Ana Paula Vieira Colombo

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.

Actinomyces Species, Streptococci, and Enterococcus faecalis in Primary Root Canal Infections

The purpose of this study was to evaluate the prevalence of Actinomyces species, streptococci, and Enterococcus faecalis in primary root canal infections by using a molecular genetic method. Samples were obtained from 53 infected teeth, of which 27 cases were diagnosed as acute periradicular abscesses. DNA was extracted to evaluate the occurrence of 13 bacterial species by using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Polymerase chain reaction using an ubiquitous bacterial primer was undertaken to check the presence of bacterial DNA in clinical samples. All root canal samples contained bacteria as demonstrated by polymerase chain reaction. The checkerboard DNA-DNA hybridization assay allowed the detection of streptococci in 22.6% of the samples, Actinomyces species in 9.4%, and E. faecalis in 7.5%. The most prevalent species were members of the Streptococcus anginosus group. With regard to the asymptomatic lesions, the most prevalent species were S. intermedius (11.5% of the cases), E. faecalis (11.5%), and S. anginosus (7.7%). S. constellatus was the most prevalent species in pus samples (25.9% of the cases). The other most prevalent species in abscessed teeth were A. gerencseriae (14.8%), S. gordonii (11.1%), S. intermedius (11.1%), A. israelii (7.4%), S. anginosus (7.4%), and S. sanguis (7.4%). S. constellatus was the only species positively associated with acute periradicular abscess (p < 0.01).

Association of red complex, A. actinomycetemcomitans and non-oral bacteria with periodontal diseases

OBJECTIVE Pathogens related to systemic infections have been detected in the periodontal microbiota. The relationship amongst these pathogens, periodontal bacteria and periodontal clinical status is poorly understood. This study evaluated the association amongst red complex, A. actinomycetemcomitans (A.a) and non-oral pathogenic bacteria in subjects with good periodontal health (PH), gingivitis (G), chronic (CP) and aggressive (AP) periodontitis. METHODS Subgingival biofilm samples were obtained from 51 PH, 42 G, 219 CP and 90 AP subjects. The presence and levels of A.a, red complex (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola), Acinetobacter baumannii, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus were determined by DNA probes and DNA-DNA hybridization technique. RESULTS CP and AP subjects presented significantly higher prevalence and levels of A.a, red complex and A. baumannii than G and PH individuals (p<0.01), whereas S. aureus was detected in lower frequency and counts in AP as compared to the other groups (p<0.001). The predictor variables age, prevalence of red complex, and the presence of A. baumannii and P. aeruginosa were strongly associated with the frequency of sites with PD and CAL ≥5 mm. Increasing age (OR 1.08), high frequency of red complex (OR 6.10), and the presence of A.a with P. aeruginosa (OR 1.90) were associated with periodontal disease (p<0.001). Subjects harbouring a high prevalence of A.a, A. baumannii, and red complex with P. aeruginosa were more likely to have AP than CP (p<0.001). CONCLUSION Putative periodontal pathogens and non-oral bacteria alone or in association were strongly associated with periodontitis.

Endodontic therapy associated with calcium hydroxide as an intracanal dressing: microbiologic evaluation by the checkerboard DNA-DNA hybridization technique.

This study evaluated the predominant microbiota of infected necrotic pulps and the effects of calcium hydroxide therapy on these microorganisms by the checkerboard DNA-DNA hybridization technique. Conventional endodontic therapy associated with calcium hydroxide as intracanal dressing was performed in 12 single-rooted teeth with pulp necrosis and periradicular bone lesion. Samples were collected from the canal at baseline and 14 days after therapy, and the presence of 44 bacterial species was determined by the checkerboard method. Significant differences in the microbiota from baseline to post-therapy were sought by the paired-samples t test. The most prevalent species included F. nucleatum ss. vincentii, C. sputigena, C. ochracea, S. constellatus, V. parvula, P. gingivalis, P. melaninogenica, and S. sanguis. Most of the microorganisms were reduced after treatment, particularly A. gerencseriae, A. israelii, A. naeslundii, C. gingivalis, C. ochracea, P. gingivalis, S. noxia, S. sanguis, and S. oralis (p < 0.05). Conversely, A. actinomycetemcomitans, C. sputigena, and E. corrodens increased in numbers after therapy. These results indicate that conventional endodontic therapy with calcium hydroxide results in the reduction of pathogenic species associated with pulp necrosis. However, its use is limited, because it did not eliminate the whole spectrum of microorganisms.

Microbial signature profiles of periodontally healthy and diseased patients

AIM To determine microbial profiles that discriminate periodontal health from different forms of periodontal diseases. METHODS Subgingival biofilm was obtained from patients with periodontal health (27), gingivitis (11), chronic periodontitis (35) and aggressive periodontitis (24), and analysed for the presence of >250 species/phylotypes using HOMIM. Microbial differences among groups were examined by Mann-Whitney U-test. Regression analyses were performed to determine microbial risk indicators of disease. RESULTS Putative and potential new periodontal pathogens were more prevalent in subjects with periodontal diseases than periodontal health. Detection of Porphyromonas endodontalis/Porphyromonas spp. (OR 9.5 [1.2-73.1]) and Tannerella forsythia (OR 38.2 [3.2-450.6]), and absence of Neisseria polysaccharea (OR 0.004 [0-0.15]) and Prevotella denticola (OR 0.014 [0-0.49], p < 0.05) were risk indicators of periodontal disease. Presence of Aggregatibacter actinomycetemcomitans (OR 29.4 [3.4-176.5]), Cardiobacterium hominis (OR 14.9 [2.3-98.7]), Peptostreptococcaceae sp. (OR 35.9 [2.7-483.9]), P. alactolyticus (OR 31.3 [2.1-477.2]), and absence of Fretibacterium spp. (OR 0.024 [0.002-0.357]), Fusobacterium naviforme/Fusobacterium nucleatum ss vincentii (OR 0.015 [0.001-0.223]), Granulicatella adiacens/Granulicatella elegans (OR 0.013 [0.001-0.233], p < 0.05) were associated with aggressive periodontitis. CONCLUSION There were specific microbial signatures of the subgingival biofilm that were able to distinguish between microbiomes of periodontal health and diseases. Such profiles may be used to establish risk of disease.

Systemic Antimicrobials Adjunctive to a Repeated Mechanical and Antiseptic Therapy for Aggressive Periodontitis: A 6-Month Randomized Controlled Trial

BACKGROUND The purpose of this study is to compare the additional benefit of systemic antimicrobials versus placebos to a repeated mechanical instrumentation combined with comprehensive local chemical plaque control for the periodontal treatment of generalized aggressive periodontitis (GAgP). METHODS This was a 6-month randomized, double-masked, placebo-controlled clinical trial. All GAgP patients received full-mouth disinfection followed by staged scaling and root planing without (placebo group; n = 17) or with (test group; n = 18) systemic antimicrobials (500 mg amoxicillin [AMX] + 250 mg metronidazole [MET]; three times a day for 10 days). Clinical parameters were measured at baseline and 3 and 6 months post-therapy. Significant differences between groups at baseline were sought by using the Mann-Whitney U test, whereas comparisons over time were examined by using a general linear model repeated measures procedure. RESULTS Both groups demonstrated similar improvements in most parameters over time. The test group presented a greater mean probing depth (PD) reduction and clinical attachment level (CAL) gain at sites with initially moderate PD at 6 months (P <0.03). No differences were seen between groups regarding mean reductions and mean gains, respectively, for PD and CAL initially ≥7 mm. The test group presented a higher percentage of sites that improved ≥2 mm and ended up with PD ≤4 mm or a lower percentage of sites that worsened ≥2 mm and remained with PD >4 mm at 3 months (P <0.01). No differences were noticed between groups for these parameters at 6 months. CONCLUSION AMX + MET brought additional clinical effects to the repeated mechanical and antiseptic treatment of GAgP in a very short time (3 months), which tended to fade away over time (6 months).

Comparison of 16S rDNA-based PCR and checkerboard DNA-DNA hybridisation for detection of selected endodontic pathogens.

Molecular methods have been used recently to investigate the bacteria encountered in human endodontic infections. The aim of the present study was to compare the ability of a 16S rDNA-based PCR assay and checkerboard DNA-DNA hybridisation in detecting Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Peptostreptococcus micros, Porphyromonas endodontalis, Por. gingivalis and Treponema denticola directly from clinical samples. Specimens were obtained from 50 cases of endodontic infections and the presence of the target species was investigated by whole genomic DNA probes and checkerboard DNA-DNA hybridisation or taxon-specific oligonucleotides with PCR assay. Prevalence of the target species was based on data obtained by each method. The sensitivity and specificity of each molecular method was compared with the data generated by the other method as the reference--a value of 1.0 representing total agreement with the chosen standard. The methods were also compared with regard to the prevalence values for each target species. Regardless of the detection method used, T. denticola, Por. gingivalis, Por. endodontalis and B. forsythus were the most prevalent species. If the checkerboard data for these four species were used as the reference, PCR detection sensitivities ranged from 0.53 to 1.0, and specificities from 0.5 to 0.88, depending on the target bacterial species. When PCR data for the same species were used as the reference, the detection sensitivities for the checkerboard method ranged from 0.17 to 0.73, and specificities from 0.75 to 1.0. Accuracy values ranged from 0.6 to 0.74. On the whole, matching results between the two molecular methods ranged from 60% to 97.5%, depending on the target species. The major discrepancies between the methods comprised a number of PCR-positive but checkerboard-negative results. Significantly higher prevalence figures for Por. endodontalis and T. denticola were observed after PCR assessment. There was no further significant difference between the methods with regard to detection of the other target species.

Kinin Danger Signals Proteolytically Released by Gingipain Induce Fimbriae-Specific IFN-γ- and IL-17-Producing T Cells in Mice Infected Intramucosally with Porphyromonas gingivalis

Porphyromonas gingivalis, a Gram-negative bacterium that causes periodontitis, activates the kinin system via the cysteine protease R-gingipain. Using a model of buccal infection based on P. gingivalis inoculation in the anterior mandibular vestibule, we studied whether kinins released by gingipain may link mucosal inflammation to T cell-dependent immunity through the activation of bradykinin B2 receptors (B2R). Our data show that P. gingivalis W83 (wild type), but not gingipain-deficient mutant or wild-type bacteria pretreated with gingipain inhibitors, elicited buccal edema and gingivitis in BALB/c or C57BL/6 mice. Studies in TLR2−/−, B2R−/−, and neutrophil-depleted C57BL/6 mice revealed that P. gingivalis induced edema through the sequential activation of TLR2/neutrophils, with the initial plasma leakage being amplified by gingipain-dependent release of vasoactive kinins from plasma-borne kininogens. We then used fimbriae (Fim) Ag as a readout to verify whether activation of the TLR2→PMN→B2R axis (where PMN is polymorphonuclear neutrophil) at early stages of mucosal infection had impact on adaptive immunity. Analyzes of T cell recall responses indicated that gingipain drives B2R-dependent generation of IFN-γ-producing Fim T cells in submandibular draining lymph nodes of BALB/c and C57BL/6 mice, whereas IL-17-producing Fim T cells were generated only in BALB/c mice. In summary, our studies suggest that two virulence factors, LPS (an atypical TLR2 ligand) and gingipain, forge a trans-cellular cross-talk between TLR2 and B2R, thus forming an innate axis that guides the development of Fim-specific T cells in mice challenged intrabuccally by P. gingivalis. Ongoing research may clarify whether kinin-driven modulation of T cell responses may also influence the severity of chronic periodontitis.

Subgingival microbial profiles of generalized aggressive and chronic periodontal diseases

OBJECTIVE The aim of this study was to distinguish between generalized aggressive (GAgP) and chronic periodontitis (CP) based on the subgingival microbial profiles predominant in these diseases. METHODS Two-hundred and sixty subjects, 75 with GAgP and 185 with CP were recruited. Full-mouth clinical measurements were recorded. Individual subgingival plaque samples were taken from 7 sites per subject and analyzed for the prevalence and levels of 51 species by chequerboard. Differences between groups were examined by the Mann-Whitney test. Associations between bacterial species and GAgP were examined by logistic regression analysis. RESULTS Actinomyces gerensceriae, Actinomyces israelli, Eubacterium nodatum and Propionibacterium acnes were detected in significantly greater counts in GAgP, whereas Capnocytophaga ochracea, Fusobacterium periodonticum, Staphylococcus aureus and Veillonella parvula were more predominant in CP patients (adjusted p < 0.001). E. nodatum (at mean levels ≥4 × 10(5)) increased significantly the probability of a subject being diagnosed with GAgP than CP (OR 2.44 [0.96-6.20]), whereas P. gingivalis (OR 0.34 [0.11-0.93]) and T. denticola (OR 0.35 [0.11-0.94]) were associated with CP. CONCLUSIONS Very few subgingival species differed in prevalence and/or levels between GAgP and CP in this sample population. In particular, E. nodatum was strongly related to GAgP, whereas P. gingivalis and T. denticola were associated with CP.

Detection of Helicobacter pylori, Enterococcus faecalis, and Pseudomonas aeruginosa in the subgingival biofilm of HIV-infected subjects undergoing HAART with chronic periodontitis

This study compared the frequency of Helicobacter pylori, Enterococcus faecalis, and Pseudomonas aeruginosa in the subgingival microbiota of HIV-seropositive and HIV-seronegative subjects with periodontitis or clinically healthy periodontal tissues. Fifty-four subjects were distributed into two HIV-seropositive groups (chronic periodontitis [HCP = 13] and periodontal health [HH = 10]) and two HIV-seronegative groups (chronic periodontitis [CP = 17] and periodontal health [H = 14]). The detection of bacterial species was carried out by polymerase chain reaction (PCR). CP patients showed significantly more periodontal destruction, inflammation, and supragingival plaque than HCP patients (P < 0.05). All species were detected at a higher prevalence in CP and HCP than H individuals (P < 0.01). In the HIV groups, H. pylori was significantly more prevalent in periodontitis compared to healthy patients (P < 0.01). A higher frequency of E. faecalis and P. aeruginosa was observed in the subgingival biofilm of HH than H subjects (P < 0.01). Moreover, E. faecalis was detected significantly more often in HIV-seropositive compared to HIV-seronegative patients, regardless of periodontal status (P < 0.01). These data indicate that H. pylori is frequently detected in the subgingival microbiota of periodontitis subjects. In contrast, HIV-seropositive patients with either periodontitis or periodontal health present a high prevalence of E. faecalis.