Made available in DSpace on 2014-06-12T23:02:15Z (GMT). No. of bitstreams: 2 arquivo969_1.pdf: 1672515 bytes, checksum: 57a925ef24be8c7dd06dd31514a160d4 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O presente trabalho teve por objetivo avaliar a presença dos periodontopatógenos que formam o complexo vermelho (Tannerella forsythia (Tf), Porphyromonas gingivalis (Pg) e Treponema denticola (Td)) e o Aggregatibacter actinomycetemcomitans (Aa) em pacientes portadores de periodontite crônica. A amostra foi constituída de 29 pacientes com diagnóstico clínico e radiográfico de periodontite crônica de acordo com os critérios da AAP (2000). Todos os dentes foram sondados em seis sítios para registro de profundidade, perda de inserção clínica e sangramento após sondagem. As amostras para análise microbiológica foram coletadas dos 4 sítios com maior profundidade de sondagem para cada paciente, totalizando 116 amostras. Estas amostras foram processadas através da técnica de PCR convencional e foram observados os seguintes resultados: 46,6% apresentaram resultado positivo para a bactéria Pg; 41,4% para Tf; 33,6% para Td e 27,6% para Aa. Não se verificou associação significante entre a presença dos periodontopatógenos e as variáveis faixa etária, sexo e sangramento à sondagem. Para a bactéria Pg verificou-se associação significante (p<0,05) com a variável placa visível, e a presença das bactérias Pg e Tf foi mais prevalente (p < 0,05) em bolsas periodontais &#8805; 8 mm. Nos sítios com profundidade &#61619; 8 mm foram observadas com maior freqüência as combinações Pg + Tf (23,2%) e Pg + Tf + Td (20,0%). Foram estatisticamente significantes (p < 0,05) as associações entre a presença simultânea das bactérias Aa + Pg, Aa+ Tf, Pg + Tf e entre Tf + Td. Concluiu-se que as bactérias analisadas, principalmente as do complexo vermelho, estiveram fortemente relacionadas com a periodontite crônica, e que as bactérias Pg e Tf foram mais frequentes em bolsas periodontais profundas

Das parodontopathogene Bakterium Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) exprimiert zahlreiche Virulenzfaktoren. In dieser Studie wurden die Gene für die Virulenzfaktoren Leukotoxin (LtxA), Cytolethal Distending Toxin (CDT) und Fimbriae-assoziiertes Protein (Flp1) in 99 A. actinomycetemcomitans-Isolaten aus der Plaque von Parodontitispatienten aus vier deutschen Universitätskliniken untersucht. Die Proben wurden serotypisiert. Die Entnahme erfolgte mit sterilen Papierspitzen aus der jeweils tiefsten Tasche jedes Quadranten. Es wurden von den Patienten Sondierungstiefe (PD) und Attachmentlevel (AL) an sechs Stellen pro Zahn gemessen und ebenfalls die Tiefen an den vier Entnahmestellen notiert. Außerdem wurden ethnische Herkunft der Eltern, Geschlecht und Raucherstatus erfragt. Lediglich zwei A. actinomycetemcomitans-Isolate aus Frankfurt/Main wiesen das ltx-Gen mit Deletion auf. Diese zeigten signifikant höhere PD an den vier Entnahmestellen. Die übrigen 97 Proben hatten das ltx-Gen ohne Deletion in der DNA-Promotorregion ihrer A. actinomycetemcomitans-Stämme. Probanden mit Genlokus für das cdtB-Gen, mit drei cdt-Genen oder insgesamt fünf Genen für Virulenzfaktoren litten signifikant häufiger an aggressiver Parodontitis. A. actinomycetemcomitans-Isolate mit cdtA-Gen, cdtB-Gen, cdtCGen, drei cdt-Gene oder flp-1-Gen wiesen signifikant häufiger Serotyp b oder c auf. Probanden ohne cdtC-Gen oder flp-1-Gen in der DNA ihrer isolierten A. actinomycetemcomitans-Stämme zeigten am häufigsten Serotyp e. Probanden mit Genlokus für das cdtB-Gen oder drei cdt-Gene in den isolierten A. actinomycetemcomitans-Proben oder mit aggressiver Parodontitis stammten signifikant häufiger aus dem Ausland. Es wurde kein signifikanter Zusammenhang zwischen Vorkommen der Gene für Virulenzfaktoren und PD bzw. AL im gesamten Gebiss gefunden.

Abstract  Aggregatibacter aphrophilus, a Gram negative bacterium, found in the oral cavity, causing cerebral abscesses and infective endocarditis, has been shown to be serum resistant in previous studies. Bacterial secretion systems are important for bacteria as they transfer virulence factors into other bacteria or host cells as an attack. A. aphrophilus encodes a type VI secretion system, which is a spike-like membrane protein, mainly consisting of a hemolysin co-regulated protein (Hcp). In this work, it was tested if Hcp would contribute to serum resistance of A. aphrophilus. Firstly, to assess Hcp contribution to serum resistance, a bacterial serum killing assay-method was used and data was collected from three independent experiments. Two strains of A. aphrophilus were used in the experiments: the laboratory strain HK83 and a HK83 hcp mutant strain. The results showed that Hcp provided no significant effect on serum resistance of A. aphrophilus. Secondly, optical density measurements were made for growth curve analysis, to determine if the HK83 hcp mutant strain had an impact in growth compared to HK83. The results indicated that the HK83 hcp mutant strain had a somewhat reduced growth compared to its parental strain.

Objectives: Microbial infection and the host response to the infection play a significant role in the etiology of periodontal diseases. Previous studies reported a relatively high prevalence of periodontitis among adolescents in Morocco. The importance of the composition of subgingival plaque and the presence and proportion of Aggregatibacter actinomycetemcomitans to the total plaque bacteria in the pathogenesis of periodontitis is not well understood. The purpose of this study is to compare the relative abundance of A. actinomycetemcomitans in subgingival dental plaque from young Moroccans with aggressive periodontitis, chronic periodontitis, and those without periodontitis, and to construct a multivariable model to investigate the effect of demographic attributes of age, gender and relative ratio of A. actinomycetemcomitans in dental plaque with periodontal disease status. Methods: Sample population includes 984 subjects, aging from 12-20 years old, who were surveyed and examined for periodontal disease status. 82 subjects were selected consisting of 26% aggressive periodontitis, 12% chronic periodontitis, and 62% without periodontitis. Subgingival plaque was collected from these 82 subjects. Whole DNA was extracted and purified, and real-time PCR was used employing a primer for eubacteria, and specific primer for A. actinomycetemcomitans. PCR assays confirmed the amplification and quantification of DNA of total bacterial and A. actinomycetemcomitans. Results: 73% of the subjects harbored A. actinomycetemcomitans: 63% in aggressive periodontitis, 90% in chronic periodontitis, and 73% in controls. The percentage A. actinomycetemcomitans to total bacterial load increased with age, was similar among males and females, and was somewhat higher in persons with periodontitis than the controls. Using a logistic regression analysis that included age, gender and A. actinomycetemcomitans ratio showed that only age is significantly correlated with the diagnosis of periodontitis in this population. Conclusions: A. actinomycetemcomitans is prevalent among this young Moroccan group and is somewhat more prevalent in subjects with periodontitis than the controls. However, the presence and ratio of this species to the total bacteria in subgingival plaque explained only a small proportion of the variance in periodontitis in this group. / Oral Biology

Aggregatibacter actinomycetemcomitans is a Gram negative bacterium found exclusively in the mammalian oral cavity where it resides in the gingival crevice, the space between the tooth and gum tissue. Though it has historically been considered a common commensal organism, it is now appreciated that A. actinomycetemcomitans is an opportunistic pathogen associated with the diseases periodontitis and endocarditis. To cause infection, A. actinomycetemcomitans must interact and compete with neighboring bacteria for space and nutrients, though little is known about the physiology it employs within the gingival crevice. Using A. actinomycetemcomitans grown in a chemically defined medium containing carbon sources found in vivo, I use transcriptome analyses and growth studies to show that A. actinomycetemcomitans preferentially utilizes lactate over the phosphotransferase system (PTS) sugars glucose and fructose. Additionally, the presence of lactate or pyruvate inhibits the transport and metabolism of these sugars in a post-transcriptionally controlled process I have termed PTS substrate exclusion. Since lactate is an energetically inferior carbon source, PTS substrate exclusion appears to be a carbon resource partitioning mechanism that allows A. actinomycetemcomitans to avoid competition for energetically favorable sugars with other species, and I propose a model to describe this phenomenon. To begin to understand the mechanism of PTS substrate exclusion, I examine the first step of the proposed model by purifying and characterizing the L-lactate dehydrogenase (LctD) from A. actinomycetemcomitans. I demonstrate that, unlike other studied lactate dehydrogenases, the LctD from A. actinomycetemcomitans does not exhibit feedback inhibition in the presence of physiologically relevant concentrations of pyruvate, which supports my hypothesis that elevated intracellular pyruvate levels inhibit the PTS. The results of my studies provide insight into a new regulatory mechanism governing carbon utilization in this bacterium. / text

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>

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans is primarily associatedwith aggressive forms of periodontal disease. Additionally, it has occasionally been found to causemetastatic infections in non-oral sites. This requires the ability to evade the bactericidal activity ofthe complement system of the humoral immune system. Outer membrane proteins, namely,Omp100 and OmpA have been connected to normal human serum resistance for several bacteriaspecies. The objective of this study was to investigate if serum-resistant ompA mutants can beobtained, and to detect changes in OMP expression. We used A. actinomycetemcomitansserotype a strain D7SS and D7SS ompA knockouts. The strains were incubated in 50 % NHS.This resulted in a substantial decrease of survival among D7SS ompA knockouts. D7SS ompAknockouts were exposed to 50 % NHS once more to confirm stable serum resistance. 13 out of14 tested clones showed growth, indicating that serum resistant ompA mutants could begenerated. SDS-PAGE gel of extracted outer membrane vesicles revealed an additional proteinband of approximately 34 kDa in at least 4 of 5 tested serum resistant ompA mutants. This proteinband has been analyzed in the laboratory, and according to LC-MS/MS it contains an OmpAhomologue, which has been named OmpA2. We conclude that OmpA2 expression might be amajor mechanism for serum survival in A. actinomycetemcomitans serotype a strain D7SS ompAknockouts.