entre eles, existem palavras-tripletes que representam sítios de ligação de proteínas universais, que incluem importantes assinaturas procarióticas, além de sítios estrategicamente colocados em regiões críticas relacionados com a formação do complexo 5S ribonucleoproteína (RNP). Nestes sítios, onde a pressão GC promove substituições, as alterações não afetaram seu significado biológico. Sítios formados por GGY (ou mais raramente GER), jogam um papel importante como receptores de proteínas ribossomicas rpL18 e rpL5. Os dados também sugerem que ao contrário de aumentar a variabilidade molecular, esperada pela diversidade em espécies e habitats ocupados pelo grupo, GC funciona como um mecanismo para diversidade inter-específica.