Antimicrobial activity of violacein against oral bacteria associated with halitosis: an in vitro study

Introduction: violacein is a natural purple pigment produced by environmental bacteria that presents antimicrobial activity, particularly against Gram-positive bacteria. Intraoral halitosis (IOH) is a condition defined by the unpleasant odor emanating from the mouth, whose main source are volatile sulfur compounds, produced by Gram-negative oral bacteria on the tongue coating. In IOH treatment, antimicrobials have been indicated as chemical adjuncts, including natural products. Objective: thus, this study tested the antimicrobial activity of a violacein extract on key IOH-related bacteria (Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, Prevotella intermedia, Solobacterium moorei). Materials and Methods: bacteria were cultured in fastidious anaerobe blood agar in anaerobiosis, and 109 cells/ml suspensions were plated. Crude extract of violacein obtained from Chromobacterium violaceum was diluted in a 25% ethanol aqueous solution to 8, 4, 2, 1, 0.5 and 0.25 mg/ml. Using the disk agar diffusion method, 10 µl aliquots of each dilution were deposited on the seeded plates. Chlorohexidine (0.1%) and 25% ethanol solution were used as controls. Plates were incubated in anaerobiosis at 37°C for 72h, and the inhibition halos were recorded. Results: although chlorhexidine showed higher inhibition halos than the violacein extract, most species were inhibited at 4 and 8 mg/ml concentrations (p<0.05). P. gingivalis followed by F. nucleatum were the most affected species in relation to the other bacteria, although statistical significance was only reached for P. gingivalis (p<0.05). Conclusion: crude violacein extract from C. violaceum demonstrated antimicrobial activity against IOH-associated oral bacteria, being a potential antimicrobial to be studied as an adjunct in the control of IOH.

Introdução: a violaceína é um pigmento roxo natural produzido por bactérias ambientais que apresenta ação antimicrobiana, particularmente contra bactérias Gram-positivas. A halitose intraoral (HIO) é uma condição definida pelo odor desagradável que emana da boca, cuja principal fonte são os compostos sulfurados voláteis produzidos por bactérias Gram-negativas da saburra lingual. No tratamento da HIO, antimicrobianos têm sido indicados como adjuvantes, incluindo produtos naturais. Objetivo: assim, este estudo avaliou o potencial antimicrobiano de um extrato de violaceína em patógenos-chave da HIO (Porphyromonas gingivalis, Porphyromonas endodontalis, Fusobacterium nucleatum, Prevotella intermedia, Solobacterium moorei). Materiais e Métodos: bactérias foram cultivadas em meio ágar sangue para fastidiosos, em anaerobiose, e suspensões de 109 células/ml foram semeadas. O extrato bruto de violaceína obtido de Chromobacterium violaceum foi diluído em solução aquosa com 25% de etanol nas concentrações de 8, 4, 2, 1, 0,5 e 0,25 mg/ml. Através do método de disco difusão, 10 µl de cada diluição foram depositados nas placas semeadas. A clorexidina (0,1%) e a solução etanólica a 25% foram usadas como controles. As placas foram incubadas em anaerobiose a 37°C por 72h, e os halos de inibição foram registrados. Resultados: embora a clorexidina tenha apresentado os maiores halos de inibição do do que o extrato, a maioria das espécies foi inibida nas concentrações de 4 e 8 mg/ml (p<0,05). P. gingivalis e F. nucleatum foram as espécies mais afetadas em relação às outras bactérias, porém só foi observada significância estatística para P. gingivalis (p<0,05). Conclusão: o extrato bruto de violaceína de C. violaceum demonstrou atividade antimicrobiana contra bactérias orais associadas a HIO, sendo um potencial antimicrobiano a ser estudado como adjuvante no controle da HIO.

Colonization dynamics of subgingival microbiota in recently installed dental implants compared to healthy teeth in the same individual: a 6-month prospective observational study

Abstract Objectives To evaluate the colonization dynamics of subgingival microbiota established over six months around newly installed dental implants in periodontally healthy individuals, compared with their corresponding teeth. Methodology Seventeen healthy individuals assigned to receive single dental implants participated in the study. Subgingival biofilm was sampled from all implant sites and contralateral/ antagonist teeth on days 7, 30, 90, and 180 after implant installation. Microbiological analysis was performed using the Checkerboard DNA-DNA hybridization technique for detection of classical oral taxa and non-oral microorganisms. Significant differences were estimated by Mann-Whitney and Friedman tests, while associations between implants/teeth and target species levels were assessed by linear regression analysis (LRA). Significance level was set at 5%. Results Levels of some species were significantly higher in teeth compared to implants, respectively, at day 7 ( V.parvula , 6 × 10 5 vs 3 × 105 ; Milleri streptococci , 2 × 10 6 vs 6 × 10 5 ; Capnocytophaga spp., 2 × 10 6 vs 9 × 10 5 ; E.corrodens , 2 × 10 6 vs 5 × 10 5 ; N. mucosa , 2 × 10 6 vs 5 × 10 5 ; S.noxia , 2 × 10 6 vs 3 × 10 5 ; T.socranskii , 2 × 10 6 vs 5 × 10 5 ; H.alvei , 4 × 10 5 vs 2 × 10 5 ; and Neisseria spp., 6 × 10 5 vs 4 × 10 4 ), day 30 ( V.parvula , 5 × 10 5 vs 10 5 ; Capnocytophaga spp., 1.3 × 10 6 vs 6.8 × 10 4 ; F.periodonticum , 2 × 10 6 vs 10 6 ; S.noxia , 6 × 10 5 vs 2 × 10 5 ; H.alvei , 8 × 10 5 vs 9 × 10 4 ; and Neisseria spp., 2 × 10 5 vs 10 6 ), day 120 ( V.parvula , 8 × 10 5 vs 3 × 10 5 ; S.noxia , 2 × 10 6 vs 0; and T.socranskii , 3 × 10 5 vs 8 × 10 4 ), and day 180 ( S.enterica subsp. enterica serovar Typhi, 8 × 10 6 vs 2 × 10 6 ) (p<0.05). Implants showed significant increases over time in the levels of F.nucleatum , Gemella spp., H.pylori , P.micra , S.aureus , S.liquefaciens , and T.forsythia (p<0.05). LRA found that dental implants were negatively correlated with high levels of S. noxia and V. parvula (β=-0.5 to -0.3; p<0.05). Conclusions Early submucosal microbiota is diverse and only a few species differ between teeth and implants in the same individual. Only 7 days after implant installation, a rich microbiota can be found in the peri-implant site. After six months of evaluation, teeth and implants show similar prevalence and levels of the target species, including known and new periodontopathic species.

Periodontal disease severity is associated to pathogenic consortia comprising putative and candidate periodontal pathogens

Abstract Based on a holistic concept of polymicrobial etiology, we have hypothesized that putative and candidate periodontal pathogens are more frequently detected in consortia than alone in advanced forms of periodontal diseases (PD). Objective To correlate specific consortia of periodontal pathogens with clinical periodontal status and severity of periodontitis. Methodology Subgingival biofilm was obtained from individuals with periodontal health (113, PH), gingivitis (91, G), and periodontitis (209, P). Genomic DNA was purified and the species Aggregatibacter actinomycetemcomitans (Aa), Aa JP2-like strain, Porphyromonas gingivalis (Pg), Dialister pneumosintes (Dp), and Filifactor alocis (Fa) were detected by PCR. Configural frequency and logistic regression analyses were performed to correlate microbial consortia and PD. Results Aa + Pg in the presence of Dp (phi=0.240; χ2=11.9, p<0.01), as well as Aa JP2 + Dp + Fa (phi=0.186, χ2=4.6, p<0.05) were significantly more associated in advanced stages of P. The consortium Aa + Fa + Dp was strongly associated with deep pocketing and inflammation (p<0.001). The best predictors of disease severity (80% accuracy) included older age (OR 1.11 [95% CI 1.07 - 1.15], p<0.001), Black/African-American ancestry (OR 1.89 [95% CI 1.19 - 2.99], p=0.007), and high frequency of Aa + Pg + Dp (OR 3.04 [95% CI 1.49 - 6.22], p=0.002). Conclusion Specific microbial consortia of putative and novel periodontal pathogens, associated with demographic parameters, correlate with severe periodontitis, supporting the multifactorial nature of PD.

Long-term evaluation of the antimicrobial susceptibility and microbial profile of subgingival biofilms in individuals with aggressive periodontitis

This study evaluates the antimicrobial susceptibility and composition of subgingival biofilms in generalized aggressive periodontitis (GAP) patients treated using mechanical/antimicrobial therapies, including chlorhexidine (CHX), amoxicillin (AMX) and metronidazole (MET). GAP patients allocated to the placebo (C, n = 15) or test group (T, n = 16) received full-mouth disinfection with CHX, scaling and root planning, and systemic AMX (500 mg)/MET (250 mg) or placebos. Subgingival plaque samples were obtained at baseline, 3, 6, 9 and 12 months post-therapy from 3–4 periodontal pockets, and the samples were pooled and cultivated under anaerobic conditions. The minimum inhibitory concentrations (MICs) of AMX, MET and CHX were assessed using the microdilution method. Bacterial species present in the cultivated biofilm were identified by checkerboard DNA-DNA hybridization. At baseline, no differences in the MICs between groups were observed for the 3 antimicrobials. In the T group, significant increases in the MICs of CHX (p < 0.05) and AMX (p < 0.01) were detected during the first 3 months; however, the MIC of MET decreased at 12 months (p < 0.05). For several species, the MICs significantly changed over time in both groups, i.e., Streptococci MICs tended to increase, while for several periodontal pathogens, the MICs diminished. A transitory increase in the MIC of the subgingival biofilm to AMX and CHX was observed in GAP patients treated using enhanced mechanical therapy with topical CHX and systemic AMX/MET. Both protocols presented limited effects on the cultivable subgingival microbiota.

Antimicrobial efficacy of the EndoVac system plus PDT against intracanal Candida albicans: an ex vivo study

This study evaluated the ex vivoantimicrobial efficacy of the EndoVac system and the photodynamic therapy (PDT) associated with chemomechanical debridement (CMD) and intracanal medication on Candida albicans. Seventy-eight sterile premolars were contaminated withC. albicans (ATCC 21433) for 30 days. The teeth were randomly assigned into four groups: Control (CMD with conventional irrigation); Endovac (CMD with EndoVac system); PDT (CMD with conventional irrigation and PDT); and Endovac + PDT (CMD with EndoVac and PDT). After the therapies, intracanal dressing (calcium hydroxide) was applied to all teeth for seven days. Samples were obtained before (T1) and after the therapeutic procedures (T2), and after intracanal medication (T3), plated onto BHI agar and incubated (37°C, 48 h) to determine the colony-forming units (CFU)/mL. The overall mean level ofC. albicans at baseline was relatively high (1.85 x 106 ± 2.7 x 106 CFU mL-1). A significant reduction of C. albicans(p < 0.05) was observed over time (T1 to T2 and T1 to T3) in all groups. An additional significant reduction from T2 to T3 was observed only in the Endovac group (p < 0.05). No differences in mean reduction of C. albicans were observed among groups. However, the Endovac group presented the lowest mean counts of C. albicans at T3, whereas the PDT group had the highest counts of this microorganism (p < 0.05). The EndoVac system of irrigation/aspiration associated with CMD was the most effective therapeutic protocol for reducing intracanal levels of C. albicans. PDT showed a very limited efficacy against this species.

Estudo comparativo da atividade antimicrobiana "in vivo" de seis antissépticos bucais sobre a microbiota da saliva / A comparative in vitro study of antimicrobial activity of six commercial mouthwashes against salivary bacteria

Introdução: Soluções de antissépticos bucais podem desempenhar importante papel no controle químico do biofilme dentário. No entanto, os procedimentos de controle de qualidade relacionados com a atividade antimicrobiana destes enxaguatórios contra bactérias da cavidade oral não são bem divulgados. Objetivo: Avaliar a atividade antimicrobiana in vivo de seis soluções de antissépticos bucais disponíveis no mercado brasileiro, empregadas como enxaguatórios contra bactérias da saliva humana. Material e métodos: Um estudo in vivo foi desenvolvido com indivíduos voluntários (8 do sexo masculino e 7 do sexo feminino, variando de 18 a 63 anos de idade ), independente do estado de saúde bucal. Os seguintes produtos comerciais foram testados durante 2 horas após um único procedimento de bochecho: 1) Plax®, 2) Listerine®, 3) Periogard®, 4) Cepacol®, 5) Sanifill Premium® e 6) Oral B®.Os resultados foram analisados pelo teste de ANOVA de medidas repetidas e ANOVA one-way com um nível de significância de 5%. Resultados: Houve diferença significativa (p <0,05) observada na diminuição da carga microbiana para Plax® entre o início (antes anti-séptico bucal) e imediatamente após o bochecho (T0); para Periogard® entre os valores iniciais e T60 (60 minutos após o bochecho), na linha de base e T120 (120 minutos após o bochecho) e B® Oral entre os valores iniciais e T-30 (30 minutos após o bochecho). Periogard® apresentou a maior redução da carga microbiana salivares. Conclusão: Dos seis bochechos testados, Plax®, Oral B® e Periogard ® apresentou atividade antibacteriana imediata. Periogard® foi o anti-séptico bucal que mostrou a atividade mais prolongada contra bactérias anaeróbias salivares (AU).

Introduction: Mouthwashes solutions can play an important role in the chemical control of dental biofilm. However, quality control procedures related to antimicrobial activity of these solutions against oral bacteria are not well known. Objective: To evaluate in vivo antimicrobial activity of six mouthwashes solutions available in the Brazilian market against anaerobic salivary bacteria. Material and methods: An in vivo study was developed in human volunteers (8 male and 7 female, ranging from 18 to 63 years old), despite their oral health status. The following commercial products were tested after 2 hours of a single mouthwash procedure: 1) Plax®, 2) Listerine®, 3) Periogard®, 4) Cepacol®, 5) Sanifill Premium® and 6)Oral B®. Data were analyzed by ANOVA to repeated measures and ANOVA one-way with a significance level of 5%. Results: Statistically significant difference (p<0.05) was observed in the decrease of microbial counts to Plax® between baseline (before mouthwash) and immediately after mouthwash (T0); to Periogard® between baseline and T60 (60 minutes after mouthwash), baseline and T120 (120 minutes after mouthwash) and to Oral B® between baseline and T-30 (30 minutes after mouthwash). Periogard® showed the highest and delayed reduction of salivary microbial counts. Conclusion: Out of six tested mouthwashes, Plax®, Oral B® and Periogard ® showed immediate antibacterial activity. Periogard® was the oral anti-septic that showed the best delayed activity against salivary anaerobic bacteria (AU).

Methicillin-resistant Staphylococcus aureus in HIV patients: Risk factors associated with colonization and/or infection and methods for characterization of isolates - a systematic review

Staphylococcus aureus is an important cause of infections and HIV-infected individuals are frequently susceptible to this pathogen. The aim of this study was to perform a systematic review to identify both the risk factors associated with colonization/infection by methicillin-resistant S. aureus in HIV patients and the methods used for characterization of isolates. An electronic search of articles published between January 2001 and December 2013 was first conducted. Among 116 studies categorized as being at a quality level of A, B or C, only 9 studies were considered to have high methodological quality (level A). The majority of these studies were retrospective (4/9 studies). The risk factors associated with colonization/infection by S. aureus were use of antimicrobials (4/9 studies), previous hospitalization (4/9 studies) and low CD4+ T lymphocyte counts (<200 cells/μl) (3/9 studies). Culture in mannitol salt agar (3/9 studies) and the latex agglutination test (5/9 studies) were the main methods used for bacterial phenotypic identification. Genotypic profiles were accessed by pulsed-field gel electrophoresis (6/9 studies) and USA300 was the most prevalent lineage (5/9 studies). Most isolates were resistant to erythromycin (3/9 studies) and susceptible to vancomycin (4/9 studies). Ultimately, use of antimicrobials and previous hospitalization were the main risk factors for colonization/infection by methicillin-resistant S. aureus in HIV-infected individuals. However, the numbers of evaluated patients, the exclusion and inclusion criteria and the characterization of the S. aureus isolates were not uniform, which made it difficult to establish the characteristics associated with HIV patients who are colonized/infected by S. aureus.

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.

Effect of non-surgical periodontal treatment on the subgingival microbiota of patients with chronic kidney disease

This study investigated the effect of non-surgical periodontal therapy on the composition of subgingival microbiota of patients with chronic kidney disease (CKD). Sixteen CKD pre-dialysis individuals (CKD) and 14 individuals without clinical evidence of kidney disease (C) presenting chronic periodontitis were treated by scaling and root planing. Subgingival samples were collected from each patient and analyzed for their composition by checkerboard at baseline and 3 months post-therapy. Significant differences between groups at baseline were sought by the Mann-Whitney and χ² tests. Changes over time were examined by the Wilcoxon test. At baseline, the CKD group had significantly lower counts of E. faecalis compared to the C group (p < 0.05). After treatment, the levels of a greater number of species were reduced in the C group. Higher levels of A. israelii, C. rectus, F. periodonticum, P. micra, P. nigrescens, T. forsythia, N. mucosa, and S. anginosus (p < 0.05) were found in the CKD group compared to the C group. Also, non-responsive sites in CKD individuals harbored significantly higher levels of pathogenic species (T. forsythia, P. gingivalis, T. denticola, Fusobacterium spp., D. pneumosintes, E. faecalis and S. aureus; p < 0.05) than sites that responded to therapy, as well as non-responsive sites in the C group. The periodontitis-associated subgingival microbiota of CKD and systemically healthy individuals was similar in composition. However, high levels of pathogenic species persisted in the subgingival microbiota of patients with CKD after treatment.

Análise ultra-estrutural e microbiológica da associação de clorexidina e hidroxiapatita: potencial material para terapia de doença periodontal / Ultrastructural analysis of the association of chlorhexidine and hydroxyapatite: potential material for periodontal disease therapy

O objetivo deste trabalho foi realizar as avaliações ultra-estrutural e microbiológica de um biomaterial constituído de microesferas de hidroxiapatita associadas à clorexidina visando à aplicação no tratamento das periodontopatias. Foram desenvolvidos estudos sobre associação hidroxiapatita e clorexidina e feitas caracterizações físicas e morfológicas. Ensaios microbiológicos foram realizados para avaliação da capacidade de microesferas de hidroxiapatita contendo clorexidina de inibir o crescimento de E. faecalis. Os resultados demonstraram uma ligação estável entre hidroxiapatita e clorexidina sem prejudicar a estrutura cristalina da hidroxiapatita após a adsorção de clorexidina. A clorexidina presente na hidroxiapatita manteve atividade antimicrobiana inibindo o crescimento de E. faecalis. Os resultados obtidos sinalizam para a realização de testes in vivo e a aplicação do material obtido em estudos pré-clínicos.

The aim of this study was the ultrastructural and microbiological evaluations of a biomaterial composed of hydroxyapatite microspheres associated with chlorhexidine designing their application in the treatment of periodontal diseases. Studies were conducted on hydroxyapatite and chlorhexidine association and made physical and morphological characterizations. Microbiological tests were conducted to evaluate the ability of hydroxyapatite microspheres containing chlorhexidine to inhibit the proliferation of E. faecalis. The results demonstrated a stable Bond between hydroxyapatite and chlorhexidine, with maintenance of the crystalline structure of hydroxyapatite after adsorption of chlorhexidine. Chlorhexidine present in hydroxyapatite maintained antimicrobial activity inhibiting the growth of E. faecalis. The results point out the in vivo testing and application of the material obtained in preclinical studies.

Influence of IL-1 gene polymorphism on the periodontal microbiota of HIV-infected Brazilian individuals

This study investigated the association of IL-1A (+4845) and IL-1B (+3954) gene polymorphism with the subgingival microbiota and periodontal status of HIV-infected Brazilian individuals on highly active antiretroviral therapy (HAART). One hundred and five subjects were included in the study, distributed into 2 HIV groups [29 chronic periodontitis (CP+) and 30 periodontally healthy (H+)]; and 2 non-HIV groups (29 CP- and 17 H- patients). IL-1A and B were genotyped by PCR and restriction enzyme digestion. Thirty-three bacterial species were detected by checkerboard. Overall, we observed a prevalence of the allele 2 in the IL1-A and IL-1B polymorphism at 30.5 percent and 25.7 percent, respectively. Only 11.4 percent of all patients were composite genotype-positive, and 75 percent of those were HIV-infected. No significant associations between polymorphism of the IL-1 gene and periodontitis or HIV infection were observed. Likewise, no significant differences in the frequency and counts of any bacterial species were found between individuals with and without allele 2 (IL-1A or IL-1B). The data indicated that the IL-1 gene polymorphism is neither associated with periodontal destruction nor with high levels of subgingival species, including putative periodontal pathogens in HIV Brazilian individuals on HAART.

Lack of association between the TNF-α -308 (G/A) genetic polymorphism and periodontal disease in Brazilians

This study evaluated the frequency of the tumor necrosis factor-alpha (TNF-α) -308 G/A polymorphism in Brazilians with periodontal health (PH = 51), chronic periodontitis (CP = 74) and generalized aggressive periodontitis (GAgP = 38). Human DNA was obtained from mouthwash samples and TNF-α genotyping was performed by PCR and RFLP analyses. Differences in clinical and genetic parameters among groups were sought by Kruskal-Wallis, χ² and Fisher's exact tests. The allele -308G was detected in 91.7 percent, whereas the allele -308A was found in 35.4 percent of all subjects. No significant differences were observed in the frequency of these alleles (χ² = 2.610, p > 0.05) and the genotypes G/G, G/A, and A/A (χ² = 2.547, p = 0.636) among groups. The data suggest that the TNF-α -308 G/A polymorphism is not associated with periodontitis in this Brazilian population.

Prevalence of leukotoxic genotypes of Actinobacillus actinomycetemcomitans in Brazilians with chronic periodontitis

Actinobacillus actinomycetemcomitans is considered a major etiologic agent of aggressive periodontitis but this species has also been associated with other forms of periodontal disease. Further, highly leukotoxic strains are related to severity of disease. This investigation determined the prevalence of A. actinomycetemcomitans and the occurrence of the leukotoxin gene 530-bp deletion in Brazilian subjects with chronic periodontitis. Twenty periodontally healthy and 20 chronic periodontitis subjects were selected. Full-mouth clinical examination was carried out at 6 sites/tooth. Subgingival biofilm samples were collected from the 3 deepest sites and 3 healthy sites from periodontitis subjects, as well as from 3 sites of individuals with periodontal health. A. actinomycetemcomitans and the genetic deletion were determined by the polymerase chain reaction. Significant differences were sought by Mann-Whitney, Chi-square, and Wilcoxon sign tests. Periodontitis subjects presented a higher prevalence (75 percent) of A. actinomycetemcomitans than individuals with health (45 percent) (p = 0.053). A mean frequency of 57.5 percent of A. actinomycetemcomitans - positive sites was observed in the periodontitis group. Of those, 75 percent were diseased, whereas 40 percent were healthy sites (p = 0.0001). Healthy subjects showed a mean frequency of 35 percent of positive sites. In contrast, the genetic deletion was detected only in 4 diseased sites from 2 chronic periodontitis patients. A high prevalence of A. actinomycetemcomitans was observed in Brazilians with chronic periodontitis. However, the leukotoxin gene 530-bp deletion was rarely detected in the subgingival biofilm of these subjects.

Actinobacillus actinomycetemcomitans tem sido associado com diferentes formas de doenças periodontais, mas tal espécie é considerada o principal agente etiológico da doença periodontal agressiva. Algumas cepas de A. actinomycetemcomitans apresentam uma deleção de 530 pb na região promotora do operon do gene da leucotoxina, produzindo assim maiores quantidades desta toxina. Tal fato pode ter um importante papel na patogênese das doenças periodontais. A proposta do presente estudo foi determinar a prevalência do A. actinomycetemcomitans em amostras de biofilme subgengival de indivíduos brasileiros com periodontite crônica e saúde periodontal; bem como avaliar a distribuição do tipo genético leucotóxico desta espécie nestes indivíduos. Vinte pacientes com periodontite crônica e 20 controles com saúde periodontal foram selecionados. O exame clínico periodontal foi realizado em 6 sítios/dente em todos os dentes. Amostras de biofilme subgengival foram coletadas de 3 sítios com a maior profundidade de bolsa e 3 sítios sem doença dos pacientes com periodontite, assim como de 3 sítios aleatórios dos controles. A detecção do A. actinomycetemcomitans e a ocorrência da deleção genética foram realizadas utilizando a técnica de PCR diretamente nas amostras de biofilme. Pacientes com periodontite crônica apresentaram uma alta prevalência de A. actinomycetemcomitans (75 por cento) quando comparados aos indivíduos sem doença periodontal (45 por cento), porém essa diferença não foi significativa (p = 0.053). Foi observada uma freqüência média de 57.5 por cento de sítios com A. actinomycetemcomitans no grupo com periodontite. Destes sítios, 75 por cento eram sítios com doença, enquanto 40 por cento eram sítios saudáveis (p = 0.0001). Indivíduos sem doença periodontal apresentaram uma freqüência média de 35 por cento de sítios com A. actinomycetemcomitans. A deleção de 530-pb foi encontrada somente em 4 sítios doentes de 2 pacientes com periodontite crônica. Entretanto...

Prevalence of "non-oral" pathogenic bacteria in subgingival biofilm of subjects with chronic periodontitis

The oral cavity may act as a reservoir for several pathogens related to systemic infections. Therefore, the purpose of this study was to determine the prevalence and levels of pathogenic bacteria in the subgingival biofilm of chronic periodontitis lesions and healthy periodontal sites using the checkerboard DNA-DNA hybridization technique. 200 samples of subgingival biofilm from sites with periodontitis (probing pocket depth > 4 mm and /or clinical attachment level > 4 mm) and 200 samples from healthy sites of 14 patients with chronic periodontitis, as well as 200 samples from 3 periodontally healthy patients were obtained. The presence and levels of 11 pathogenic bacteria were determined using whole genomic DNA probes and the checkerboard method, computed for each site and then across sites within each subject group. Significance of differences in clinical and microbiological parameters among groups were examinated using the Mann-Whitney and Wilcoxon sign tests. The predominant species in all 600 samples included Corynebacterium diphtheriae, Enterococcus faecalis, Staphylococcus aureus,Acinetobacter baumannii and Escherichia coli. In general, most of the species were detected in greater prevalence and levels in sites with and without disease from patients with periodontitis in comparison to the periodontally healthy group. In particular, C. diphtheriae, E. coli, E. faecalis, P. aeruginosa and S. aureus were significantly more prevalent and detected in higher counts in diseased sites of patients with periodontal disease compared to healthy subjects (p < 0.05). Clinical signs of disease presented a positive correlation with the species A. baumannii, Streptococcus pyogenes, E. coli, S. aureus and P. aeruginosa. In conclusion, "non-oral" pathogenic bacteria are detected in high prevalence and levels in periodontal sites of chronic periodontitis patients.

Apesar da extensa literatura sobre a associação de bactérias orais e doenças sistêmicas, tem se dado pouca atenção à cavidade oral como um reservatório de bactérias patogênicas "não-orais". A microbiota oral é constituída de mais de 300 espécies bacterianas já caracterizadas, além de organismos não cultiváveis que vêm sendo descobertos através de técnicas moleculares. O objetivo do presente estudo foi determinar a prevalência e os níveis de 11 bactérias patogênicas "não-orais" em sítios periodontais de pacientes com periodontite e de pacientes saudáveis, utilizando a técnica do "Checkerboard DNA-DNA hybridization". 200 amostras de biofilme subgengival de sítios doentes (PBS > 4mm e/ou NCI > 4mm), 200 amostras de sítios saudáveis de 14 pacientes com periodontite e 200 amostras de sítios de 3 pacientes saudáveis foram selecionadas. A prevalência ( por cento de sítios colonizados) e os níveis de cada espécie bacteriana foram computados para cada sítio e dentro de cada grupo. Diferenças clínicas e microbiológicas entre os grupos foram avaliadas através dos testes de Mann-Whitney e Wilcoxon. As espécies que predominaram nas 600 amostras analisadas incluíram Corynebacterium diphtheriae, Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumannii e Escherichia coli. Em geral, a maioria das espécies estudadas foi detectada com maior freqüência e em níveis elevados nos sítios com e sem doença nos pacientes com periodontite em relação ao grupo com saúde periodontal. Em particular, C. diphtheriae, E. coli, E. faecalis, Pseudomonas. aeruginosa e S. aureus foram significantemente mais prevalentes e detectadas em maior número nos sítios doentes de pacientes com periodontite em relação aos sítios de indivíduos com saúde periodontal (p < 0,05). Sinais clínicos de doença periodontal apresentaram uma correlação positiva com as espécies A. baumannii, Streptococcus pyogenes, E. coli, S. aureus e P. aeruginosa. Além disso, as espécies S. pyogenes...

Analysis of leukotoxin gene types of Actinobacillus actinomycetemcomitans in brazilians with aggressive periodontitis

Actinobacillus actinomycetemcomitans (Aa) tem sido associado com diferentes formas de doenças periodontais, mas tal espécie é considerada o principal agente etiológico da doença periodontal agressiva. Algumas cepas de Aa apresentam uma deleção de 530 pb na região promotora do operon do gene da leucotoxina, produzindo assim maiores quantidades de leucotoxina. Tal fato pode ter um importante papel na patogênese das doenças periodontais. A proposta do presente estudo foi determinar a prevalência do Aa e a ocorrência da deleção genética da leucotoxina em pacientes com periodontite agressiva generalizada (PAG) de uma amostra da população brasileira. Trinta indivíduos com saúde periodontal e 29 pacientes com PAG participaram do estudo. Profundidade de bolsa à sondagem (PBS), nível clínico de inserção (NCI), presença de placa supragengival (PL) e sangramento à sondagem (SAS) foram avaliados em 6 sítios/dente de todos os pacientes. Amostras de saliva foram coletadas para isolamento do DNA bacteriano. A detecção do Aa e a ocorrência da deleção genética foram realizadas através da técnica de PCR diretamente nas amostras. Diferenças nos parâmetros clínicos e microbiológicos entre os grupos foram avaliadas através dos testes de Mann-Whitney, Fisher e Qui-quadrado. Associações entre os parâmetros clínicos e microbiológicos foram testadas através do teste de Pearson. Aa foi detectado com maior frequência em pacientes com PAG (96,6 por cento) do que pacientes saudáveis (76,7 por cento) (c2 = 4,9; p < 0,05). A deleção genética foi observada em 16 dos 28 (57,1 por cento) pacientes com PAG que foram positivos para Aa. Porém, nenhuma das amostras de indivíduos com saúde periodontal apresentaram a deleção (c2 = 19,15; p < 0,001). Correlações significantes entre a presença da deleção e os parâmetros clínicos PBS (r=0,312, p<0,05), NCI (r=0,406, p<0,01), PL (r=0,278, p<0,05) e SAS (r=0,409, p<0,01) foram observadas. Uma alta prevalência de Aa foi observada em brasileiros com PAG e...