Dentro do contexto do novo paradigma da doença periodontal, alguns estudos têm sugerido que a doença periodontal poderia influenciar o desenvolvimento de doenças sistêmicas, incluindo os acidentes vasculares cerebrais. O objetivo deste estudo foi avaliar as condições periodontais de pacientes com acidente vascular cerebral (AVC), comparativamente à amostra populacional sem AVC, bem como identificar e quantificar o nível de bactérias periodontopatogênicas presentes nas áreas de bolsa periodontal com a finalidade de investigar a correlação da doença periodontal com acidente vascular cerebral. Para tanto, foram selecionados 80 pacientes de ambos os sexos, com idade entre 30-80 anos. O grupo experimental foi constituído por 20 pacientes internados em ambiente hospitalar devido à ocorrência de AVC. O grupo controle foi constituído de 60 pacientes provenientes da amostra populacional da cidade de Bauru que não apresentavam sinais e sintomas clínicos ou história familiar de AVC. Um questionário de saúde investigando as possíveis causas do AVC e outras condições sistêmicas foi aplicado a todos os pacientes. Os dois grupos foram avaliados periodontalmente quanto às medidas de profundidade de sondagem, nível de inserção, sangramento à sondagem e índice de placa. Para identificar e quantificar as bactérias periodontopatogênicas, Porphyromonas gingivalis e Aggregatibacter (Actinobacillus) actinomycetemcomitans, em ambos os grupos, foi coletada amostra de placa dentomicrobiana subgengival dos dois sítios com maior profundidade de sondagem de todos os pacientes, por meio da introdução de tira de papel esterilizada (PerioPaper) no sulco gengival. A análise qualitativa e quantitativa dessas bactérias foi realizada por meio de PCR convencional e em tempo real. Os dados obtidos foram analisados por meio de análise de variância (ANOVA) complementado pelo método de Tukey, teste de correlação de Spearman, teste \"t\" de Student, Mann-Whitney, Qui-Quadrado e \"Odds Ratio\" para avaliar a correlação entre os diferentes parâmetros clínicos periodontais com o AVC e os resultados obtidos pelo PCR convencional e PCR em tempo-real, com nível de confiança de 95%. Os resultados obtidos mostraram maior prevalência da doença periodontal no grupo teste (95%) do que no controle (28,3%). O nível de inserção à sondagem, índice de placa e índice gengival estavam significativamente aumentados nos pacientes com AVC (p<0,001). Entretanto, as medidas de profundidade de sondagem não mostraram diferenças significantes entre os grupos (p=0,051), embora estivessem aumentadas para o grupo teste. A presença e quantidade da P.gingivalis foi estatisticamente maior no grupo teste do que no controle (p<0,05). Não foi encontrado A.actinomycetemcomitans em nenhum dos grupos estudados. Do total de pacientes com AVC, 65% desenvolveram AVC-I e 35% AVC-H. Pacientes com AVC-H abrigavam maiores níveis de P.gingivalis do que pacientes com AVC-I. Entretanto, nesse grupo, houve correlação positiva entre bolsas mais profundas e contagem de bactérias (p<0,05), o que não foi observado para AVC-H. A análise de risco por \"odds ratio\" identificou que pacientes com doença periodontal apresentam risco elevado de desenvolvimento de AVC (OR=48,06, IC=95%). Esses achados indicam que a doença periodontal é mais prevalente e severa em pacientes com AVC-I ou AVC-H, com grande quantidade de bactérias, especialmente P. gingivalis, presente em bolsas periodontais mais profundas, sugerindo que a doença periodontal poderia atuar como fator de risco ao desenvolvimento de acidentes vasculares cerebrais. / Inside of the context of the new paradigm of the periodontal disease, some studies have suggested that the periodontal disease could influence the development of systemics diseases, including vascular cerebral accident. The aim of this study was to evaluate the periodontal conditions of patients with vascular cerebral accidents (VCAs), comparatively to the population sample without VCA, as well as identifying and quantifying the level of periodontopathic bacteria presents in the areas of periodontal pocket in order to investigate the correlation of the periodontal disease with vascular cerebral accident. For this study, it had been selected 80 patients of both genders, with age between 30-80 years. The experimental group consisted of 20 hospitalized patients presenting VCA. The control group was consisted of 60 patients proceeding from the population sample of Bauru who did not present clinical signs and symptoms or family history of VCA. A health questionnaire investigating the possible causes of the VCA and others systemics conditions was applied to all patients. The both groups were periodontally evaluated according to probing depth measures; bleed on probing and plaque index. In other to identify and quantify the periodontopathic bacteria Porphyromonas gingivalis and Aggregatibacter (Actinobacillus) actinomycetemcomitans, in both groups, a sample of subgengival plaque was collected from the two deepest sites of all patients by the introduction of sterilized paper strip (PerioPaper). The qualitative and quantitative analysis of these bacteria was performed by conventional PCR and Real Time PCR. Data were analyzed by variance analysis test (ANOVA) complemented by the Tukey test, Pearson correlation test, Student \"t\" test, Mann-Whitney test, Qui-Square test and Odds Ratio to evaluate the different correlations between the different periodontal clinical parameters VCA, and the results obtained from the Real Time PCR, with a 95% confidence level. The analysis of the results showed significantly bigger prevalence of the periodontal disease in the test group (95%) than the control group (28,3%). The level of insertion, plaque index and bleeding on probing were significantly increased in the patients with VCA (p<0,001). However, the probing depth measures had not shown significant differences between the groups (p=0,051), even so were increased for the test group. The presence and amount of the P.gingivalis were statistically bigger in the test group than the control group (p<0,05). A.actinomycetemcomitans was not found in any of the studied groups. Considering all patients with VCA, 65% developed VCA-I and 35% VCA-H. Patients with VCA-H showed greater levels of P.gingivalis than patient with VCA-I. However, in this group, it had positive correlation between deeper pockets and number of bacteria (p<0,05), what it was not observed for VCA-H. The analysis of risk for \"odds ratio\" identified that patient with periodontal disease present high risk of VCA development (OR=48,06, IC=95%). These findings show that the periodontal disease is more prevalent and severe in patients with VCA-I or VCA-H, with great amount of bacteria, especially P. gingivalis, present in deeper periodontal pockets, suggesting that the periodontal disease could play as risk factor on the development of vascular cerebral accident.

Traditionally, bacterial culture has been used for bacterial detection, allowing study of living microorganisms. Molecular methods are rapid and allow simultaneous identification of numerous species and uncultivated phylotypes. The objective of this doctoral thesis was to investigate the role of the oral microbiota, including poorly characterized and uncultivated bacteria, in dental caries and periodontitis, by comprehensive molecular, clinical, and statistical methods. The microbiota of 275 pre-school children (75 with caries and 200 caries-free) was examined by whole genomic DNA probes, 16S rDNA cloning and sequencing, and PCR. Streptococcus mutans, exhibiting a combined association with Streptococcus sobrinus, was significantly associated with Early Childhood Caries (ECC). Plaque from children with Severe Early Childhood Caries (S-ECC) was diverse with 138 identified and 107 unidentified taxa, which possibly included novel phylotypes. Other species/phylotypes associated with childhood caries included Lactobacillus gasseri (p&lt;0.01), Lactobacillus fermentum, Actinomyces israelii, and Actinomyces odontolyticus (all p&lt;0.05, ECC), Veillonella parvula (p&lt;0.01), Veillonella atypica (p&lt;0.05), and Veillonella sp. HOT-780 (p&lt;0.01, S-ECC). Lactobacillus acidophilus and Lactobacillus reuteri, both used as probiotic therapy species, were detected more frequently in caries-free children than those with ECC. Fastidious periodontal species, including Parvimonas micra, Aggregatibacter actinomycetemcomitans, Eubacterium brachy, Filifactor alocis (all p &lt;0.05), and Porphyromonas gingivalis (p&lt;0.01), were also more frequently detected in children with dental caries than in caries-free children. Other variables associated with ECC were race, dental visit, snacking (all p&lt;0.05), and visible dental plaque (p&lt;0.01). The oral microbiota of early periodontitis in young adults (N=141) was analyzed by whole genomic and oligonucleotide DNA probes, and PCR. Species detected more frequently in early periodontitis than periodontal health included Treponema denticola, F. alocis, Porphyromonas endodontalis, Bacteroidetes sp. HOT-274 (oral clone AU126), and A. odontolyticus (p&lt;0.01) by oligonucleotide DNA probes, and P. gingivalis (p&lt;0.001) and T. forsythia (p=0.03) by PCR. Subgingival samples exhibited a higher prevalence of periodontitis-associated species than samples from tongue surface, including A. actinomycetemcomitans, T. denticola, T. forsythia (all p&lt;0.05), and uncultivated TM7, Treponema, and Actinobaculum clones (all p&lt;0.05). P. gingivalis (p&lt;0.01) by PCR was associated with periodontal disease progression. Early periodontitis was associated with older age (p=0.01), male gender (p=0.04), and cigarette smoking (p=0.05). The role of bacterial subgroups in periodontitis was examined by studying the serotypeability of 313 genotyped clinical A. actinomycetemcomitans isolates (189 subjects). A total of 95 strains (30 subjects) remained non-serotypeable, although PCR revealed presence of the serotype- specific genes. The absence of the immunodominant serotype-specific antigen was confirmed by immunoblot assays. No major DNA rearrangement in the studied serotype-specific gene clusters was found. In summary, detection of previously cultured species and uncultivated phylotypes revealed the diversity of the oral microbiota in dental diseases and health already early in life. Bacterial species have insufficiently characterized subgroups that may have attributes to evade the host response. Molecular approaches used in this study enable comprehensive, culture-independent characterization of the oral microbiome that may in the future lead to identification of diagnostic bacterial profiles for dental diseases.

One of the greatest threats to human health is increasing antimicrobial resistance among pathogens, and finding alternatives for treatment of bacterial infections is of highest importance together with a more controlled use of antibiotics. Porphyrins and heme have both been shown to be a promising class of compounds for inactivation of bacteria; porphyrins by their excellent properties to act as a photosensitizer, and heme by its importance as an iron source during a bacterial infection in vertebrates. This thesis describes the development of analytical methods for the identification and determination of porphyrins and heme using liquid chromatography coupled to tandem mass spectrometry. Subsequently, these developed methods were applied to bacterial samples to investigate different culture conditions and additives effect to the intracellular porphyrin and heme composition. Singlet oxygen production of three naturally occurring porphyrins have been determined together with the photosensitivity for blue light and the porphyrin content in E. coli. Toothbrushes equipped with a LED, emitting light with a wavelength of 450 nm, were used in an eight week randomized clinical trial to investigate any positive periodontal effect of blue light. Porphyrin and heme content in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were highly affected by the different cultivation conditions. The culture age of A. actinomycetemcomitans affected the porphyrin profile, while only small changes were observed for P. gingivalis during growth. A large change of the porphyrin profile could be observed when the bacteria were passaged onto a new growth medium. Additional porphyrins were detected and the total porphyrin content increased up to 28 times. These findings highlight the need for more standardized cultivation procedures when performing in vitro experiments. Heme content in Escherichia coli was affected when different additives related to biosynthesis of heme were added to the growth medium. The uptake of heme could be reduced with 52% when a compound that chemically looks similar to heme was added to the growth medium. Since heme acquisition is important for many pathogens, this could be a promising target for antimicrobial drugs. E. coli showed no sensitivity for 405 nm light using light doses up to 172.8 J/cm2 and only low concentrations of porphyrins could be quantified. By adding a porphyrin precursor to E. coli the intracellular concentration of porphyrins increased remarkably and a light dose of 57.6 J/cm2 reduced the bacterial number with &gt; 5 log10 steps. This shows that E. coli can be killed due to their endogenous porphyrins. In the clinical study we could see a weak trend that the 450 nm LED toothbrush possessed a phototherapeutic effect for three clinical indices. All indices were decreased in the intervention group, but there were no statistically significant difference compared to the control group. However, four inflammation markers were significantly decreased in the intervention group while only one decreased significantly in the control group. In conclusion, this thesis has shown that porphyrins and heme are produced endogenously in microorganisms and that the porphyrin profiles vary depending on culture conditions and different additives. Furthermore, porphyrins may be used as endogenous photosensitizers to inactivate bacteria, but more research is necessary to determine if there is a specific porphyrin that contributes more to the photosensitivity. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

Oral health is a part of general health and the two are connected in many ways as well as impact each other. Oral diseases are some of the most common chronic diseases of mankind. Diseases such as caries and periodontitis are two of the most common ones affecting the oral cavity. Bacteria associated with these two diseases are Streptococcus mutans and Aggregatibacter actinomycetemcomitans respectively. Our hypothesis is that extracts from guava leaf or matcha tea affect adhesion of A. actinomycetecomitans or S. mutans to human epithelium. The aim of this study is to investigate a DNA-based method for studying attachment of bacteria to epithelial cells. Two different concentrations of the two bacterial species, high and low, were treated with matcha or guava leaf extract and adhesion on the human epithelial cell cultures was analyzed. The results were then analyzed using qPCR-based methods to test the amount of bacterial adhesion to the epithelial cells. Furthermore, the results showed that matcha was more effective for inhibition of bacterial adhesion than guava leaf.In conclusion, our results show that bacterial adhesion of A. actinomycetecomitans and S. mutans to human epithelial cells can be quantified by DNA-based methods, and the adhesion altered by plant extracts.

Actinobacillus actinomycetemcomitans is present in elevated proportions and numbers in dental bacterial biofilms of patients with localized aggressive periodontitis. This variant of periodontal disease, occurring in adolescents and young adults, is characterized by rapid and severe destruction of the connective tissues and bone supporting the teeth, eventually culminating in tooth loss. The cytolethal distending toxin (Cdt) is a newly discovered bacterial protein toxin, uniquely present in A. actinomycetemcomitans among all known to-date oral bacterial species. The Cdt has the capacity to inhibit mammalian cell growth, but its putative role in the pathogenesis of the disease is unclear. The aim of this in vitro work has been to study the effects of A. actinomycetemcomitans on periodontal connective tissue cell cultures, and to evaluate the possible involvement of its Cdt. A. actinomycetemcomitans inhibited the proliferation of gingival and periodontal ligament fibroblasts, as a result of a combined arrest at the G1 and G2/M phases of the cell cycle. This growth inhibition was non-lethal and the cells remained metabolically active, although their DNA synthesis was reduced. The intoxicated cells exhibited increased size and irregular structure, characterized by distension and elongation. This cellular enlargement occurred in both G1 and G2/M phase arrested cells. The Cdt of A. actinomycetemcomitans was responsible for the observed growth inhibition, as well as the concomitant morphological alterations. The possible induction of inflammatory cytokines related to bone resorption was investigated in response to A. actinomycetemcomitans, and the involvement of Cdt was evaluated. Extensive focus was given to the study of receptor activator of NF-κB ligand (RANKL) expression, a membrane-bound ligand that signals osteoclast progenitors to differentiate and fuse into mature osteoclasts, activating bone resorption. It was demonstrated that A. actinomycetemcomitans induced RANKL mRNA and protein expression in the cells studied, but did not affect the expression of its decoy receptor, osteoprotegerin. This induction was solely attributed to its Cdt, as demonstrated by the use of a cdt-knockout A. actinomycetemcomitans strain, purified recombinant Cdt, and antibodies blocking the Cdt. In addition, this event was not mediated by pro-inflammatory cytokines known to stimulate RANKL. Interleukin-6 mRNA and protein expression were also enhanced by A. actinomycetemcomitans, but Cdt had limited involvement in this enhancement. In conclusion, two distinct mechanisms by which A. actinomycetemcomitans Cdt may be involved in the pathogenesis of localized aggressive periodontitis are proposed. Firstly, the growth arrest of the resident fibroblasts may impair the physiological connective tissue remodelling equilibrium and lead to connective tissue attachment loss. Secondly, the induction of RANKL by these cells, residing in the proximity of the alveolar bone, may locally stimulate osteoclastogenesis and promote alveolar bone resorption. This work also provides further insights to the understanding of Cdt mechanisms of action, contributing to the global characterization of the toxin’s virulence.