Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Early childhood caries (ECC) is considered a serious public health issue among children all over the world. However, many aspects should be explored about the oral microbiota related to the ECC progression and how the bacterial community modifies according to the dentine lesion activity. This thesis, composed by two chapters, aimed to investigate and quantify with quantitative polymerase chain reaction (qPCR) the following bacteria Actinomyces naeslundii, Bifidobacterium spp., Lactobacillus acidophilus, Streptococcus gordonii, Streptococcus mutans, as well as members of the groups Lactobacillus casei and Mitis on biofilm from pre-school children with different stages of early childhood caries progression (Chapter 1) and on active and inactive dentine carious lesions (Chapter 2), and also to verify the association of these microorganisms on the process of health or disease. The sample consisted on preschool children aged between 2 and 5 years from nurseries and public preeschools in Fortaleza-CE. The children were examined for caries diagnosis with ICDAS II index (International Caries Detection Assessment System), and the Nyvad criteria, in order to evaluate prevalence and caries activity. The supragingival biofilm collection was taken from 75 children, who were divided in three groups according to the ICDAS II: CF (caries free) (n=20), ECL (presence of enamel caries lesion) (n=17) and DCL (presence of dentine caries lesion) (n=38). Samples of carious dentine were collected under rubber dam isolation of 56 lesions of dentine affected by caries, being 17 inactive and 39 active. The DNA of all the collected samples was extracted and purified, then tested for the presence of the formerly mentioned bacterial species/groups through qPCR. The quantity of bacteria was compared through the Kruskal-Wallis and Mann-Whitney tests. Besides, the association between the presence of bacteria and ECC was analyzed through the Chi-square test, with a 5% significance level and the multiple logistic regression was applied. Bacteria from the group L. casei and L. acidophilus presented low detection on biofilm of all evaluated groups. The presence of S. mutans and Bifidobacterium spp. showed a strong association with dental caries progression on the biofilm from children with dentine lesions with odds ratio of 21,5 and 5,9; respectively. On active dentine lesions, concentrations of Bifidobacterium spp. and species from the Lactobacillus casei group were significantly higher when compared to the inactive lesions (p<0.05). The levels of Actinomyces naeslundii, Streptococcus gordonni and species from Mitis group were not significantly different among biofilm groups as well as comparing dentine lesions. In conclusion, the microbial profile from biofilm samples presented differences on the proportion of acidogenic and aciduric bacteria with dental caries progression. The presence of Bifidobacterium spp. and S. mutans presented a strong association with the development of the more advanced stages of ECC. Regarding the activity of dentine lesions, higher detection levels of the group L. casei and Bifidobacterium spp. showed an important role of these bacteria in the dentine caries activity. / A cÃrie precoce da infÃncia (CPI) Ã considerada um grave problema de saÃde pÃblica em crianÃas prÃ-escolares em todo mundo. No entanto, muitos aspectos ainda devem ser explorados acerca da microbiota oral relacionada com a progressÃo da CPI e como a comunidade bacteriana se modifica de acordo com a atividade da lesÃo dentinÃria. Esta tese, constituÃda de dois capÃtulos, teve como objetivo identificar e quantificar atravÃs da tÃcnica de reaÃÃo em cadeia da polimerase quantitativa (qPCR) as bactÃrias Actinomyces naeslundii, Bifidobacterium spp., Lactobacillus acidophilus, Streptococcus gordonii, Streptococcus mutans, bem como espÃcies dos grupos Lactobacillus casei e Mitis em biofilme de crianÃas prÃ-escolares com diferentes estÃgios de progressÃo da cÃrie precoce da infÃncia (CapÃtulo 1) e em lesÃes cariosas dentinÃrias ativas e inativas (CapÃtulo 2) e ainda verificar a associaÃÃo destes microrganismos ao processo de saÃde ou de doenÃa. A amostra consistiu em prÃ-escolares com idade entre 2 e 5 anos de idade que frequentavam creches e escolas pÃblicas de Fortaleza-CE. As crianÃas foram examinadas com o uso de Ãndices visuais ICDAS II (International Caries Detection Assessment System) e Nyvad, a fim de avaliar a prevalÃncia e atividade de cÃrie. A coleta de biofilme supragengival foi realizada em 75 crianÃas, as quais foram agrupadas de acordo com Ãndice ICDAS II em trÃs grupos: CF (livres de cÃrie) (n=20), ECL (presenÃa de lesÃes de cÃrie em esmalte) (n=17) e DCL (presenÃa de lesÃes de cÃrie em dentina) (n=38). Amostras de dentina cariada foram coletadas sob isolamento absoluto de 56 lesÃes cariosas dentinÃrias, sendo 17 inativas e 39 ativas. O DNA de todas as amostras coletadas foi extraÃdo e purificado e, em seguida, testado para a presenÃa das espÃcies/grupos bacterianos acima citados atravÃs de qPCR. A quantidade das bactÃrias foi comparada pelos testes Kruskal-Wallis e Mann-Whitney. AlÃm disso, a associaÃÃo da presenÃa de bactÃrias e CPI foi analisada atravÃs do teste Qui-quadrado, com nÃvel de significÃncia de 5% e aplicado a regressÃo logÃstica mÃltipla. BactÃrias L. acidophilus e do grupo L. casei apresentaram baixa detecÃÃo no biofilme de todos os grupos avaliados. A presenÃa de S. mutans e Bifidobacterium spp. mostrou forte associaÃÃo com a progressÃo da doenÃa no biofilme de crianÃas com lesÃes dentinÃrias com âodds ratioâ de 21,5 e 5,9, respectivamente. Em lesÃes dentinÃrias ativas, concentraÃÃes de Bifidobacterium spp. e bactÃrias do grupo L. casei foram significativamente maiores quando comparadas Ãs lesÃes inativas (p<0.05). Os nÃveis de A. naeslundii, bactÃrias do grupo Mitis e S. gordonni nÃo apresentaram diferenÃa significativa entre os grupos de biofilme, assim como nas lesÃes dentinÃrias. Conclui-se que as amostras de biofilme apresentaram alteraÃÃo na proporÃÃo de bactÃrias acidogÃnicas e acidÃricas com a progressÃo da doenÃa cÃrie. A presenÃa de Bifidobacterium spp. e S. mutans apresentou forte associaÃÃo com os estÃgios mais avanÃados da CPI. Em relaÃÃo Ãs lesÃes dentinÃrias, o aumento da concentraÃÃo de bactÃrias Bifidobacterium spp. e do grupo L. casei evidenciou um papel importante destas bactÃrias na atividade de lesÃes dentinÃrias.

Actinomycosis is an infrequent bacterial infection involving Actinomyces spp and related organisms which may occur at many body sites. It can also be found in the microbiota. Actinomyces spp are described as gram-positive bacilli whereas some species grow strictly anaerobically and some facultative. Culturing is a standardized method when suspecting actinomycosis and can be a diagnostic challenge because of inhibition of microbiota and slow growth. Enriched agar plates are used when culturing fastidious bacteria and may be more selective when including antibiotics. The aim of this project was to evaluate if using antibiotic disc facilitates identification of Actinomyces spp when mixed with microbiota. A mix of microbiota was made by pooling together several species. The susceptibility of different isolates and microbiota was analysed using antibiotic discs to determine which disc to use in a trial. A trial was done by inoculating the isolates with the microbiota on agar plates, dispensing ciprofloxacin, and trimethoprim discs. A control group without antibiotic discs were also tested. Results showed variance for most isolates susceptibility. No disc performed superior in the trial, but ciprofloxacin on FAA plates incubated anaerobically gave slightly higher recovery. Both discs facilitated identification of some isolates by supressing much microbiota. Considering that the isolates had varying susceptibility it may be problematic to find one common disc. This study has given new insights on what may facilitate identification. Further studies are needed to determine if antibiotic discs could facilitate identification of Actinomyces and needs testing on clinical samples using larger sample size.

Aktinomyzeten sind ein Teil der residenten Flora des menschlichen Verdauungstraktes, des Urogenitalsystems und der Haut. Die zeitraubende Isolation und Identifikation der Aktinomyzeten durch konventionelle Methoden stellt sich häufig als sehr schwierig dar. In den letzten Jahren hat sich jedoch die Matrix-unterstützte Laser-Desorption/Ionisation-Flugzeit-Massenspektrometrie (MALDI-TOF-MS) als Alternative zu etablierten Verfahren entwickelt und stellt heutzutage eine schnelle und simple Methode zur Bakterienidentifikation dar. Unsere Studie untersucht den Nutzen dieser Methode für eine schnelle und zuverlässige Identifizierung von oralen Aktinomyzeten, die aus dem subgingivalen Biofilm parodontal erkrankter Patienten isoliert wurden. In dieser Studie wurden elf verschiedene Referenzstämme aus den Stammsammlungen ATCC und DSMZ und 674 klinische Stämme untersucht. Alle Stämme wurden durch biochemische Methoden vorab identifiziert und anschließend ausgehend von den erhobenen MALDI-TOF-MS-Daten durch Ähnlichkeitsanalysen und Klassifikationsmethoden identifiziert und klassifiziert. Der Genotyp der Referenzstämme und von 232 klinischen Stämmen wurde durch Sequenzierung der 16S rDNA bestimmt. Die Sequenzierung bestätigte die Identifizierung der Referenzstämme. Diese und die zweifelsfrei durch 16S rDNA Sequenzierung identifizierten Aktinomyzeten wurden verwendet, um eine MALDI-TOF-MS-Datenbank zu erstellen. Methoden der Klassifikation wurden angewandt, um eine Differenzierung und Identifikation zu ermöglichen. Unsere Ergebnisse zeigen, dass eine Kombination aus Datenerhebung mittels MALDI-TOF-MS und deren Verarbeitung mittels SVM-Algorithmen eine gute Möglichkeit für die Identifikation und Differenzierung von oralen Aktinomyzeten darstellt.

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Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

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