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.
| Identifer | oai:union.ndltd.org:usp.br/oai:teses.usp.br:tde-07012011-095622 |
| Date | 25 November 2010 |
| Creators | Matarazzo, Flávia |
| Contributors | Mayer, Marcia Pinto Alves |
| Publisher | Biblioteca Digitais de Teses e Dissertações da USP |
| Source Sets | Universidade de São Paulo |
| Language | Portuguese |
| Detected Language | English |
| Type | Tese de Doutorado |
| Format | application/pdf |
| Rights | Liberar o conteúdo para acesso público. |
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