Renata Souto

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.

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.

Prevalence of potential bacterial respiratory pathogens in the oral cavity of hospitalised individuals

OBJECTIVE To assess the prevalence of oral colonisation by bacterial respiratory pathogens in hospitalised patients. METHODS Thirty patients undergoing myocardium revascularisation surgery were evaluated. At baseline (pre-operative phase), full-mouth clinical periodontal assessment was performed. Saliva and biofilm samples were obtained from subjects at baseline and at the post-operative phase, after orotracheal extubation. DNA was extracted from samples and species of Acinetobacter, Pseudomonas, Staphylococcus aureus and Dialister pneumosintes were detected by PCR or culture (for staphylococci isolates). RESULTS Most of the subjects were males, with history of hypertension and smoking. Thirteen were edentulous (ED) and 17 were dentate (DE), with moderate chronic periodontitis. The most prevalent bacteria in saliva were Staphylococcus spp. (85.7%), Pseudomonas spp. (83.8%), and Acinetobacter spp. (53.3%). There was a trend for D. pneumosintes to be more frequently detected in DE (43.7%) than ED (11.5%) patients. In plaque samples, DE with >14 teeth showed a higher prevalence of Pseudomonas spp. (100%) than individuals with < or =14 teeth (69.1%; p=0.048). Conversely, P. aeruginosa was more prevalent in subjects with fewer teeth (35.5%) than with >14 teeth (5.7%; p=0.037). All staphylococci isolates were coagulase-negative, and about 11% were positive for the mecA gene. These mecA-positive isolates showed a tendency to increase in all samples, whereas P. aeruginosa reduced after surgery. A strong correlation between the presence of Acinetobacter spp. and Pseudomonas spp. was observed (rho=0.886, p<0.05). CONCLUSIONS The oral cavity of hospitalised patients harbours high frequencies of bacterial respiratory pathogens, supporting its potential role as a reservoir for these species.

Ex vivo antimicrobial efficacy of the EndoVac® system plus photodynamic therapy associated with calcium hydroxide against intracanal Enterococcus faecalis

AIM To evaluate the ex vivo efficacy of the EndoVac system and photodynamic treatment (PDT) as adjuncts to chemomechanical debridement associated with calcium hydroxide (CaOH2 ) in reducing the levels of intracanal Enterococcus faecalis. METHODOLOGY One hundred and twenty-five sterile premolar teeth were conventionally accessed, prepared and then contaminated with E. faecalis (ATCC 29212) for 30 days. Teeth were randomly divided into 4 groups: Control (chemomechanical debridement with conventional irrigation); Endovac (chemomechanical debridement with EndoVac system); PDT (chemomechanical debridement with conventional irrigation and PDT) and Endovac+PDT (chemomechanical debridement with EndoVac and PDT). The irrigants used in all groups were 5.25% sodium hypochlorite and 17% EDTA. After treatment, an intracanal dressing (CaOH2 ) was applied in all canals for 7 days. Samples were obtained before (T1) and after the therapeutic procedures (T2) and, after intracanal medication (T3), plated onto BHI media and incubated (37 °C, 48 h) to determine the colony-forming units (CFU mL(-1) ). RESULTS The overall mean cell counts (CFU mL(-1) ) of E. faecalis were high at the initial contamination (T1). A significant reduction (P < 0.05) of E. faecalis mean counts was observed in all groups from baseline (T1) to both post-therapy samplings (T2 and T3); no differences amongst the groups were detected. No significant change in bacterial counts from T2 to T3 was detected. CONCLUSION The adjunctive use of the EndoVac system and the photodynamic treatment, in combination or not, was as effective as the conventional chemomechanical debridement associated with CaOH2 on reducing the counts of intracanal E. faecalis.

Prevalence of Pseudomonas aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with chronic periodontal infection

P. aeruginosa and Acinetobacter spp. are important pathogens associated with late nosocomial pneumonia in hospitalized and institutionalized individuals. The oral cavity may be a major source of these respiratory pathogens, particularly in the presence of poor oral hygiene and periodontal infection. This study investigated the prevalence of P. aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with periodontal disease or health. Samples were obtained from 55 periodontally healthy (PH) and 169 chronic periodontitis (CP) patients. DNA was obtained from the samples and detection of P. aeruginosa and Acinetobacter spp. was carried out by multiplex and nested PCR. P. aeruginosa and Acinetobacter spp. were detected in 40% and 45% of all samples, respectively. No significant differences in the distribution of these microorganisms between men and women, subgingival biofilm and saliva samples, patients ≤ 35 and > 35 years of age, and smokers and non-smokers were observed regardless periodontal status (p > 0.05). In contrast, the frequencies of P. aeruginosa and Acinetobacter spp. in saliva and biofilm samples were significantly greater in CP than PH patients (p < 0.01). Smokers presenting P. aeruginosa and high frequencies of supragingival plaque were more likely to present CP than PH. P. aeruginosa and Acinetobacter spp. are frequently detected in the oral microbiota of CP. Poor oral hygiene, smoking and the presence of P. aeruginosa are strongly associated with periodontitis.

The Association Between Detectable Plasmatic Human Immunodeficiency Virus (HIV) Viral Load and Different Subgingival Microorganisms in Brazilian Adults With HIV: A Multilevel Analysis

BACKGROUND This study investigates the association between detectable plasmatic human immunodeficiency virus (HIV) viral load (HVL) and high levels of periodontal- and non-periodontal-related microorganisms in the subgingival microbiota of individuals with HIV. METHODS Thirty-seven individuals with HIV were divided into two groups: 1) detectable HVL (n = 15); and 2) undetectable HVL (n = 22). Subgingival biofilm samples were obtained, and the levels of 35 microbial species were determined by the checkerboard DNA-DNA hybridization method. Periodontal clinical measures and laboratory and sociodemographic data were also registered. χ(2) test, Fisher exact test, and Mann-Whitney U test were used to compare groups. Multilevel ordinal regression models were used to test the association between HVL and the levels of 35 microbial species in subgingival biofilm, adjusted for confounders. RESULTS Of the 35 species studied, 11 (31.4%) showed higher mean levels in the detectable HVL group than undetectable HVL group (P <0.001). These species included Actinomyces naeslundii II, Actinomyces israelii, Actinomyces odontolyticus, Veillonella parvula, Capnocytophaga gingivalis, Eikenella corrodens, Campylobacter concisus, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Candida albicans. Significant associations between detectable HVL and high levels of microorganisms, adjusted for confounders, were observed for A. naeslundii I, Actinomyces gerencseriae, C. gingivalis, E. corrodens, C. concisus, Prevotella nigrescens, T. forsythia, and Dialister pneumosintes. CONCLUSION Detectable plasmatic HVL in individuals with HIV was associated with elevated levels of known periodontal pathogens, such as P. nigrescens, T. forsythia, and E. corrodens, as well as C. concisus, C. gingivalis, and D. pneumosintes in the subgingival biofilm.

Adsorption of chlorhexidine on synthetic hydroxyapatite and in vitro biological activity

The kinetic of chlorhexidine digluconate (CHXDG) uptake from aqueous solution by hydroxyapatite (HA) was investigated by ultraviolet (UV) analysis performed in HA powder (UV-solid) after the CHX adsorption. Adsorption isotherm of chlorhexidine (CHX) uptake was modeled by a combination of Languimir and Langmuir-Freundlich mechanisms. Strong molecule-molecule interactions and positive cooperativity predominated in the surface when CHX concentration was above 8.6 μg(CHX)/mg(HA). UV-solid spectra (shape, intensity and band position) of CHX bound to HA revealed that long-range molecular structures, such as aggregates or micelles, started to be formed at low CHX concentrations (1.52 μg(CHX)/mg(HA)) and predominated at high concentrations. Grazing-incidence X-ray diffraction (GIXRD) analysis from synchrotron radiation discarded the formation of crystalline structures on HA surface or precipitation of CHX crystalline salts, as suggested in previous works. The effect of the HA/CHX association on HA in vitro bioactivity, cytotoxicity and CHX antimicrobial activity was evaluated. It was shown that CHX did not inhibit the precipitation of a poorly crystalline apatite at HA/CHX surface after soaking in simulating body fluid (SBF). Cell viability studies after exposure to extracts of HA and HA/CHX showed that both biomaterials did not present significant in vitro toxicity. Moreover, HA/CHX inhibited Enterococcus faecalis growth for up to 6 days, revealing that binding to HA did not affect antimicrobial activity of CHX and reduced bacterial adhesion. These results suggested that HA/CHX association could result in a potential adjuvant antimicrobial system for clinical use.