However, non-oral pathogens internalized in oral epithelial cells and their relationship with periodontal status are poorly understood. The purpose of this study was to detect opportunistic species within buccal and gingival crevice epithelial cells collected from subjects with periodontitis or individuals with good periodontal health, and to associate their prevalence with periodontal clinical status. Quantitative detection of total bacteria and Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis in oral epithelial cells was determined by quantitative real-time PCR using universal and species-specific primer sets. Intracellular bacteria were visualized by confocal microscopy and fluorescence in situ hybridization. Overall, 33 % of cell samples from patients with periodontitis contained at least one opportunistic species, compared with 15 % of samples from healthy individuals. E. faecalis was the most prevalent species found in oral epithelial cells (detected in 20.6 % of patients with periodontitis, P50.03 versus healthy individuals) and was detected only in cells from patients with periodontitis. Quantitative real-time PCR showed that high levels of P. aeruginosa and S. aureus were present in both the periodontitis and healthy groups. However, the proportion of these species was significantly higher in epithelial cells of subjects with periodontitis compared with healthy individuals (P50.016 for P. aeruginosa and P50.047 for S. aureus). Although E. faecalis and P. aeruginosa were detected in 57 % and 50 % of patients, respectively, with probing depth and clinical attachment level ¢6 mm, no correlation was found with age, sex, bleeding on probing or the presence of supragingival biofilm. The prevalence of these pathogens in epithelial cells is correlated with the state of periodontal disease. INTRODUCTIONThe oral cavity harbours a complex microbiota that is commonly related to oral health. Nonetheless, it is common knowledge that only some microbial consortia are associated with carious lesions and periodontal disease (Aas et al., 2005;Socransky & Haffajee, 2005). On the other hand, classically pathogenic micro-organisms can find favourable environmental conditions within and occupy specific niches of the oral cavity (Fourrier et al., 1998). Bacterial species commonly associated with nosocomial infections and multiresistance to antimicrobials, such as Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Enterococcus faecalis and the Enterobacteriaceae family, have been detected in high proportions and levels in the subgingival biofilm of individuals with periodontitis (Slots et al., 1990;Colombo et al., 2002) and in the endodontic canal (Brito et al., 2007). Furthermore, there seems to be a strong association between the presence of these pathogens in dental plaque and respiratory infections in hospitalized patients (El-Solh et al., 2004;Didilescu et al., 2005;Bahrani-Mougeot et al., 2007).Abbreviations: BOP, bleeding on probing; CAL, clinical attachment level; FISH, fluo...

It is well known that strain and virulence diversity exist within the population structure of Porphyromonas gingivalis. In the present study we investigate intra- and inter-species variability in biofilm formation of Porphyromonas gingivalis and partners Prevotella intermedia and Prevotella nigrescens. All strains tested showed similar hydrophobicity, except for P. gingivalis W83 which has roughly half of the hydrophobicity of P. gingivalis ATCC33277. An intraspecies variability in coaggregation of P. gingivalis with P. intermedia was also found. The association P. gingivalis W83/P. intermedia 17 produced the thickest biofilm and strain 17 was prevalent. In a two-compartment system P. gingivalis W83 stimulates an increase in biomass of strain 17 and the latter did not stimulate the growth of P. gingivalis W83. In addition, P. gingivalis W83 also stimulates the growth of P. intermedia ATCC25611 although strain W83 was prevalent in the association with P. intermedia ATCC25611. P. gingivalis ATCC33277 was prevalent in both associations with P. intermedia and both strains of P. intermedia stimulate the growth of P. gingivalis ATCC33277. FISH images also showed variability in biofilm structure. Thus, the outcome of the association P. gingivalis/P. intermedia seems to be strain-dependent, and both soluble factors and physical contact are relevant. The association P. gingivalis-P. nigrescens ATCC33563 produced larger biomass than each monotypic biofilm, and P. gingivalis was favored in consortia, while no differences were found in the two-compartment system. Therefore, in consortia P. gingivalis-P. nigrescens physical contact seems to favor P. gingivalis growth. The intraspecies variability found in our study suggests strain-dependence in ability of microorganisms to recognize molecules in other bacteria which may further elucidate the dysbiosis event during periodontitis development giving additional explanation for periodontal bacteria, such as P. gingivalis and P. intermedia, among others, to persist and establish chronic infections in the host.

The image for Fig 3 is missing Fig 3B and 3C. Please see the corrected Fig 3 here. Table 1 has been corrected for improved readability. Please see the corrected Table 1 here.

Ao "Pai Maior" que para mim constitui o princípio e a finalidade de minhas tarefas. Às pessoas que fizeram parte da construção da minha tese ofereço meu agradecimento sincero. À minha orientadora Profa. Regina pelo acolhimento e por me conduzir e amparar nos momentos mais delicados. Obrigada de coração. Ao meu co-orientador Paulo Henrique que de coadjuvante, nada possui. Obrigada por me ensinar na prática a ser um ser humano melhor e por me conduzir com maestria nos caminhos da ciência. À querida amiga e conselheira Profa. Andréa Colombo por seu grande incentivo, bom humor e por dividir comigo seus conhecimentos. Às queridas amigas do laboratório Daniela Higashi e Luciana Kfouri, companheiras do "chão de fábrica", das risadas e das angústias. os amigos Melissa, Juliana, Vitor e Janaína pela ajuda indispensável no laboratório e pela alegria de sua juventude. Às Profas. Márcia Mayer, Elisabete José seus laboratórios. A todos os amigos do laboratório 110: Ericka, Priscila, Dione, Sílvia, Ellen e Lucas, por dividirem seu espaço de trabalho e p momentos de necessidade. Aos amigos Léo e João por seu amparo técnico. À Rosana Duarte por sua dedicação e paciência co Ao Roberto, do Departamento de Biologia Celular e do Desenvolvim imagens de microscopia confocal. Às secretárias Alice, Gisele, Beth e Naíde por to À Gláubia e Maria pelo apoio técnico ao meu lar. À minha família pela sustentação e amparo fu Vitor,