La pericoronaritis es una patologia infecciosa que suele producirse con frecuencia alrededor del tercer molar, cuando este erupciona. Suele manifestarse con mayor frecuencia en molares inferiores, cursando clinicamente con dolor y exsudado, traendo implicaciones y complicaciones severas al paciente si el diagnóstico y el enfoque terapeutico no sea el correcto. Por su naturaleza polimicrobiana la pericoronaritis tiene un caracter patogeno puesto de manifiesto onde se describen con mayor problemas de cultivo los Streptococcus responsábles por esta patologia, o sea, el grupo milleri. Por todo esto se ha empleado la tecnica molecular de PCR para comprovar si los cebadores diseñados, en el estudio empirico anteriormente presentado, serian especificos para cada una de las cepas bacterianas descritas. Se ha extraido el DNA de las colonias aisladas, mediante un protocolo previamente elaborado. Se ha ajustado la PCR a cada par de cebadores, empleando las cepas de Streptococcus grupo milleri como controles positivos. Para comprovar la especificidad empírica de los cebadores diseñados se realizaron pruebas cruzadas de DNA aislado de otras cepas del género Streptococcus y otras bacterias orales. Por fin, se llevó a cabo la analisis de muestras clínicas de casos de pericoronaritis y controles, analizando por duplicado cada par de cebadores en cada una de ellas. Cada bacteria ha sido examinada con dos juegos de primers, con el fin de reducir la posibilidad de falsos negativos derivados de una variabilidad en la secuencia genética. La viasualizacion de resultados se ha realizado por electoforesis en gel de agarosa a 1% con gel red.

O objetivo do presente estudo foi testar a hipótese de que polimorfismo na região que codifica o sistema de dois componentes FimSR e seus níveis de transcrição influenciaria expressão de fímbrias e a capacidade de adesão de Porphyromonas gingivalis. Foram avaliadas 21 cepas clínicas e as cepas de referência 33277 (fimbriada) e W83 (não fimbriada). A presença de cápsula foi determinada em 13/21 cepas e de fímbrias em 15/21. Todas as cepas foram capazes de aderir às células (eficiência de 1,2 a 6,1%). Não houve correlação entre os genótipos fimA, presença de fímbrias / cápsula e perfis de macrorestrição por PFGE. Diferenças na transcrição de fimA não podem ser atribuídas a diferenças na região promotora de fimSR. fimA foi regulado positivamente após interação com células epiteliais na maioria das cepas. Os dados indicam que a regulação de fimA é cepa específica. Cepas não-fimbriadas podem apresentar outras estratégias para aderir às células, sugerindo que, além das fímbrias, outras estruturas poderiam desempenhar um papel na interação dessa espécie com as células. / The aim of this study was to test the hypothesis that polymorphism in genes encoding the two-component system FimSR and their transcription levels would influence the expression of fimbriae and adhesion of Porphyromonas gingivalis. We evaluated 21 clinical strains and reference strains 33277 (fimbriate) and W83 (non fimbriated). The presence of a capsule was determined in 13/21 strains and the presence of fimbriae in 15/21. All strains were able to adhere to the cells (efficiency from 1.2 to 6.1%). There was no correlation between genotypes fimA, presence of fimbriae / capsule and macrorestriction profiles by PFGE. Differences in transcription of fimA could not be attributed to differences in the promoter region of fimS. fimA was upregulated after interaction with epithelial cells in most strains. The data indicate that regulation of fimA is strain specific. Non-fimbriated strains may have other strategies to adhere to epithelial cells, suggesting that in addition to fimbriae, other structures could play a role in the interaction of that specie with cells.

O objetivo deste estudo foi determinar a diversidade bacteriana no biofilme subgengival de indivíduos portadores de doença periodontal agressiva (PA). 12 indivíduos com PA e 30 indivíduos com saúde periodontal (PH) foram selecionados. Amostras de placa subgengival foram coletadas de 9 sítios por indivíduo para análise por Hibridação DNA-DNA e de um sítio para a análise clonal 16S. Os patógenos periodontais T. forsythia, P. gingivalis e T. denticola foram encontrados em alta proporção no grupo PA (p<0,001) em comparação ao grupo PH. 120 espécies foram identificadas pela análise clonal de 16SrRNA, sendo 70 destas mais prevalentes. 57% destas espécies são não cultiváveis. Espécies de Selenomonas e Streptococcus foram detectadas em alta prevalencia. Selenomonas sputigena, foi a espécie mais comumente detectada. A microbiota subgengival do grupo PA diferiu marcantemente da do grupo PH. Outras espécies, particularmente do gênero Selenomonas, podem fazer parte da microbiota subgengival em alta proporção em pacientes com PA / The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with aggressive periodontitis (AgP). 12 subject with AgP and 30 periodontally healthy (PH) subjects were selected. Subgingival plaque samples were collected from 9 sites per subject for using in the Checkerboard DNA-DNA technique and one sample by 16S cloning analysis. Periodontal pathogens, such as T. forsythia, P. gingivalis and T. denticola were found in higher proportions AgP groups (p<0.001) than in PH subjects. 120 species were identified by 16S rRNA cloning analyses, therefore 70 species was most prevalent. 57% of the species were not cultivable. Several species of Selenomonas and Streptococcus were found in high prevalence. Selenomonas sputigena, the specie most commonly detected. The subgingival microbiota of AgP markedly differed from PH subjects. Other species, notably species of Selenomonas, may be present in higher proportion in subjects with aggressive periodontitis.

O objetivo do presente estudo foi testar a hipótese de que polimorfismo na região que codifica o sistema de dois componentes FimSR e seus níveis de transcrição influenciaria expressão de fímbrias e a capacidade de adesão de Porphyromonas gingivalis. Foram avaliadas 21 cepas clínicas e as cepas de referência 33277 (fimbriada) e W83 (não fimbriada). A presença de cápsula foi determinada em 13/21 cepas e de fímbrias em 15/21. Todas as cepas foram capazes de aderir às células (eficiência de 1,2 a 6,1%). Não houve correlação entre os genótipos fimA, presença de fímbrias / cápsula e perfis de macrorestrição por PFGE. Diferenças na transcrição de fimA não podem ser atribuídas a diferenças na região promotora de fimSR. fimA foi regulado positivamente após interação com células epiteliais na maioria das cepas. Os dados indicam que a regulação de fimA é cepa específica. Cepas não-fimbriadas podem apresentar outras estratégias para aderir às células, sugerindo que, além das fímbrias, outras estruturas poderiam desempenhar um papel na interação dessa espécie com as células. / The aim of this study was to test the hypothesis that polymorphism in genes encoding the two-component system FimSR and their transcription levels would influence the expression of fimbriae and adhesion of Porphyromonas gingivalis. We evaluated 21 clinical strains and reference strains 33277 (fimbriate) and W83 (non fimbriated). The presence of a capsule was determined in 13/21 strains and the presence of fimbriae in 15/21. All strains were able to adhere to the cells (efficiency from 1.2 to 6.1%). There was no correlation between genotypes fimA, presence of fimbriae / capsule and macrorestriction profiles by PFGE. Differences in transcription of fimA could not be attributed to differences in the promoter region of fimS. fimA was upregulated after interaction with epithelial cells in most strains. The data indicate that regulation of fimA is strain specific. Non-fimbriated strains may have other strategies to adhere to epithelial cells, suggesting that in addition to fimbriae, other structures could play a role in the interaction of that specie with cells.

O objetivo deste estudo foi determinar a diversidade bacteriana no biofilme subgengival de indivíduos portadores de doença periodontal agressiva (PA). 12 indivíduos com PA e 30 indivíduos com saúde periodontal (PH) foram selecionados. Amostras de placa subgengival foram coletadas de 9 sítios por indivíduo para análise por Hibridação DNA-DNA e de um sítio para a análise clonal 16S. Os patógenos periodontais T. forsythia, P. gingivalis e T. denticola foram encontrados em alta proporção no grupo PA (p<0,001) em comparação ao grupo PH. 120 espécies foram identificadas pela análise clonal de 16SrRNA, sendo 70 destas mais prevalentes. 57% destas espécies são não cultiváveis. Espécies de Selenomonas e Streptococcus foram detectadas em alta prevalencia. Selenomonas sputigena, foi a espécie mais comumente detectada. A microbiota subgengival do grupo PA diferiu marcantemente da do grupo PH. Outras espécies, particularmente do gênero Selenomonas, podem fazer parte da microbiota subgengival em alta proporção em pacientes com PA / The purpose of this study was to determine the bacterial diversity in the subgingival plaque of subjects with aggressive periodontitis (AgP). 12 subject with AgP and 30 periodontally healthy (PH) subjects were selected. Subgingival plaque samples were collected from 9 sites per subject for using in the Checkerboard DNA-DNA technique and one sample by 16S cloning analysis. Periodontal pathogens, such as T. forsythia, P. gingivalis and T. denticola were found in higher proportions AgP groups (p<0.001) than in PH subjects. 120 species were identified by 16S rRNA cloning analyses, therefore 70 species was most prevalent. 57% of the species were not cultivable. Several species of Selenomonas and Streptococcus were found in high prevalence. Selenomonas sputigena, the specie most commonly detected. The subgingival microbiota of AgP markedly differed from PH subjects. Other species, notably species of Selenomonas, may be present in higher proportion in subjects with aggressive periodontitis.

Archaea ainda não foi reconhecido como patógeno de doença humana. O objetivo deste estudo foi determinar a prevalência, diversidade, níveis e proporções de Archaea no biofilme subgengival de indivíduos com periodontite agressiva (PA) e saúde periodontal (SP). Sessenta indivíduos foram selecionados para este estudo (n=30/grupo). A análise de diversidade foi realizada em 10 indivíduos/grupo. Quatro sítios/indivíduo do grupo PA e 2 sítios/indivíduo do grupo SP foram analisados por qPCR. A freqüência de Archaea foi de 60% dos indivíduos/ 15,2% dos sítios em PA e de 63,3% dos indivíduos/ 15,6% dos sítios em SP (p>0,05). Um a três filotipos foi identificado por amostra. O número de cópias e a proporção de Archaea e Bacteria foram menores no grupo SP do que no grupo PA (p<0,05). Archaea são encontrados no biofilme subgengival de indivíduos com PA e SP. Methanobrevibacter oralis é o filotipo mais prevalente, podendo ser considerado residente da cavidade bucal. A alteração ecológica na microbiota de indivíduos com PA inclui o aumento dos níveis e proporções de Archaea. / Membrers of Archaea domain may be detected in the microbiota of mucous surfaces of human and animals, but their association with diesase have not been yet stablished Some studies have suggested that Archaea domain may be indirectly associated with pathogenesis of periodontitis, since they are found restrict to subgingival sites with severe periodontal destruction. The aim of this study was to determine the prevalence, diversity, levels and proportions of microorganisms of Archaea domain in subgingival biofilm of aggressive periodontitis and periodontally healthy subjects. Thirty generalized aggressive periodontitis (GAgP) and 30 periodontally healthy (PH) subjects were selected. Archaea detection was performed by PCR using domain-specific primers in 9 subgingival samples taken from each subject. Archaea diversity was determined by evaluating a single positive sample per subject, randomly selected from 10 GAgP and 10 PH subjects. Archaeal 16S rRNA gene library were constructed to each sample and the identity of phylotypes were determined for the comparison of unrecognized sequences with gene database. The levels and proportions of Archaea in relation to total microbial load were analysed by quantitative PCR (qPCR) in 4 sites per GAgP subject and 2 sites per PH subject. A total of 540 subgingival samples were analysed to Archaea presence. This domain were detected in 18 GAgP (60%) and in 19 PH (63.3%) subjects. Forty-one (15.2%) and 42 (15.6%) samples were positive for this domain in GAgP and PH subjects, respectively. There was not difference in prevalence of this domain between subjects from GAgP and PH groups, as well as there was not difference in prevalence between sites with different probing depthsin GAgP group. Thenumber of 16S rRNA clones available to identification per sample varies from 33 to 47 in GAgP group, and from 15 to 23 in PH group, with a mean of 42.8+3.9 e 20.2 +2.2, respectively. The analysis of 629 sequencies permits an identification of 1 to 3 phylotypes of Archaea domain per sample. Methanobrevibacter oralis was detected in all archaeal positive samples, being the single detected specie of this domain in 5 subjects from GAgP group, and in 3 subjects from PH group. Methanobacterium curvum/congolense was detected in 3/10 GAgP and 6/10 PH samples, whereas Methanosarcina mazeii was detected in 4/10 samples from both groups. Archaea analysis by qPCR was carried out in 103 sites and 28 GAgP subjects and in 60 sites and 30 PH individuals. Archaea has been detected in 27/28 subjects and in 68% of studied sites in GAgP group and in 26/30 subjects and 58,3% of total analysed sites in PH group. Archaeal and bacterial 16S rRNA mean levels were smaller in PH group than in GAgP group (Mann Whitney, p<0.05). There was no statistic significance of difference in the levels of Archaea in regard to probing depth categories in GAgP group (p>0.05). Moreover, the proportion of Archaea in relation to total microbial load (Archaea + Bacteria) was 0.02% and 0.08% in PH and GAgP group (p<0.05), respectively. These data suggest that Archaea is commonly found in the subgingival biofilm of humans, and that M. oralis may be considered a member of the resident microbiota of subgingival sites. The ecological shift in the microbiota of aggressive periodontitis subjects includes the increase of levels and proportions of Archaea domain.

Archaea ainda não foi reconhecido como patógeno de doença humana. O objetivo deste estudo foi determinar a prevalência, diversidade, níveis e proporções de Archaea no biofilme subgengival de indivíduos com periodontite agressiva (PA) e saúde periodontal (SP). Sessenta indivíduos foram selecionados para este estudo (n=30/grupo). A análise de diversidade foi realizada em 10 indivíduos/grupo. Quatro sítios/indivíduo do grupo PA e 2 sítios/indivíduo do grupo SP foram analisados por qPCR. A freqüência de Archaea foi de 60% dos indivíduos/ 15,2% dos sítios em PA e de 63,3% dos indivíduos/ 15,6% dos sítios em SP (p>0,05). Um a três filotipos foi identificado por amostra. O número de cópias e a proporção de Archaea e Bacteria foram menores no grupo SP do que no grupo PA (p<0,05). Archaea são encontrados no biofilme subgengival de indivíduos com PA e SP. Methanobrevibacter oralis é o filotipo mais prevalente, podendo ser considerado residente da cavidade bucal. A alteração ecológica na microbiota de indivíduos com PA inclui o aumento dos níveis e proporções de Archaea. / Membrers of Archaea domain may be detected in the microbiota of mucous surfaces of human and animals, but their association with diesase have not been yet stablished Some studies have suggested that Archaea domain may be indirectly associated with pathogenesis of periodontitis, since they are found restrict to subgingival sites with severe periodontal destruction. The aim of this study was to determine the prevalence, diversity, levels and proportions of microorganisms of Archaea domain in subgingival biofilm of aggressive periodontitis and periodontally healthy subjects. Thirty generalized aggressive periodontitis (GAgP) and 30 periodontally healthy (PH) subjects were selected. Archaea detection was performed by PCR using domain-specific primers in 9 subgingival samples taken from each subject. Archaea diversity was determined by evaluating a single positive sample per subject, randomly selected from 10 GAgP and 10 PH subjects. Archaeal 16S rRNA gene library were constructed to each sample and the identity of phylotypes were determined for the comparison of unrecognized sequences with gene database. The levels and proportions of Archaea in relation to total microbial load were analysed by quantitative PCR (qPCR) in 4 sites per GAgP subject and 2 sites per PH subject. A total of 540 subgingival samples were analysed to Archaea presence. This domain were detected in 18 GAgP (60%) and in 19 PH (63.3%) subjects. Forty-one (15.2%) and 42 (15.6%) samples were positive for this domain in GAgP and PH subjects, respectively. There was not difference in prevalence of this domain between subjects from GAgP and PH groups, as well as there was not difference in prevalence between sites with different probing depthsin GAgP group. Thenumber of 16S rRNA clones available to identification per sample varies from 33 to 47 in GAgP group, and from 15 to 23 in PH group, with a mean of 42.8+3.9 e 20.2 +2.2, respectively. The analysis of 629 sequencies permits an identification of 1 to 3 phylotypes of Archaea domain per sample. Methanobrevibacter oralis was detected in all archaeal positive samples, being the single detected specie of this domain in 5 subjects from GAgP group, and in 3 subjects from PH group. Methanobacterium curvum/congolense was detected in 3/10 GAgP and 6/10 PH samples, whereas Methanosarcina mazeii was detected in 4/10 samples from both groups. Archaea analysis by qPCR was carried out in 103 sites and 28 GAgP subjects and in 60 sites and 30 PH individuals. Archaea has been detected in 27/28 subjects and in 68% of studied sites in GAgP group and in 26/30 subjects and 58,3% of total analysed sites in PH group. Archaeal and bacterial 16S rRNA mean levels were smaller in PH group than in GAgP group (Mann Whitney, p<0.05). There was no statistic significance of difference in the levels of Archaea in regard to probing depth categories in GAgP group (p>0.05). Moreover, the proportion of Archaea in relation to total microbial load (Archaea + Bacteria) was 0.02% and 0.08% in PH and GAgP group (p<0.05), respectively. These data suggest that Archaea is commonly found in the subgingival biofilm of humans, and that M. oralis may be considered a member of the resident microbiota of subgingival sites. The ecological shift in the microbiota of aggressive periodontitis subjects includes the increase of levels and proportions of Archaea domain.