published_or_final_version / Dentistry / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 1993. / Includes bibliographical references.

Orientador: Ana Cláudia Pavarina / Resumo: O objetivo deste estudo foi avaliar a eficácia da terapia fotodinâmica antimicrobiana associada (aPDT) ao metronidazol (MTZ) em biofilmes periodontopatogênicos. Para tal finalidade, foram realizadas as seguintes etapas: (1) determinação do tempo de adesão (24 e 48 horas) e formação de biofilme mono e duo-espécie (3, 5 e 7 dias) de Fusobacterium nucleatum (NCTC 11326) e Porphyromonas gingivalis (ATCC 33277); (2) aplicação da aPDT mediada por PDZ associada ao MTZ em biofilmes mono-espécie de F. nucleatum e P. gingivalis. Foram avaliadas diferentes concentrações do PDZ (50, 75 e 100 mg/L) e dose de luz de 50 J/cm2 (660nm). Após a aplicação da aPDT, os biofilmes foram incubados com diferentes concentrações do MTZ (MIC, 50x MIC e 100x MIC) por 24 horas. Os grupos controles positivos (L-F-) não receberam fotossensibilizador e não foram iluminados. A viabilidade dos microrganismos após os tratamentos foi avaliada por meio da contagem de UFC/ml. Os resultados demonstraram que o período de adesão de 24 horas, seguido de 5 dias de formação de biofilme foi satisfatório para a obtenção de biofilmes maduros mono-espécie. Para F. nucleatum, os resultados demonstraram que aPDT 75 mg/mL associado com MTZ 100x MIC e aPDT 100 mg/L associado com MTZ nas concentrações de 50x MIC e 100x MIC reduziu significativamente o número de UFC/mL, 2,99; 2,9 e 3,94 Log10 respectivamente. Para P. gingivalis, a redução mais significativa de UFC/mL foi obtida quando a associação de aPDT 100 mg/L e MTZ 100x MIC ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the efficacy of metronidazole (MTZ) associated antimicrobial photodynamic therapy (aPDT) on periodontopathogenic biofilms. For this purpose, the following steps were performed: (1) determination of adhesion period (24 and 48 hours) and single and duo species biofilm formation (3, 5 and 7 days) of Fusobacterium nucleatum (NCTC 11326) and Porphyromonas gingivalis (ATCC 33277); (2) Photodithazine ® (PDZ)- mediated aPDT in association with MTZ in single-specie biofilms of F. nucleatum and P. gingivalis. Different concentrations of PDZ (50, 75 e 100 mg/L) and light dose of 50 J / cm2 (660nm) were evaluated. After application of aPDT, the biofilms were incubated with different concentrations of MTZ (MIC, 50x MIC and 100x MIC) for 24 hours. Positive control groups (L-F-) received no photosensitizer and were also not illuminated. The viability of the microorganisms after the treatments was evaluated by counting CFU/ml. The results demonstrated that the 24 hours adhesion period followed by 5 days of biofilm formation was satisfactory for obtaining a mature biofilm in single-specie. For F. nucleatum, the results demonstrated that 75 mg/L aPDT associated with MTZ 100x and 100 mg/mL aPDT associated with MTZ at 50x MIC and 100x MIC concentrations significantly reduced the number of CFU/mL, 2.99; 2.9 and 3.94 Log10 respectively. For P. gingivalis, the greatest reduction of CFU/mL was obtained when the association of aPDT 100 mg/L and MTZ 100x MIC was p... (Complete abstract click electronic access below) / Doutor

The need for a non-invasive, accurate, easy-to-use, and cost-effective colorectal cancer (CRC) detection device is apparent in the low survival rates seen in late-stage diagnoses. Once CRC has progressed past stage I, the 5-year survival rate drops significantly, and treatment options become less favourable. The best way to treat CRC is to catch it early. The development of an RNA-cleaving fluorogenic DNAzyme (RFD) holds the potential to remediate this deficiency. A DNAzyme, called RFD-FN1, was identified from a synthetic random-sequence DNA library to selectively bind to an unknown target associated with Fusobacterium nucleatum, which has been found to be overabundant in pre- and cancerous colorectal tissue and stool. Target recognition by the DNAzyme induces the cleavage of a fluorogenic substrate and generates a fluorescent signal to indicate the presence of the bacterium. This thesis outlines the efforts made towards functionalizing the F. nucleatum-responsive probe in stool samples to create a non-invasive screening test. RFD-FN1 is selective towards a heat-stable F. nucleatum protein, but its limit of detection is only 10^7 CFU/mL. Although able to detect spiked concentrations of F. nucleatum cells in processed stool samples, the use of heat, filtering, centrifugation, antibiotics, culturing or serial dilutions are not sufficient to detect the F. nucleatum that is naturally present in the diseased samples. Experiments designed to enrich the target concentration revealed that the target is not produced consistently in any growing condition tested. Size exclusion chromatography and mass spectrometry analysis identified five potential targets that RFD-FN1 may be responding to. Three candidate targets were cloned and purified, but they failed to induce RFD-FN1’s activity. Due to the COVID-19 pandemic, the purification of the final two proteins was not completed. Purifying the two candidate targets and testing their ability to induce RFD-FN1 represents future research efforts. If the target for the DNAzyme is confirmed, a reselection for a more sensitive DNAzyme, that can function in human stool, can be attempted. / Thesis / Master of Health Sciences (MSc)

Aptamers are nucleic acid ligands that bind to a specific target molecule. They are discovered by in-vitro selection, whereby binding sequences are selected from a large library of random sequences through iterative affinity steps. Aptamers are used as molecular recognition elements in aptamer-based, as such, creating aptamers with high affinity and specificity to their targets is important to the field. Ligands with two binding sites have been reported to have enhanced binding affinity than ligands with one binding site. To improve the quality of aptamers for downstream applications, multidomain selection is proposed as a new method for selecting aptamers compatible with dimerization. Here, we applied the multidomain selection approach to Fusobacterium nucleatum DnaK and produced aptamers that target the N-terminal domain (NTD) and the C-terminal domain (CTD) of DnaK. The top aptamer for DnaK-NTD had a Kd of 59.7 nM, and for DnaK-CTD had a Kd of 202.0 nM. However, the aptamers did not bind to the full-length DnaK and could not be dimerized. Multiple-site binding offers greater flexibility in the design of detection systems, which could provide higher selectivity and sensitivity than aptamers found through standard approaches. Validation of a method to discover aptamers compatible with dimerization would result in the development of a targeted approach to discover high-quality aptamers for bacterial proteins that can be used in bacteria-detection techniques. / Thesis / Master of Science (MSc)

Les parodontites sont des maladies inflammatoires de nature infectieuse affectant les tissus de soutien de la dent. La présence de bactéries parodontopathogènes dans le sillon gingival représente le facteur étiologique primaire responsable du déclenchement de la parodontite. La réponse immunitaire de l'hôte face à l'agression par ces parodontopathogènes détermine l'évolution de la maladie vers la destruction tissulaire ou la guérison. En effet, la stimulation des cellules immunitaires et mucosales par les bactéries et leurs facteurs de virulence engendre une forte production de médiateurs inflammatoires et de métalloprotéinases matricielles. Ce phénomène a pour conséquence d'activer diverses voies de dégradation tissulaire et osseuse. Le système du complément est l'un des éléments importants de la réaction immunitaire puisqu'il permet l'élimination de microorganismes pathogènes. Pour éviter un effet néfaste du système du complément, les cellules de mammifères expriment à leur surface des protéines régulatrices du complément (CRPs) qui neutralisent les composantes du complément. Le but de cette étude était d'investiguer la capacité des bactéries parodontopathogènes à déjouer le système de défense de l'hôte et/ou de contribuer à l'inflammation et à la destruction tissulaire, en interagissant avec les CRPs. D'une part, nous avons démontré que Porphyromonas gingivalis, une bactérie anaérobie stricte à Gram négatif fortement associée à la parodontite chronique, peut induire le largage de la protéine CD46 de la surface des cellules épithéliales buccales. Une fois détachée des cellules, cette protéine est dégradée par les enzymes protéolytiques sécrétées par P. gingivalis. D'autre part, Fasobacterhim nucleatum, une bactérie cohabitant avec P. gingivalis, a montré une capacité à lier à sa surface la protéine CD46 soluble. F. nucleatum couvert de la protéine CD46 s'est avéré capable d'induire une sécrétion de cytokines proinflammatoires par les cellules épithéliales. Enfin, une stimulation des cellules épithéliales par le lipopolysaccharide des parodontopathogènes a révélé une surexpression des gènes codant pour les CRPs, dont CD46, CD55 et CD59. L'ensemble de ces résultats ont mené à une meilleure compréhension des interactions des interactions des bactéries parodontopathogènes avec le système du complément et des mécanismes contribuant à l'inflammation et à la destruction tissulaire.

Aggregatibacter actinomycetemcomitans e Fusobacterium nucleatum são microrganismos gram-negativos presentes nos processos inflamatórios e nas diferentes formas da doença periodontal. Ambos formam parte da microbiota residente da cavidade bucal humana podendo levar ao desenvolvimento de infecções endógenas ou exógenas. Neste estudo, as avaliações qualitativa, quantitativa e genotípica de A. actinomycetemcomitans e F. nucleatum isolados de pacientes com gengivite, periodontite crônica e indivíduos sadios foram realizadas. Biofilmes subgengivais de 70 pacientes com gengivite, 75 com periodontite crônica e 95 indivíduos saudáveis foram avaliados. A. actinomycetemcomitans foi isolado em 2 (2,8%) pacientes com gengivite, 4 (5,3%) com periodontite e 5 (5,3%) sadios; e F. nucleatum em 13 (18,6%) pacientes com gengivite, 20 (26,6%) com periodontite crônica e 19 (20%) sadios. Ambos os microrganismos foram isolados em 5 (7,1%) pacientes com gengivite, 9 (12%) com periodontite crônica e 3 (3,15%) sadios. Por PCR, os DNA de A. actinomycetemcomitans foram detectados em 23 (32,8%) pacientes com gengivite, 20 (26,6%) com periodontite crônica e 38 (40%) indivíduos saudáveis; e de F. nucleatum em 17 (24,3%) pacientes com gengivite, 11 (14,6%) com periodontite e 19 (20%) sadios. Em associação esses microrganismos foram detectados em 23 (32,8%) pacientes com gengivite, 40 (53,3%) com periodontite crônica e 17 (17,8%) sadios. A. actinomycetemcomitans isolados de pacientes com gengivite pertenceram aos biotipos I, II, IV, V e X, e aos sorotipos a, c, e e. Em pacientes com periodontite foram encontrados os biotipos II, VI e X, e os sorotipos a, b, e c, sendo o sorotipo c o mais predominante (80%); e em indivíduos sadios os biotipos II e X, e os sorotipos b e c. Os valores quantitativos de A. actinomycetemcomitans para os três grupos analisados variaram em número de cópias de 0 a 1,14 x 108, e de F. nucleatum de 0 a 3,98 x 106. Os resultados obtidos por AP-PCR mostram a heterogeneidade dos isolados de A. actinomycetemcomitans e de F. nucleatum nos diferentes grupos clínicos de pacientes avaliados. Esses resultados comparativos poderão ser levados em consideração pelos clínicos para melhor direcionar o tratamento da doença periodontal, colaborando de forma efetiva para o seu monitoramento. / Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are gram-negative microorganisms observed in inflammatory processes and different forms of periodontal disease. Both are part of the human oral resident microbiota which may cause endogenous or exogenous infections. In this study, a qualitative, quantitative and genotypic analysis of A. actinomycetemcomitans and F. nucleatum isolated from patients with gingivitis, chronic periodontitis and healthy subjects were determined. Subgingival biofilms of 70 patients with gingivitis, 75 with chronic periodontitis and 95 healthy subjects were evaluated. A. actinomycetemcomitans was isolated in 2 (2,8%) patients with gingivitis, 4 (5,3%) with periodontitis and 5 (5,3%) healthy individuals; and F. nucleatum in 13 (18,6%) patients with gingivitis, 20 (26,6%) with chronic periodontitis and 19 (20%) healthy. Both microorganisms were identified in 5 (7,1%) patients with gingivitis, 9 (12%) with chronic periodontitis and 3 (3,15%) healthy. By PCR, DNA of A. actinomycetemcomitans were detected in 23 (32,8%) patients with gingivitis, 20 (26,6%) with chronic periodontitis and 38 (40%) healthy individuals; and F. nucleatum 17 (24,3%) patients with gingivitis, 11 (14,6%) with periodontitis and 19 (20%) healthy. In association, microorganisms were detected in 23 (32,8%) patients with gingivitis, 40 (53,3%) with chronic periodontitis and 17 (17,8%) healthy. A. actinomycetemcomitans isolated from patients with gingivitis belonged to biotype I, II, IV, V, X, and serotypes a, c and e. In patients with periodontitis biotypes II, VI and X and serotypes a, b, and c were found and serotype c was the most predominant (80%); and healthy individuals biotypes II and X, and serotypes b and c. Quantitative values for A. actinomycetemcomitans in the three patients groups were ranged from 0 to 1.14 x 108 and F. nucleatum 0 to 3.98 x 106. The results of this study by AP-PCR showed the heterogeneity of A. actinomycetemcomitans and F. nucleatum in the different clinical status. These comparative results can be considered by dentists for the treatment of periodontal disease and its effective monitoring.

A periodontite é uma resposta inflamatória desencadeada por um complexo biofilme constituído por diversos microrganisnos, particularmente por bactérias anaeróbias, tais como Porphyromonas spp. e Fusobacterium spp. Essas bactérias produzem vários fatores de virulência capazes de atuar no início e progressão da doença. Cem amostras subgengivais foram analisadas, sendo 50 de cães com periodontite e 50 de cães sadios, obtendo-se 144 isolados bacterianos identificados como pertencentes aos gêneros Porphyromonas e Fusobacterium. A produção de hemolisinas e hemaglutininas, susceptibilidade aos soros humano, canino e equino, e susceptibilidade a dez antibióticos foram avaliadas, assim como a presença dos genes prtC (colagenase), fimA (fímbrias), tetQ e tetM (resistência à tetraciclina). Igualmente, a capacidade quimiotática de neutrófilos e a produção de IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945, IL-11 e IL-17, foram avaliadas. <font face=\"Symbol\">&#946-hemólise foi produzida por P. gulae, P. crevioricanis, P. cangingivalis, P. gingivicanis e P. circumdentaria. Dos 144 isolados, 21 P. gulae e 2 F. nucleatum aglutinaram eritrócitos humanos. A maioria dos isolados foi resistente à ação dos soros humano, equino ou canino. Todos os 144 isolados foram sensíveis à amoxicilina, clindamicina, tetraciclina, amoxicilina/clavulanato, cefoxitina e penicilina G. Três P. gulae, 2 P.macacae e 2 P. canifelinum abrigaram o gene tetQ e apenas um F. nucleatum e um P. catoniae foram positivos para a presença do gene tetM. As espécies P. gulae, P. cangingivalis e P. circumdentaria abrigaram o gene prtC. A maioria dos isolados abrigou o gene fimA tipo I e nenhum deles abrigou o gene fimA tipo V. Somente nas P. gulae foi observada a presença de cápsula pela microscopia eletrônica de transmissão. Todos os isolados estimularam quimiotaxia dos neutrófilos. Interleucinas IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945 e IL-11 foram detectadas após estímulo com as espécies de Porphyromonas. As espécies de Fusobacterium não estimularam a produção de IL-11 e P. gulae induziu elevada concentração dessa citocina. Nenhum dos isolados estimulou a liberação de IL-17. / Periodontitis is an inflammatory response caused by a complex microbial biofilm, including anaerobic bacteria such as Porphyromonas spp. and Fusobacterium spp. Those species present several virulence factors which act on early step and during the disease evolution. One hundred of subgingival samples were analyzed, obtained from 50 dogs with and 50 without periodontitis. One hundred and forty four bacterial species were isolated belonging to both Porphyromonas and Fusobacterium genus. Hemolysin and hemagglutinin production, susceptibility to human, equine and canine sera, and antimicrobial susceptibility to 10 antibiotics were evaluated. In addition, the presence of gene prtC (collagenase), fimA (fimbriae), tetQ and tetM (tetracycline resistance), as well as neutrophils chemotaxis and IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945, IL-11 and IL-17 production were also determined. <font face=\"Symbol\">&#946-hemolysis was produced by P. gulae, P. crevioricanis, P. cangingivalis, P. gingivicanis and P. circumdentaria. Of the 144 isolates, 21 P. gulae and 2 F. nucleatum were able to agglutinate human erythrocytes. Most of the isolates were resistant to the action of human, equine or canine serum. All isolates were susceptible to amoxicillin, clindamycin, tetracycline, amoxicillin/clavulanate, cefoxitin and penicillin G. Three P. gulae, 2 P.macacae and 2 P. canifelinum harbored tetQ, and only one F. nucleatum and one P. catoniae were tetM positive. P. gulae, P. cangingivalis and P. circumdentaria harbored prtC. Most of the isolates harbored fimA I gene and none of them harbored fimA V. Only P. gulae showed capsule through transmission electron microscopy. All isolates induced neutrophils chemotaxis and IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945 and IL-11 were produced when neutrophils were stimulated by Porphyromonas spp. Fusobacterium spp. did not stimulate IL-11 production and P. gulae induced high concentration of cytokine. None of the isolates stimulated IL-17.

Aggregatibacter actinomycetemcomitans e Fusobacterium nucleatum são microrganismos gram-negativos presentes nos processos inflamatórios e nas diferentes formas da doença periodontal. Ambos formam parte da microbiota residente da cavidade bucal humana podendo levar ao desenvolvimento de infecções endógenas ou exógenas. Neste estudo, as avaliações qualitativa, quantitativa e genotípica de A. actinomycetemcomitans e F. nucleatum isolados de pacientes com gengivite, periodontite crônica e indivíduos sadios foram realizadas. Biofilmes subgengivais de 70 pacientes com gengivite, 75 com periodontite crônica e 95 indivíduos saudáveis foram avaliados. A. actinomycetemcomitans foi isolado em 2 (2,8%) pacientes com gengivite, 4 (5,3%) com periodontite e 5 (5,3%) sadios; e F. nucleatum em 13 (18,6%) pacientes com gengivite, 20 (26,6%) com periodontite crônica e 19 (20%) sadios. Ambos os microrganismos foram isolados em 5 (7,1%) pacientes com gengivite, 9 (12%) com periodontite crônica e 3 (3,15%) sadios. Por PCR, os DNA de A. actinomycetemcomitans foram detectados em 23 (32,8%) pacientes com gengivite, 20 (26,6%) com periodontite crônica e 38 (40%) indivíduos saudáveis; e de F. nucleatum em 17 (24,3%) pacientes com gengivite, 11 (14,6%) com periodontite e 19 (20%) sadios. Em associação esses microrganismos foram detectados em 23 (32,8%) pacientes com gengivite, 40 (53,3%) com periodontite crônica e 17 (17,8%) sadios. A. actinomycetemcomitans isolados de pacientes com gengivite pertenceram aos biotipos I, II, IV, V e X, e aos sorotipos a, c, e e. Em pacientes com periodontite foram encontrados os biotipos II, VI e X, e os sorotipos a, b, e c, sendo o sorotipo c o mais predominante (80%); e em indivíduos sadios os biotipos II e X, e os sorotipos b e c. Os valores quantitativos de A. actinomycetemcomitans para os três grupos analisados variaram em número de cópias de 0 a 1,14 x 108, e de F. nucleatum de 0 a 3,98 x 106. Os resultados obtidos por AP-PCR mostram a heterogeneidade dos isolados de A. actinomycetemcomitans e de F. nucleatum nos diferentes grupos clínicos de pacientes avaliados. Esses resultados comparativos poderão ser levados em consideração pelos clínicos para melhor direcionar o tratamento da doença periodontal, colaborando de forma efetiva para o seu monitoramento. / Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are gram-negative microorganisms observed in inflammatory processes and different forms of periodontal disease. Both are part of the human oral resident microbiota which may cause endogenous or exogenous infections. In this study, a qualitative, quantitative and genotypic analysis of A. actinomycetemcomitans and F. nucleatum isolated from patients with gingivitis, chronic periodontitis and healthy subjects were determined. Subgingival biofilms of 70 patients with gingivitis, 75 with chronic periodontitis and 95 healthy subjects were evaluated. A. actinomycetemcomitans was isolated in 2 (2,8%) patients with gingivitis, 4 (5,3%) with periodontitis and 5 (5,3%) healthy individuals; and F. nucleatum in 13 (18,6%) patients with gingivitis, 20 (26,6%) with chronic periodontitis and 19 (20%) healthy. Both microorganisms were identified in 5 (7,1%) patients with gingivitis, 9 (12%) with chronic periodontitis and 3 (3,15%) healthy. By PCR, DNA of A. actinomycetemcomitans were detected in 23 (32,8%) patients with gingivitis, 20 (26,6%) with chronic periodontitis and 38 (40%) healthy individuals; and F. nucleatum 17 (24,3%) patients with gingivitis, 11 (14,6%) with periodontitis and 19 (20%) healthy. In association, microorganisms were detected in 23 (32,8%) patients with gingivitis, 40 (53,3%) with chronic periodontitis and 17 (17,8%) healthy. A. actinomycetemcomitans isolated from patients with gingivitis belonged to biotype I, II, IV, V, X, and serotypes a, c and e. In patients with periodontitis biotypes II, VI and X and serotypes a, b, and c were found and serotype c was the most predominant (80%); and healthy individuals biotypes II and X, and serotypes b and c. Quantitative values for A. actinomycetemcomitans in the three patients groups were ranged from 0 to 1.14 x 108 and F. nucleatum 0 to 3.98 x 106. The results of this study by AP-PCR showed the heterogeneity of A. actinomycetemcomitans and F. nucleatum in the different clinical status. These comparative results can be considered by dentists for the treatment of periodontal disease and its effective monitoring.

A periodontite é uma resposta inflamatória desencadeada por um complexo biofilme constituído por diversos microrganisnos, particularmente por bactérias anaeróbias, tais como Porphyromonas spp. e Fusobacterium spp. Essas bactérias produzem vários fatores de virulência capazes de atuar no início e progressão da doença. Cem amostras subgengivais foram analisadas, sendo 50 de cães com periodontite e 50 de cães sadios, obtendo-se 144 isolados bacterianos identificados como pertencentes aos gêneros Porphyromonas e Fusobacterium. A produção de hemolisinas e hemaglutininas, susceptibilidade aos soros humano, canino e equino, e susceptibilidade a dez antibióticos foram avaliadas, assim como a presença dos genes prtC (colagenase), fimA (fímbrias), tetQ e tetM (resistência à tetraciclina). Igualmente, a capacidade quimiotática de neutrófilos e a produção de IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945, IL-11 e IL-17, foram avaliadas. <font face=\"Symbol\">&#946-hemólise foi produzida por P. gulae, P. crevioricanis, P. cangingivalis, P. gingivicanis e P. circumdentaria. Dos 144 isolados, 21 P. gulae e 2 F. nucleatum aglutinaram eritrócitos humanos. A maioria dos isolados foi resistente à ação dos soros humano, equino ou canino. Todos os 144 isolados foram sensíveis à amoxicilina, clindamicina, tetraciclina, amoxicilina/clavulanato, cefoxitina e penicilina G. Três P. gulae, 2 P.macacae e 2 P. canifelinum abrigaram o gene tetQ e apenas um F. nucleatum e um P. catoniae foram positivos para a presença do gene tetM. As espécies P. gulae, P. cangingivalis e P. circumdentaria abrigaram o gene prtC. A maioria dos isolados abrigou o gene fimA tipo I e nenhum deles abrigou o gene fimA tipo V. Somente nas P. gulae foi observada a presença de cápsula pela microscopia eletrônica de transmissão. Todos os isolados estimularam quimiotaxia dos neutrófilos. Interleucinas IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945 e IL-11 foram detectadas após estímulo com as espécies de Porphyromonas. As espécies de Fusobacterium não estimularam a produção de IL-11 e P. gulae induziu elevada concentração dessa citocina. Nenhum dos isolados estimulou a liberação de IL-17. / Periodontitis is an inflammatory response caused by a complex microbial biofilm, including anaerobic bacteria such as Porphyromonas spp. and Fusobacterium spp. Those species present several virulence factors which act on early step and during the disease evolution. One hundred of subgingival samples were analyzed, obtained from 50 dogs with and 50 without periodontitis. One hundred and forty four bacterial species were isolated belonging to both Porphyromonas and Fusobacterium genus. Hemolysin and hemagglutinin production, susceptibility to human, equine and canine sera, and antimicrobial susceptibility to 10 antibiotics were evaluated. In addition, the presence of gene prtC (collagenase), fimA (fimbriae), tetQ and tetM (tetracycline resistance), as well as neutrophils chemotaxis and IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945, IL-11 and IL-17 production were also determined. <font face=\"Symbol\">&#946-hemolysis was produced by P. gulae, P. crevioricanis, P. cangingivalis, P. gingivicanis and P. circumdentaria. Of the 144 isolates, 21 P. gulae and 2 F. nucleatum were able to agglutinate human erythrocytes. Most of the isolates were resistant to the action of human, equine or canine serum. All isolates were susceptible to amoxicillin, clindamycin, tetracycline, amoxicillin/clavulanate, cefoxitin and penicillin G. Three P. gulae, 2 P.macacae and 2 P. canifelinum harbored tetQ, and only one F. nucleatum and one P. catoniae were tetM positive. P. gulae, P. cangingivalis and P. circumdentaria harbored prtC. Most of the isolates harbored fimA I gene and none of them harbored fimA V. Only P. gulae showed capsule through transmission electron microscopy. All isolates induced neutrophils chemotaxis and IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945 and IL-11 were produced when neutrophils were stimulated by Porphyromonas spp. Fusobacterium spp. did not stimulate IL-11 production and P. gulae induced high concentration of cytokine. None of the isolates stimulated IL-17.