Detecção do polimorfismo genetico de Streptococcus mutans isolados de individuos livres de carie (CPOD=0)

Orientador : Reginaldo Bruno GonçalvesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: A presente pesquisa teve por objetivo analisar a variabilidade infraespecífica de cepas de Streptococcus mutans isoladas de indivíduos livres de cárie. Foram coletadas amostras de saliva total não estimulada, placa dental e de dorso de língua de indivíduos que apresentavam índice CPOD igual a zero. Após dispersão e diluição seriada das amostras, alíquotas de cada diluição foram inoculadas em meio Mitis Salivarius Bacitracina Agar (MSB). Após incubação em estufa de pC02 10% a 37°C, foram isoladas do meio colônias com morfologia típica de estreptococos, para posterior identificação por provas bioquímicas. As amostras foram incubadas em caldo de Infusão de Cérebro e Coração (BHI), centrifugadas e lavadas, sendo os sedimentos obtidos submetidos ao processo de extração das proteínas intracelulares. Os extratos protéicos foram separados por eletroforese em gel de amido e corados especificamente para a detecção do polimorfismo isoenzimático. Os géis foram analisados através dos sistemas enzimáticos: leucina aminopeptidase (LAP), manitol 1-fosfato desidrogenase (MIP), manose fosfato isomerase (MPI), nucleosídio fosforilase (NSP), fenilalanil leucina peptidase (PLP) e transaminase glutâmico-oxalacética (GOT). A análise comparativa dos perfis da eletroforese de isoenzimas (Multilocus Enzyme Electrophoresis-MLEE) dos isolados, acessada pela soma de todos sistemas enzimáticos, permitiu observar a ocorrência de dois ou mais clones de S. mutans nos diferentes sítios nestes voluntários. Os resultados obtidos mostram que pode ser encontrado mais de um tipo genético de S. mutans colonizando os diferentes sítios em um mesmo indivíduoAbstract: The current research had as aim to analyze the infra-specific variability of Streptococcus mutans strains obtained from caries-free individuals. Non-stimulated whole saliva, dental biofilm and tongue dorsum samples were collected from subjects with DMFT = O. After dispersing and decimal dilutions, aliquots from each dilution were inoculated in Mitis Salivarius Bacitracin (MSB) agar plates, in duplicate. After growing at pCO2 100,/0 and 37°C, it was taken some S. mutans suspect colonies that were biochemically identified. S. mutans-positive colonies were grown in Brain Heart Infusion (BHI) broth, they were centrifuged and washed. The pellets were submitted to cell disruption and protein extraction. Protein extracts were separated by starch gel electrophoresis and specifically stained in order to obtain the isoenzymic polymorphism. The gels were analyzed for enzymatic systems: leucine amino peptidase (LAP), mannitol-l-phosphate dehydrogenase (MIP), mannose phosphate isomerase (MPI), nucleoside phosphorylase (NSP), phenylalanyl leucine peptidase (PLP) e glutamic-oxalacetic transaminase (GOT). The comparative analysis of MLEE profiles assessed by the sum of all enzymatic systems allowed the observation of occurrence of one or more S. mutans dones, varying in the different intra oral sites among these volunteers. The obtained results showed that it may be found more than one genetic type of such S. mutans colonizing different sites in a same subjectMestradoMestre em Biologia e Patologia Buco-Denta

Caracterização clonal de cepas de Streptococcus mutans por eletroforese de enzimas codificadas por multilocus

Twenty-one Streptococcus mutans strains were clustered by Multilocus Enzyme Electrophoresis (MLEE). Six isoenzymes showed strong infra-specific discriminatory power (M1P, MPI, PLP, NSP, GOT, and LAP). MLEE is a robust technique that may be used to explore clonal diversity of S. mutans isolates in epidemiological surveys.Vinte e uma cepas de Streptococcus mutans foram agrupadas pela eletroforese de enzimas codificadas por multilocus (MLEE). Seis isoenzimas apresentaram forte poder discriminatório (M1P, MPI, PLP, NSP, GOT e LAP). A MLEE é uma técnica robusta que pode ser empregada no estudo da diversidade clonal de cepas de S. mutans, em estudos epidemiológicos.171

The proposition of an inexpensive chemically defined culture medium (MycoDef) for yeast studies

Chemically defined culture media (CDCMs) have their compositions qualitatively and quantitatively known. They are constituted of components able to meet the nutritional requirements of microorganisms. This study evaluated the employability of a multivitamin as the basis for the elaboration of a CDCM for experiments with yeasts. Candida albicans reference strains ATCC®90028™ and SC5314 were used. YNB® without amino acid (Difco Co.) was used as the standard in evaluations. For the preparation of the experimental culture medium (MycoDef), commercial multivitamin tablets had their coatings removed and were crushed until obtaining a fine powder; missing constituents were added. Comparisons were carried out evaluating aerobic and anaerobic planktonic growth rate, biofilm formation rate, whole-cell protein patterns by SDS-PAGE, minimum inhibitory concentrations and minimal fungicidal concentrations for clotrimazole, fluconazole, nystatin, and griseofulvin. Growth in MycoDef and YNB® did not differ between them (p>0.05) in both normoxic and anoxic conditions. Regarding employability for sensitivity testing, MycoDef showed performance like YNB®. The protein profiles of cells grown on both media did not differ in the number and positioning of bands. The results obtained allowed us to infer that MycoDef is a reliable low-cost culture medium useful for trials involving C. albicans