Frequency of putative periodontal pathogens among type 1 diabetes mellitus: a case-control study.

OBJECTIVE: The aim of the present study is to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria viz., Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens. To fulfil the above objective, polymerase Chain reaction using the primers targeting 16S rRNA gene of the bacterial species was performed with the subgingival plaque collected from the permanent first molars of type 1 diabetic children and age matched healthy children. RESULTS: The prevalence of periodontal pathogens in diabetic and healthy children was 6% and 16% for E. corrodens, 18% and 36% for C. rectus, 2% and 2% for P. intermedia, 4% and 0%, for P. nigrescens respectively. Statistically, significant difference was not observed for the prevalence of all the four periodontal pathogens between type 1 diabetic and healthy children (P = 1.00). The results of the present study thus reveal a negative correlation of type I diabetes to periodontitis in association to Eikenella corrodens, Campylobacter rectus, Prevotella intermedia and Prevotella nigrescens.

Phenotypic identification of periodontal Prevotella intermedia/nigrescens group isolates validated by MALDI-TOF mass spectrometry.

The accuracy of a phenotypic scheme to recognize periodontal Prevotella intermedia/nigrescens group clinical isolates on primary isolation culture plates was assessed with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 84 fresh subgingival isolates from 23 chronic periodontitis patients were presumptively recognized on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. intermedia/nigrescens based on their dark-pigmented colony morphology, brick-red autofluorescence under long-wave ultraviolet light, and a negative fluorescence test for lactose production. The presumptive P. intermedia/nigrescens clinical isolates were subjected to MALDI-TOF MS analysis using Bruker MALDI Biotyper analytic software containing mass spectra for P. intermedia and Prevotella nigrescens in its reference library of bacterial protein profiles. Using a ≥1.7 log score agreement threshold, 60 (71.4%) of the presumptive P. intermedia/nigrescens clinical isolates were confirmed as either P. intermedia (25 isolates) or P. nigrescens (35 isolates). All isolates with a <1.7 log score were also identified as P. intermedia or P. nigrescens from the top choice designated on the MALDI Biotyper most likely species identification list. These MALDI-TOF MS findings document the ability of the phenotypic scheme to correctly recognize most periodontal P. intermedia/nigrescens group clinical isolates on primary isolation culture plates.

Population-Genomic Insights into Variation in Prevotella intermedia and Prevotella nigrescens Isolates and Its Association with Periodontal Disease.

High-throughput sequencing has helped to reveal the close relationship between Prevotella and periodontal disease, but the roles of subspecies diversity and genomic variation within this genus in periodontal diseases still need to be investigated. We performed a comparative genome analysis of 48 Prevotella intermedia and Prevotella nigrescens isolates that from the same cohort of subjects to identify the main drivers of their pathogenicity and adaptation to different environments. The comparisons were done between two species and between disease and health based on pooled sequences. The results showed that both P. intermedia and P. nigrescens have highly dynamic genomes and can take up various exogenous factors through horizontal gene transfer. The major differences between disease-derived and health-derived samples of P. intermedia and P. nigrescens were factors related to genome modification and recombination, indicating that the Prevotella isolates from disease sites may be more capable of genomic reconstruction. We also identified genetic elements specific to each sample, and found that disease groups had more unique virulence factors related to capsule and lipopolysaccharide synthesis, secretion systems, proteinases, and toxins, suggesting that strains from disease sites may have more specific virulence, particularly for P. intermedia. The differentially represented pathways between samples from disease and health were related to energy metabolism, carbohydrate and lipid metabolism, and amino acid metabolism, consistent with data from the whole subgingival microbiome in periodontal disease and health. Disease-derived samples had gained or lost several metabolic genes compared to healthy-derived samples, which could be linked with the difference in virulence performance between diseased and healthy sample groups. Our findings suggest that P. intermedia and P. nigrescens may serve as "crucial substances" in subgingival plaque, which may reflect changes in microbial and environmental dynamics in subgingival microbial ecosystems. This provides insight into the potential of P. intermedia and P. nigrescens as new predictive biomarkers and targets for effective interventions in periodontal disease.

Copaifera reticulata oleoresin: Chemical characterization and antibacterial properties against oral pathogens.

Oral infections such as periodontitis and tooth decay are the most common diseases of humankind. Oleoresins from different copaifera species display antimicrobial and anti-inflammatory activities. Copaifera reticulata is the commonest tree of this genus and grows abundantly in several Brazilian states, such as Pará, Amazonas, and Ceará. The present study has evaluated the chemical composition and antimicrobial potential of the Copaifera reticulata oleoresin (CRO) against the causative agents of tooth decay and periodontitis and has assessed the CRO cytotoxic potential. Cutting edge analytical techniques (GC-MS and LC-MS) aided the chemical characterization of CRO. Antimicrobial assays included determination of the Minimum Inhibitory Concentration (MIC), determination of the Minimum Bactericidal Concentration (MBC), determination of the Minimum Inhibitory Concentration of Biofilm (MICB50), Time Kill Assay, and Checkerboard Dilution. Conduction of XTT assays on human lung fibroblasts (GM07492-A cells) helped to examine the CRO cytotoxic potential. Chromatographic analyses revealed that the major constituents of CRO were ß-bisabolene, trans-α-bergamotene, ß-selinene, α-selinene, and the terpene acids ent-agathic-15-methyl ester, ent-copalic acid, and ent-polyalthic acid. MIC and MBC results ranged from 6.25 to 200 µg/mL against the tested bacteria. The time-kill assay conducted with CRO at concentrations between 50 and 100 µg/mL showed bactericidal activity against Fusobacterium nucleatum (ATCC 25586) and Streptococcus mitis (ATCC 49456) after 4 h, Prevotella nigrescens (ATCC 33563) after 6 h, Porphyromonas gingivalis (ATCC 33277) and Lactobacillus casei (clinical isolate) after 12 h, and Streptococcus salivarius (ATCC 25975) and Streptococcus mutans (ATCC 25175) after 18 h. The fractional inhibitory concentration indexes (FICIs) revealed antagonistic interaction for Lactobacillus casei (clinical isolate), indifferent effect for Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC 25586), Prevotella nigrescens (ATCC 33563), and Streptococcus salivarius (ATCC 25975), and additive effect for Streptococcus mutans (ATCC 25175) and Streptococcus mitis (ATCC 49456). Treatment of GM07492-A cells with CRO demonstrated that concentrations up to 39 µg/mL significantly reduced cell viability as compared to the negative control, being IC50 equal to 51.85 ± 5.4 µg/mL. These results indicated that CRO plays an important part in the search for novel sources of agents that can act against oral pathogens.

Arthritis-induced alveolar bone loss is associated with changes in the composition of oral microbiota.

Rheumatoid arthritis (RA) and periodontitis (PD) are chronic inflammatory disorders that cause bone loss. PD tends to be more prevalent and severe in RA patients. Previous experimental studies demonstrated that RA triggers alveolar bone loss similarly to PD. The aim of this study was to investigate if arthritis-induced alveolar bone loss is associated with modification in the oral microbiota. Checkerboard DNA-DNA hybridization was employed to analyze forty oral bacterial species in 3 groups of C57BL/6 mice: control (n = 12; without any challenge); Y4 (n = 8; received oral inoculation of Aggregatibacter Actinomycetemcomitans strain FDC Y4) and AIA group (n = 12; chronic antigen-induced arthritis). The results showed that AIA and Y4 group exhibited similar patterns of bone loss. The AIA group exhibited higher counts of most bacterial species analyzed with predominance of Gram-negative species similarly to infection-induced PD. Prevotella nigrescens and Treponema denticola were detected only in the Y4 group whereas Campylobacter showae, Streptococcus mitis and Streptococcus oralis were only found in the AIA group. Counts of Parvimonas micra, Selenomonas Noxia and Veillonella parvula were greater in the AIA group whereas Actinomyces viscosus and Neisseira mucosa were in large proportion in Y4 group. In conclusion, AIA is associated with changes in the composition of the oral microbiota, which might account for the alveolar bone loss observed in AIA mice.

Prevalence of eight putative periodontal pathogens in atherosclerotic plaque of coronary artery disease patients and comparing them with noncardiac subjects: A case-control study.

BACKGROUND: The study was aimed to assess the prevalence of periodontal pathogens namely Tannerella forsythia (T.f), Campylobacter rectus (C.r), Eikenella corrodens (E.c), Porphyromonas gingivalis (P.g), Treponema denticola (T.d), Prevotella nigrescens (P.n) ,Aggregatibacter actinomycetemcomitans (A.a), P.g (fi mA gene) and Prevotella intermedia (P.i), in the subgingival and the atheromatous plaque of patients with coronary artery disease (CAD), and comparing them with the noncardiac subjects thereby indicating the role of periodontal pathogenic bacteria in the progression of atherosclerosis in south Indian population. MATERIALS AND METHODS: 51 cardiac and non cardiac subjects within the age group of 40-80 years,who met the eligibility criteria, were selected and categorized as the experimental and control group respectively. Total number of teeth was recorded, and oral hygiene was evaluated using Plaque Index and Oral Hygiene Index (OHI). Periodontal pocket depth and clinical attachment level were also assessed as a part of periodontal examination. Subgingival plaque samples were collected with the help of with Gracey's curette in both the groups. In experimental group, biopsy was obtained from the atherosclerotic plaque during Coronary artery bypass grafting CABG procedure. Both, subgingival and the coronary atherosclerotic plaque samples were subjected to polymerase chain reaction (PCR) analysis for identification of the periodontal bacteria. STATISTICAL ANALYSIS: Mean, standard deviation and test of significance of quantitative variables such as periodontal parameters were compared between experimental group and control group. Kappa measures of agreement was done to analyze the relationship between the presence/absence of microorganisms in the subgingival and atherosclerotic plaque samples in the experimental group. RESULTS: The mean Plaque Index, Gingival Index, Russel's Periodontal Index, OHI, clinical attachment level, Pocket Depth Index was statistically significant in both the groups. Association of T.f, E.c, C.r, P.g, P.g (fi mA), T.d, P.i, P.n was significantly associated. A.a was absent in the control group, whereas only one patient in the experimental group was positive for the bacteria. Kappa analysis showed the significant association of periodontal bacteria T.f, C.r, P.g, P.g (fi mA), P.i and P.n in subgingival plaque and atherosclerotic plaque of the same patients of the experimental group. CONCLUSION: It is concluded that CAD subjects had higher prevalence of periodontal pathogens in subgingival biofilms as compared to the non cardiac subjects. Further, the number of bacteria was significantly associated between the subgingival and atherosclerotic plaques of the cardiac patients in south Indian population.

Bacterial profile of aggressive periodontitis in Morocco: a cross-sectional study.

BACKGROUND: Aggressive periodontitis (AgP) is one of the most severe forms of periodontal diseases. In Morocco, Aggregatibacter actinomycetemcomitans has been strongly associated with AgP, however limited knowledge is available about the implication of other periodontal pathogens in this entity. Therefore, the main aim of this study was to evaluate the composition of the subgingival microbiota in Moroccan patients with AgP. METHODS: Subgingival plaque samples were collected from 50 aggressive, 13 localized and 37 generalized periodontitis patients. Samples from 20 chronic periodontitis (ChP) patients were taken as controls. Samples collected from the four deepest periodontal pockets in each patient were pooled in pre-reduced transport fluid and examined by culture. RESULTS: A. actinomycetemcomitans was significantly more frequent (p = 0.004) in generalised AgP compared to ChP, and Porphyromonas gingivalis was less prevalent in localized AgP, when compared with generalized AgP (p = 0.040) or ChP (p = 0.016). Prevotella intermedia, Fusobacterium nucleatum and Tannerella forsythia were also frequently detected in all groups. Mean proportions of A. actinomycetemcomitans were significantly higher in AgP groups, when compared to ChP, and generalized AgP patients harbored significantly higher proportions of P. gingivalis and T. forsythia, when compared to localized AgP or ChP. CONCLUSIONS: A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia and F. nucleatum were frequently detected in this Moroccan population with AgP. Differences in frequency of detection, counts and proportions of A. actinomycetemcomitans, P. gingivalis and T. forsythia suggests the presence of distinct microbiological profiles for localized AgP, generalized AgP and ChP patients.

Proinflammatory Activity of Primarily Infected Endodontic Content against Macrophages after Different Phases of the Root Canal Therapy.

INTRODUCTION: This study investigated the presence of target bacterial species and the levels of endotoxins in teeth with apical periodontitis. Levels of inflammatory mediators (interleukin [IL]-1ß and tumor necrosis factor [TNF]-α) were determined after macrophage stimulation with endodontic content after different phases of endodontic therapy using different irrigants. METHODS: Thirty primarily infected root canals were randomly assigned into 3 groups according to the irrigant used for root canal preparation (n = 10 per group): GI: 2.5% sodium hypochlorite, GII: 2% chlorhexidine gel, and GIII (control group): saline solution. Root canal samples were taken by using paper points before (s1) and after root canal instrumentation (s2), subsequently to 17% EDTA (s3), after 30 days of intracanal medication (Ca[OH]2 + saline solution) (s4), and before root canal obturation (s5). Polymerase chain reaction (16S recombinant DNA) and limulus amebocyte lysate assay were used for bacterial and endotoxin detection, respectively. Macrophages were stimulated with the root canal contents for IL-1ß/TNF-α measurement using enzyme-linked immunosorbent assay. RESULTS: Porphyromonas gingivalis (17/30), Porphyromonas endodontalis (15/30), and Prevotella nigrescens (11/30) were the most prevalent bacterial species. At s1, endotoxins were detected in 100% of the root canals (median = 32.43 EU/mL). In parallel, substantial amounts of IL-1ß and TNF-α were produced by endodontic content-stimulated macrophages. At s2, a significant reduction in endotoxin levels was observed in all groups, with GI presenting the greatest reduction (P < .05). After a root canal rinse with EDTA (s3), intracanal medication (s4), and before root canal obturation (s5), endotoxin levels reduced without differences between groups (P < .05). IL-1ß and TNF-α release decreased proportionally to the levels of residual endotoxin (P < .05). CONCLUSIONS: Regardless of the use of sodium hypochlorite or CHX, the greatest endotoxin reduction occurs after chemomechanical preparation. Increasing steps of root canal therapy associated with intracanal medication enhances endotoxin reduction, leading to a progressively lower activation of proinflammatory cells such as macrophages.

Detection and genetic characterization of ß-lactamases in Prevotella intermedia and Prevotella nigrescens isolated from oral cavity infections and peritonsillar abscesses.

A prospective analysis on ß-lactam resistance mechanisms and ß-lactamase prevalence was conducted on Prevotella intermedia and Prevotella nigrescens recovered from patients with chronic periodontitis and peritonsillar abscesses. Both phenotypic and genotypic methods were performed to characterize the ß-lactamases, their coding genes and their genetic contexts. Overall, ß-lactamase production was observed in 64% (16/25) P. intermedia and 23.8% (5/21) P. nigrescens (p < 0.01). Besides higher ß-lactamase production rates were observed in P. intermedia (8/16) than in P. nigrescens (2/16) recovered from chronic periodontitis, almost all isolates from peritonsillar abscesses were producers (8/9 and 3/3, respectively). cfxA, but not cepA and cblA, was detected in those isolates, which were previously categorized as ß-lactamase producers. CfxA producing isolates displayed higher ß-lactam MICs than non-producers in both species. The most frequent allele was cfxA2, followed by cfxA3 and a new allelic variant named cfxA6. The analysis of the downstream flanking region in the three cfxA variants revealed the association with mobA of Tn4555, suggesting their localization in a mobilizable element. ß-lactam resistance and cfxA carriage prevalence seems to be not only related to the bacterial species but also to the infection site.

Distribution of 8 periodontal microorganisms in family members of Chinese patients with aggressive periodontitis.

OBJECTIVE: To date, no information on the distribution of periodontal microorganisms among family members of Chinese patients with aggressive peridontitis (AgP) is available. The aim of the present study was to investigate the probability of transmission of eight periodontal microorganisms between patients with aggressive periodontitis and their family members. DESIGN: Saliva and pooled subgingival plaque samples were collected from 103 participants from 41 nuclear families (including 41 AgP probands, 19 mothers, 22 fathers, 21 siblings). Eight periodontal microorganisms, including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens and Fusobacterium nucleatum were detected in these samples by the polymerase chain reaction (PCR). In addition, the distribution of fimA genotypes was assessed in P. gingivalis-positive individuals by PCR. RESULTS: P. gingivalis, T. forsythia, T. denticola, C. rectus and F. nucleatum were the most frequently detected species both in AgP probands and in their relatives. Kappa statistical analysis revealed that the detection of A. actinomycetemcomitans (Kappa = 0.503) and F. nucleatum (Kappa = 0.565) in probands was highly consistent with that in their relatives. Most probands shared the identical fimA genotype of P. gingivalis with their relatives. CONCLUSIONS: Our results suggested that the intrafamilial transmission of periodontal microorganisms may occur between Chinese patients with aggressive periodontitis and their relatives.

Macrophage cell activation with acute apical abscess contents determined by interleukin-1 Beta and tumor necrosis factor alpha production.

INTRODUCTION: This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-α) throughout the root canal treatment against macrophage cells. METHODS: Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH]2 + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-α and IL-1 beta. RESULTS: Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-α (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)2 + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). CONCLUSIONS: AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.

Detection of eight periodontal microorganisms and distribution of Porphyromonas gingivalis fimA genotypes in Chinese patients with aggressive periodontitis.

BACKGROUND: The microbiologic feature of aggressive periodontitis (AgP) in Chinese patients has not yet been determined. This study aims to investigate the prevalence of eight periodontal microorganisms and the distribution of the Porphyromonas gingivalis fimA genotype in a cohort of Chinese patients with AgP. METHODS: Saliva and pooled subgingival plaque samples were collected from 81 patients with AgP (25 with incisor-first molar type and 56 with generalized type [GAgP]) and 34 periodontally healthy controls. Eight periodontal microorganisms, including Aggregatibacter actinomycetemcomitans, P. gingivalis, Tannerella forsythia, Treponema denticola, Campylobacter rectus, Prevotella intermedia, Prevotella nigrescens, and Fusobacterium nucleatum were detected in these samples by the polymerase chain reaction (PCR). In addition, the distribution of fimA genotypes was assessed in P. gingivalis-positive individuals by PCR. RESULTS: The prevalence of P. gingivalis, T. forsythia, T. denticola, C. rectus, P. intermedia, F. nucleatum, and A. actinomycetemcomitans in patients with AgP was significantly higher than that in healthy controls. The prevalence of A. actinomycetemcomitans in patients with GAgP was relatively low (30.4%) compared with other pathogens. Results of logistic regression analysis showed that younger patients were more likely to harbor A. actinomycetemcomitans (odds ratio = 2.85). Type II was the most prevalent fimA genotype of P. gingivalis in patients with AgP. CONCLUSIONS: P. gingivalis, T. forsythia, T. denticola, C. rectus, P. intermedia, and F. nucleatum were the predominant periodontal pathogens of patients with GAgP in China. Type II of fimA was the most prevalent genotype of P. gingivalis in patients with AgP. The prevalence of A. actinomycetemcomitans in patients with GAgP was relatively low.

The effects of different orthodontic appliances upon microbial communities.

OBJECTIVES: Orthodontic appliances can promote accumulation of dental plaque, with associated enamel decalcification or gingival inflammation. The aim of this study was to examine longer-term microbiological changes during orthodontic treatment with fixed appliances. MATERIALS AND METHODS: Twenty-four orthodontic patients aged 11-14 years undergoing fixed appliance therapy were recruited into the study. Each was randomized for cross-mouth assignment of molar bands and bonded molar tubes to contralateral quadrants of the mouth. All patients received self-ligating brackets, but again using randomization, one upper lateral incisor bracket (left or right) also received an elastomeric ligature. Plaque samples from the molars and upper lateral incisors were obtained at intervals during treatment and up to 1 year after appliance removal. Denaturing gradient gel electrophoresis and 16S rDNA microarray were used to compare plaque microbial fingerprints. RESULTS: Plaque populations changed within 3 months of commencing treatment at all sites. The greatest differences in plaque composition were seen with self-ligating brackets with an elastomeric ligature. Post-treatment plaque associated with both types of molar attachment contained increased levels of periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Eubacterium nodatum, while Campylobacter rectus, Parvimonas micra, and Actinomyces odontolyticus were also elevated with bonds. CONCLUSIONS: The results suggest that orthodontic treatment may cause sustained changes in plaque microbiotas and that molar bond-associated plaque may have raised disease potential.

Asociación de Prevotella intermedia/nigrescens, bacilos entéricos gram-negativos y parámetros clínicos en periodontitis crónica1 / Association of Prevotella intermedia/nigrescens, gram-negative enteric rods and clinical parameters in chronic periodontitis

Introducción: La relación entre Prevotella intermedia/nigrescens y bacilos entéricos gram-negativos presentes en placa subgingival de pacientes con periodontitis ha recibido muy poca atención en la literatura. Objetivo: Investigar la asociación de Prevotella intermedia/nigrescens y bacilos entéricos gram-negativos con parámetros clínicos de pacientes con periodontitis crónica. Materiales y métodos: Se estudió la prevalencia de P. intermedia/nigrescens y de bacilos entéricos gram-negativos en 76 pacientes con periodontitis crónica. Se utilizaron las pruebas de chi cuadrado y Mann-Whitney para evaluar las diferencias clínicas de los pacientes con presencia y ausencia de los microorganismos estudiados. También se usó la prueba no paramétrica de Spearman para determinar la correlación entre P. intermedia/nigrescens y bacilos entéricos gram-negativos con las variables clínicas (p<0,05). Resultados y conclusiones: El 46,7% y 26,31% de los pacientes presentaron P. intermedia/nigrescens y bacilos entéricos gram-negativos, respectivamente. En el 22% de los sujetos se observaron simultáneamente los microorganismos estudiados. Se encontró una correlación positiva estadísticamente significativa entre P. intermedia/nigrescens y bacilos entéricos gram-negativos (r=0,286, p<0,0001). La presencia de los microorganismos estuvo positivamente correlacionada con profundidad de sondaje (PS), nivel de inserción clínica y presencia de sangrado (r=0,362; 0,358; 0,079; p<0,0001, respectivamente). Igualmente, los pacientes que presentaron P. intermedia/nigrescens y bacilos entéricos gram-negativos mostraron un promedio de PS y de pérdida de inserción clínica significativamente mayor. Los parámetros clínicos adversos observados en presencia de estos microorganismos podrían tener implicaciones en la patogénesis de la periodontitis y un posible impacto en los resultados de la terapia periodontal (AU)

Introduction: The relationships between Prevotella intermedia/nigrescens and gram-negative enteric rods in subgingival plaque of patients with periodontitis has received little attention in the literature. Objective: The objective of this study was to explore the relationships between P. intermedia/nigrescens, gram-negative enteric rods and clinical parameters of patients with chronic periodontitis. Materials and Methods: Clinical parameters and occurrence of P. intermedia/nigrescens and gram-negative enteric rods were examined in 76 patients with chronic periodontitis. Chi-square and Mann-Whitney tests were used to determine differences in clinical variables versus the presence or absence of both microorganisms. Correlations among microorganisms studied and clinical data were determined using Spearman rank correlation coefficient. Results and conclusions: P. intermedia/nigrescens and gram-negative enteric rods were detected in 46.7% and 26.31% individuals, respectively. A total of 22% of patients harbored the microorganisms studied. There were significantly positive correlations between P. intermedia/nigrescens and gram-negative enteric rods (r=0.286, p<0.0001). The microorganisms studied were significant and positively correlated with probing depth (PD), clinical attachment level and bleeding on probing (r=0.362; 0.358; 0.079; p<0.0001). The mean PD (mm) and clinical attachment loss of the sampled sites was significantly higher in patients with presence of P. intermedia/nigrescens and gram-negative enteric rods. The adverse clinical parameters observed in presence of these microorganisms could have implications in the pathogenesis of periodontal disease and a possible impact on outcomes after treatment (AU)

Subgingival root brushing in deep human periodontal pockets.

OBJECTIVE: The short-term clinical and microbiological effects of patient-applied subgingival root brushing were assessed on untreated deep human periodontal pockets. METHODS: Assessments of plaque, bleeding on probing, probing depth, total cultivable subgingival counts, and cultivable counts and proportions of six putative periodontal pathogens were carried out at baseline and after 14 days on two contralateral > or = 6 mm bleeding interproximal posterior sites in each of 11 adults with untreated chronic periodontitis. One of the sites was randomly assigned to daily patient-applied subgingival root brushing for 14 days, and the other to remain with the patient's pre-existing tooth brushing and flossing regimen. No other periodontal therapy was performed during the 14 test days. RESULTS: Significant reductions in plaque, bleeding on probing, probing depth, total subgingival counts, and levels of putative periodontal pathogens were found after 14 days of subgingival root brushing. Subgingival root brushing nearly eliminated bleeding on probing at test sites, reduced probing depths by a mean of 1.8 mm, and reduced cultivable subgingival proportions of six evaluated putative periodontal pathogens from a cumulative total of 14.1% to 0.8%. In comparison, no significant clinical or microbiological changes were detected after 14 days where the patient's pre-existing oral hygiene regimen remained unaltered. CONCLUSIONS: Subgingival root brushing over 14 days, in properly trained patients, induced favorable clinical and microbiological changes in deep periodontal pockets > or = 6 mm even in the absence of professional subgingival debridement.

Suppuration-associated bacteria in patients with chronic and aggressive periodontitis.

BACKGROUND: Suppuration (SUP) on probing may be an indication of active periodontal breakdown. The aim of the present study is to analyze which subgingival species are associated with SUP in patients with chronic (CP) and aggressive (AgP) periodontitis. METHODS: A total of 156 patients with CP and 66 with AgP were submitted to full-mouth periodontal examination and subgingival biofilm sampling (14 sites/patient). The counts of 44 bacterial species were determined by checkerboard. Comparisons between groups and sites were analyzed by the Mann-Whitney and Wilcoxon tests, respectively. Associations between frequency of SUP and bacterial species were analyzed by the Spearman correlation coefficient. RESULTS: The prevalence of SUP in patients with CP was 24.4%, and in patients with AgP it was 30.3%, and the percentage of SUP sites in the groups was 5.72% ± 1.06% and 6.96% ± 1.70%, respectively (P >0.05). SUP sites from patients with CP had significantly higher counts of Veillonella parvula, Dialister pneumosintes, Tannerella forsythia, and Prevotella nigrescens than SUP sites from patients with AgP (P <0.005). Significant positive correlations between high frequency of SUP and high levels of Actinomyces spp, Streptococcus spp., members of the orange complex, Acinetobacter baumannii, and Pseudomonas aeruginosa were observed in patients with CP (P <0.05). In patients with AgP, Actinomyces oris, Propionibacterium acnes, P. aeruginosa, Staphylococcus aureus, and Streptococcus sanguinis were positively associated with SUP, whereas Prevotella intermedia presented a negative association with SUP (P <0.05). CONCLUSIONS: SUP sites from patients with CP harbored significantly higher counts of several periodontal species than SUP sites from patients with AgP. Actinomyces spp., Streptococcus spp., members of the orange complex, T. forsythia, and certain non-oral pathogens were associated with a high number of sites with SUP.

Association of periodontitis with increased colonization by Prevotella nigrescens.

AIM: To estimate differences in the prevalence of Prevotella intermedia and Prevotella nigrescens in the subgingival plaque of patients with periodontitis (including aggressive and advanced chronic periodontitis) compared to healthy controls, and to search for significant associations with clinical status. METHODS: Sixteen patients and 16 healthy controls were enrolled in this study. Interproximal plaque index, oral hygiene index, gingival index, bleeding on probing, probing depth, and clinical attachment level were recorded. Samples of subgingival plaque were taken with paper points from four teeth of each individual and immediately plated on appropriate supplemented Columbia agar. Black pigmented colonies were identified with the Rapid ID32 A system, and further differentiated using matrix-assisted laser desorption ionization-time of flight mass spectrometry. For the statistical analysis, chi-squared test and the Mann-Whitney U-test were used. RESULTS: Prevotella nigrescens was isolated from 10 patients and three controls, while P. intermedia was isolated from only two patients. P. nigrescens was found more frequently in the subgingival plaque of patients (P = 0.029), and was significantly associated with high values of clinical indices (P ≤ 0.025). Significant differences for P. intermedia were not found. CONCLUSIONS: Periodontitis seems to be associated with increased colonization with P. nigrescens. Whether or not it is a major pathogen needs to be determined.

Prevalence of ß-lactamase-producing bacteria in human periodontitis.

BACKGROUND AND OBJECTIVE: Beta-lactam antibiotics prescribed in periodontal therapy are vulnerable to degradation by bacterial ß-lactamases. This study evaluated the occurrence of ß-lactamase-positive subgingival bacteria in chronic periodontitis subjects of USA origin, and assessed their in vitro resistance to metronidazole at a breakpoint concentration of 4 µg/mL. MATERIAL AND METHODS: Subgingival plaque specimens from deep periodontal pockets with bleeding on probing were removed from 564 adults with severe chronic periodontitis before treatment. The samples were transported in VMGA III and then plated onto: (i) nonselective enriched Brucella blood agar (EBBA) and incubated anaerobically for 7 d; and (ii) selective trypticase soy-bacitracin-vancomycin (TSBV) and incubated for 3 d in air + 5% CO2 . At the end of the incubation periods, the bacterial test species were identified and quantified. Specimen dilutions were also plated onto EBBA plates supplemented with 2 µg/mL of amoxicillin, a combination of 2 µg/mL of amoxicillin plus 2 µg/mL of the ß-lactamase inhibitor clavulanic acid, or 4 µg/mL of metronidazole, followed by anaerobic incubation for 7 d. Bacterial test species presumptively positive for ß-lactamase production were identified by growth on EBBA primary isolation plates supplemented with amoxicillin alone and no growth on EBBA primary isolation plates containing both amoxicillin plus clavulanic acid. A subset of such isolates was subjected to nitrocefin-based chromogenic disk testing to confirm the presence of ß-lactamase activity. In vitro resistance to 4 µg/mL of metronidazole was noted when growth of test species occurred on metronidazole-supplemented EBBA culture plates. RESULTS: Two-hundred and ninety-four (52.1%) of the study subjects yielded ß-lactamase-producing subgingival bacterial test species, with Prevotella intermedia/nigrescens, Fusobacterium nucleatum and other Prevotella species most frequently identified as ß-lactamase-producing organisms. Of the ß-lactamase-producing bacterial test species strains recovered, 98.9% were susceptible in vitro to metronidazole at 4 µg/mL. CONCLUSION: The occurrence of ß-lactamase-positive subgingival bacterial species in more than half of the subjects with severe chronic periodontitis raises questions about the therapeutic potential of single-drug regimens with ß-lactam antibiotics in periodontal therapy. The in vitro effectiveness of metronidazole against nearly all recovered ß-lactamase-producing subgingival bacterial species further supports clinical periodontitis treatment strategies involving the combination of systemic amoxicillin plus metronidazole.

Biofilms of black tooth stains: PCR analysis reveals presence of Streptococcus mutans

This study investigated the presence of the black-pigmented bacteria Prevotella nigrescens and Prevotella intermedia, the non-black-pigmented bacteria Actinomyces spp and particularly the cariogenic pathogen Streptococcus mutans in the dental biofilms of patients with or without black extrinsic tooth stains, using the multiplex polymerase chain reaction (PCR) technique. Analysis of the dental biofilms of patients with (n=26) or without (n=26) black tooth stains was performed using duplex PCR for the 16S ribosomal RNA gene (P. nigrescens, P. intermedia, Actinomyces spp) and glucosyltransferase-I gene for S. mutans. P. nigrescens and S. mutans were the most frequent bacteria detected in both groups. The least frequently detected were P. intermedia and Actinomyces spp. The similar bacterial composition of dental biofilms of black tooth stains and healthy tooth surfaces indicates that black tooth stains are not free of cariogenic bacteria.

O objetivo deste estudo foi investigar a presença das bactérias pigmentadoras de negro Prevotella nigrescens e Prevotella intermedia, da não pigmentadora de negro Actinomyces spp e particularmente a bactéria cariogênica Streptococcus mutans, no biofilme dentário de pacientes com ou sem manchas dentárias extrínsecas negras, utilizando a técnica multiplex PCR (reação em cadeia da polimerase). Análises do biofilme dentário de pacientes com manchas (n=26) e sem manchas (n=26) foram realizadas utilizando a multiplex PCR para o gene 16S RNA ribosomal (P. nigrescens, P. intermedia, Actinomyces spp) e o gene glucosiltransferase-I para S. mutans. P. nigrescens e S. mutans foram as bactérias mais frequentemente detectadas em ambos os grupos. As menos frequentemente detectadas foram P. intermedia e Actinomyces spp. A similaridade entre a composição bacteriana dos biofilmes dentários das manchas dentárias extrínsecas negras e das superfícies dentárias sem manchas indicam que as manchas dentárias extrínsecas negras não estão livres de bactérias cariogênicas.