The pathogen Streptococcus pyogenes is a significant cause of human morbidity and mortality. Most of the work in this thesis is focused on streptococcal virulence factor IdeS, but the thesis also features work on SpeB, another streptococcal virulence factor. Both IdeS and SpeB are secreted cysteine proteases and both have previously been shown to degrade human IgG. IgG is the only known substrate for IdeS while SpeB is a more promiscuous protease with a larger number of identified substrates. A significant part of the data presented in this thesis is the result of designing and optimizing methods to detect and accurately measure the proteolytic degradation of IgG. Methods aimed at measuring the binding interactions between enzyme and substrate have also been frequently utilized. I show that IdeS is a monomeric protease, as opposed to previously published data that suggested it to be dimeric. IdeS cleaves the two heavy chains of IgG in a two-step reaction and I demonstrate that the first cleavage is magnitudes faster than the second one. This means that IdeS is a more efficient enzyme than previously thought. The difference in rate cannot completely be explained by a loss of affinity between IdeS and IgG after the cleavage of the first heavy chain. The velocity of IdeS is further increased by the presence of human Cystatin C, via an unknown mechanism. Cystatin C is normally a protease inhibitor and it having an opposite effect is puzzling.The synthesis and evaluation of novel inhibitors are also described. Peptide analogues mimicking the sequence surrounding the scissile bond on IgG - with an amino acid replaced with a more rigid motif - act as specific, but low-affinity, inhibitors of IdeS. The peptide analogues’ inhibitory capacity for SpeB and papain was also assayed.When it comes to SpeB, I show that it does not have IgG as a substrate under physiological conditions, in contrast to what was previously thought. This thesis does not only present findings on the IgG degrading capacity of IdeS and SpeB but also include data on fundamental enzymatic properties for these proteases.

Streptococcus sanguinis is the second most common causative agent of bacterial infective endocarditis (IE). Risk of S. sanguinis IE is dependent on pre-disposing damage to the heart valve endothelium, which results in deposition of clotting factors for formation of a sterile thrombus (referred to as vegetation). Despite medical advances, high mortality and morbidity rates persist. Molecular characterization of S. sanguinis virulence determinants may enable development of prevention methods. In a previous screen for S. sanguinis virulence determinants by signature-tagged mutagenesis (STM) an attenuated mutant was identified with a transposon insertion in the nrdD gene, encoding an anaerobic ribonucleotide reductase. Evaluation of this mutant, as well as an nrdD in-frame deletion mutant, JFP27, by a soft-agar growth assay confirmed the anaerobic growth sensitivity of these strains. These studies suggest that an oxygen gradient occurs at the site of infection which selects for expression of anaerobic-specific genes at the nexus of the vegetation. The random STM screen failed to identify any favorable streptococcal surface-exposed prophylactic candidates. It was also apparent that additional genetic tools were required to facilitate the in vivo analyses of mutant strains. As it was desirable to insert antibiotic resistance markers into the chromosome, we identified a chromosomal site for ectopic expression of foreign genes. In vitro and in vivo analyses verified that insertion into this site did not affect important cellular phenotypes. The genetic tools developed facilitated further in vivo screening of S. sanguinis cell wall-associated (Cwa) protein mutants. A directed application of STM was employed for a comprehensive analysis of this surface protein class in the rabbit model of IE. Putative sortases, upon which Cwa proteins are dependent for cell surface localization, were also evaluated. No single S. sanguinis Cwa protein was determined essential for IE by STM screening; however competitiveness for colonization of the infection site was reduced for the mutant lacking expression of sortase A. The studies described here present a progressive picture of S. sanguinis IE, beginning with surface protein-dependent colonization of the vegetation in early IE, that later shifts to a bacterial persistence in situ dependent on condition-specific housekeeping genes, including nrdD.

O objetivo do presente estudo foi avaliar, em aparelhos disjuntores de Haas, a contaminação in situ por estreptococos do grupo mutans, sob a forma de colônias/biofilmes, nas diferentes superfícies (acrílico, fios, bandas e parafusos), com e sem o uso de bochechos com gluconato de clorexidina a 0,12%, por meio de Cultura Microbiana e Microscopia Eletrônica de Varredura (MEV). Adicionalmente, a corrosão na área da união entre o fio, a solda de prata e a banda dos aparelhos foi avaliada por meio de Estereomicroscopia Ótica, MEV e análise em Espectrometria de Energia Dispersiva (EDS). Foram selecionados 34 pacientes de 7 a 12 anos de idade, que compareceram à clínica de Ortodontia Preventiva da FORP/USP, com necessidade de correção com aparelho disjuntor de Haas por problemas transversais da maxila (mordida cruzada posterior). Em seguida, utilizando uma tabela de números randômicos, os pacientes foram aleatoriamente divididos em dois grupos de 17 indivíduos cada (Grupos I e II). Durante todo o período de permanência dos aparelhos na cavidade bucal, no Grupo I (controle; n=17) os pacientes foram orientados a utilizar dentifrício fluoretado para escovação diária e não empregar bochechos com soluções antimicrobianas. Por outro lado, no Grupo II (experimental; n=17) os pacientes foram orientados a realizar, além da escovação diária com o uso de dentifrício fluoretado, 2 bochechos por semana com solução de gluconato de Clorexidina a 0,12% (Periogard®). Decorridos cerca de 4 meses da permanência na cavidade bucal, os aparelhos foram removidos. Sendo seccionadas, aleatoriamente, partes dos aparelhos constituídas de uma banda com fio soldado, para análise em estereomicroscopia ótica, MEV e EDS. Os resultados em estereomicroscopia foram submetidos à análise estatística por meio do teste de Fisher, com nível de significância de 5%. Em seguida, os aparelhos foram submetidos ao processamento microbiológico, em meio de cultura CaSa B, para contagem das colônias/biofilmes de estreptococos do grupo mutans. O teste estatístico não-paramétrico de Mann-Whitney foi aplicado para verificação de possíveis diferenças entre os grupos, com relação à formação de colônias/biofilmes sobre a superfície das diferentes áreas (parafuso, resina acrílica, bandas, fio vestibular e fio palatino), como também para verificar se havia diferença entre a formação de colônias/biofilmes sobre as superfícies livres (voltadas para a cavidade bucal) e as superfícies não-livres (em contato direto com a mucosa palatina e sulco gengival). O nível de significância adotado foi de 5%. Após cultura microbiana, partes dos aparelhos representativas de cada grupo foram submetidas ao processamento e análise em MEV. Os resultados obtidos evidenciaram que, com relação às colônias/biofilmes das superfícies nãolivres dos Grupos I e II, não houve diferença entre os grupos (p=0,009). No entanto, quando as superfícies livres dos Grupos I e II foram comparadas, evidenciou-se diferença significante entre os 2 grupos, em todas as áreas analisadas (parafuso, resina acrílica e bandas) (p<0,001). Os resultados da cultura microbiana foram confirmados em MEV. A análise em estereomicroscopia ótica evidenciou a presença de áreas de alteração de coloração sugestivas de corrosão na região de solda em contato com a banda e com o fio, em ambos os grupos (p=1). Os picos dos elementos químicos observados em EDS também foram semelhantes em ambos os grupos. Pôde-se concluir que o uso do gluconato de clorexidina a 0,12% (Periogard®), sob a forma de bochecho, apresentou eficácia na redução da formação de colônias/biofilmes nas superfícies livres dos aparelhos disjuntores de Haas, in situ, sem elevação nos níveis de corrosão. / The purpose of this study was to evaluate, in situ, in Haas expanders, the contamination of different surfaces (acrylic resin, wires, bands and screws) by mutans group streptococci colonies/biofilms with and without prescription of 0.12% chlorhexidine gluconate mouthwashes, by means of microbial culture and scanning electron microscopy (SEM). Additionally, the corrosion in the area of union between the appliances wire, silver soldering and band was assessed by optical stereomicroscopy, SEM and energy dispersive spectroscopy (EDS). Thirty-four children aged 7 to 12 years were selected among the patients attending the Preventive Orthodontic Clinic at FORP/USP with need of corrective orthodontics with Haas expander due to transversal problems of the maxilla (posterior crossbite). Thereafter, using a table of random numbers, the patients were randomly assigned to two groups of 17 individuals each (Groups I and II). Throughout the time that the appliances remained in the oral cavity, the patients in Group I (control; n=17) were instructed to use a fluoridated dentifrice for daily toothbrushing and not to use antimicrobial mouthwash solutions. On the other hand, in Group II (experimental; n=17), in addition to daily toothbrushing with a fluoridated dentifrice, mouthwashes with an antimicrobial agent (0.12% chlorhexidine gluconate - Periogard®) were prescribed to the patients. After approximately 4 months of maintenance in the patients mouth, the appliances were retrieved. Thereafter, components of the appliances (consisting of a band with soldered wire) were sectioned at random for analysis under optical stereomicroscopy, SEM and EDS. The results of the optical stereomicroscopy were submitted to statistical analysis using Fisher\'s test at 5% significance level. The appliances were sent to microbiology processing, in CaSa B culture medium, for counting of the number of mutans streptococci colonies/biofilms. Mann-Whitney non-parametric statistical test was applied to verify possible differences between the groups with respect to the formation of colonies/biofilms on the surface of different areas (screw, acrylic resin, bands, buccal wire and palatal wire), as well as to determine whether there were differences between the formation of colonies/biofilms on free surfaces (facing the oral cavity) and non-free surfaces (in direct contact with the palatal mucosa and gingival sulcus). The level of significance was set at 5%. After microbial culture, components of the appliances that were representative of each group were submitted technical processing and SEM analysis. The obtained results showed that there was no statistically significant difference between Groups I and II (p=0.009) regarding the formation of colonies/biofilms on the non-free surfaces. However, when the free surfaces of Groups I and II were compared, statistically significant difference was observed between these groups in all analyzed areas (screw, acrylic resin and bands) (p<0.001). The results of the microbial culture were confirmed by the SEM analysis. The optical stereomicroscopic analysis showed the existence of color alteration suggestive of corrosion in the solder region in contact with the band and with the wire in both groups (p=1). The peaks of chemical elements observed in EDS were also similar in both groups. It may concluded that the use of 0.12% chlorhexidine gluconate mouthwashes (Periogard®) showed efficacy in reducing the formation of colonies/biofilms on the free surfaces of Haas expanders, in situ, without increasing the corrosion levels.

O objetivo deste estudo foi avaliar, em crianças de creches públicas e filantrópicas do município de São Paulo, a prevalência de cárie dental e os fatores de risco relacionados à presença de cárie e presença de altos níveis de estreptococos mutans (SM). Foram incluídas variáveis denominadas sociodemográficas, condições de gestação e nascimento, variáveis fisiológicas do estado nutricional, aleitamento materno e alimentação complementar, hábitos alimentares, variáveis comportamentais e odontológicas. O estudo foi desenvolvido em parceria entre a Disciplina de Odontopediatria da Faculdade de Odontologia da Universidade de São Paulo (FOUSP) e a Disciplina de Nutrologia do Departamento de Pediatria da Universidade Federal de São Paulo (UNIFESP EPM). É parte integrante do Projeto Crecheficiente - Ações de Segurança e Educação Alimentar em Creches Públicas e Filantrópicas do Município de São Paulo. A população deste estudo foi constituída de 150 e 144 crianças, que participaram da avaliação odontológica e microbiológica respectivamente, entre 12 e 24 meses, de ambos os gêneros, regularmente matriculadas em cinco creches, sendo duas públicas e três filantrópicas. A coleta dos dados foi obtida por meio de entrevistas com as mães ou responsáveis, antropometria, coleta de sangue para dosagem de hemoglobina, avaliação odontológica (índices ceo-d e ceo-d modificado) e coleta salivar das mães e das crianças para teste de estreptococos mutans. Os dados obtidos foram analisados estatisticamente utilizando análise de regressão logística univariada, considerando o nível de significância a 5%. Foram utilizadas as mesmas variáveis de risco e seus respectivos pontos de corte nas análises univariadas da avaliação odontológica e microbiológica. Das crianças examinadas 7,3% apresentaram experiência de cárie, sendo encontrada uma média do índice ceo-d igual a 0,09 e ceo-d mod de 3,37, sem diferença entre gêneros e tipo de creche. Em relação à presença de cárie, a análise de regressão univariada indicou significância dos seguintes fatores: o tempo de matrícula na creche, aleitamento materno exclusivo menor de 60 dias, idade do primeiro contato com açúcar, e níveis altos de SM que favoreceram a ocorrência da doença, o número maior de irmãos e a presença de biofilme visível foram significantes e associados com proteção. Dentre os alimentos citados encontrados na dieta dos lactentes, observa-se que o consumo de mel e o consumo de refrigerantes favoreceram a ocorrência da cárie dentária. Em relação à presença de SM, 72,9% das crianças tinham este microrganismo presente na cavidade bucal sendo que 9,5% apresentavam altos níveis de SM. As variáveis que influenciaram significantemente os altos níveis de SM foram: idade da mãe, idade de introdução do açúcar e presença de cárie dentária na criança. Em relação aos hábitos alimentares apenas o consumo de petit suisse favoreceu a quantidade elevada de SM. A contagem dos níveis salivares de SM da mãe não foi significante. / The aim of this study was to assess in children of public and philanthropic day care centers of São Paulo, the prevalence of dental carie and the risk factors associated to dental carie and high levels of mutans streptococci (MS). Were included variables denominated socio-demographic, pregnancy and birth conditions, physiological variables of nutrition status, breast feeding and complementary diet, dietary habits, behavior and dental variables. This study was the result of a partnership between the Pediatrics Department of the University of São Paulo and the Nutrology Department of Federal University of São Paulo, UNIFESP EPM. It is part of a larger study called Crecheficiente Project Security and Dietary Education actions of public and philanthropic day care centers of São Paulo city. The population of this study were consisted of 150 and 144 children that participated of the dental and microbiological evaluation respectively, between 12 and 24 years old, both gender, regularly registered in five day care centers, two publics and three philanthropic. The data collection were done by interviews with mothers or childrens responsible, anthropometry, bloody collection for hemoglobin, dental test (Knutson, dmf and modified dmf) and salivary collection from mothers and children for estreptococos mutans test analysis. Data analysis involved univariate logistic regression, The level of significance was set at 5%. The same variables presented at univariate analysis of the dental test and break point, were described into microbiological test. 7,3% of the children had dental carie. A mean of dmft index were 0,09 and modified dmft 3,37. No difference was found between gender and type of day care center. The univariate analyses indicated significance for caries experience: time of day care center admission, exclusive breast feeding least than 60 days, age of the first sugar contact, high levels of MS benefit caries, the major number of brothers and sisters, presence of dental plaque were associated to protection. The foods found in the dietary habits of the infants, honey and soft drinks exposed the occurrence of dental caries. For the streptococci mutans experience, 72,9% of the children had the microorganism present and 9,5% were high levels of MS. The variables that influence significantly this group were: mothers age, age of the first sugar contact and childrens carie experience. In the dietary habits, only the pettit suisses consume exposed to high levels of SM. The mothers mutans streptococci levels were not significant.

Este trabalho teve o objetivo de avaliar o efeito do uso de goma de mascar contendo 15% de xilitol, por 30 dias, em 12 indivíduos com altos níveis salivares (= 10 5 u.f.c./ml de saliva) de Estreptococos do grupo mutans (SM) sobre os níveis salivares de SM, os genótipos de S. mutans e a ocorrência de amostras xilitol-tolerantes (X R ) na saliva. Os voluntários foram instruídos a mascarem a goma por 5 minutos, 5 vezes ao dia, durante 30 dias, sem realizarem nenhuma alteração nos hábitos de higiene ou dieta. Amostras de saliva foram coletadas no início do experimento (antes do consumo da goma de mascar), 30 dias após a utilização da goma de mascar e 30 dias após a interrupção do uso da goma. Dois voluntários foram excluídos da última coleta devido ao uso de antibióticos. Os resultados mostraram que ocorreu redução acentuada nos níveis salivares de SM. A concentração média de SM no exame inicial foi de 9,8 x 10 5 ± 2,61 x 10 5 UFC/ml de saliva, enquanto este valor foi de 2,17 x 10 5 ± 1,94 x 10 5 após o consumo da goma, sendo a diferença estatisticamente significante (p < 0,05). Porém este efeito não foi duradouro, não apresentando diferença estatística 30 dias após a interrupção do consumo (3,5 x 10 5 ± 2,71 x 10 5 ), em relação aos valores iniciais. Foram identificados 17 genótipos distintos de S. mutans nas amostras salivares de 10 voluntários, que completaram as 3 etapas do estudo, pela técnica de RAPD-PCR, com o iniciador OPA-2. O ensaio de tolerância de S. mutans ao xylitol in vitro, em meio BHI e BHI /xilitol 1%, permitiu a identificação de 12 amostras X R entre 124 amostras estudadas, representantes dos 17 genótipos distintos. Amostras de S. mutans (X R ) foram identificadas nas 3 coletas, inclusive antes do consumo da goma. Foram identificadas amostras X R e X S com o mesmo genótipo por RAPD-PCR. A prevalência de amostras X R não apresentou relação direta com o uso de xilitol ou com os tipos genotípicos. Baseados nas condições do presente estudo, os resultados sugerem que o uso regular de gomas de mascar contendo 15 % de xilitol por 30 dias contribui para a redução dos níveis salivares de SM durante o período de consumo, porém esta redução não persiste após a interrupção do uso. Além disso, foi observado que o uso de xilitol não foi fator determinante na seleção in vivo de determinado genótipo X R / The aim of this study was to evaluate the effect of xylitol (15%) containing chewing gum, for 30 days , in 12 subjects with high mutans Streptococci (MS) salivary levels (= 10 5 c.f.u./ml) on Mutans streptococci (MS) salivary levels, on genotypes and detection of xilitol tolerant (X R ) strains of S. mutans . Volunteers were asked to chew one lozangle, for 5 minutes, five times per day, for 30 days with no change in their habitual diet or oral hygiene. Saliva samples were collected at baseline, 30 days after gum usage and 30 days after the interruption of the consumption. Two subjects were excluded from the last examination due to antibiotic use. The results showed a marked reduction in MS salivary levels. The average levels of MS in saliva in baseline was 9.8 x 10 5 ± 2.61 x 10 5 c.f.u./ml saliva and decreased to 2.17 x 10 5 ± 1.94 x 10 5 after 30 days of consumption. This difference was statistically significant (p< 0.05). However this effect was not long lasting, and 30 days after the interruption of consumption (3.5 x 10 5 ± 2.71 x 10 5 ) there was no statistically difference in relation to baseline data. Seventeen different genotypes were identified in MS salivary samples of 10 volunteers that completed the 3 sampling procedures, by RAPD-PCR using OPA-2 primer. The assay of xylitol tolerance of S. mutans in vitro, in BHI and BHI/1% xylitol allowed the identification of 12 X R isolates, among 124 tested strains representing all detected genotypes. X R strains were detected in all sampling steps, including at baseline, before chewing gum consumption. There were X R and X S isolates with the same genotypes by RAPD-PCR. The prevalence of X R strains did not show direct relation to consumption of xylitol neither to genotypes. Based on the conditions of the present study, the data suggested that the regular use of a chewing gum containing 15% xylitol for 30 days contributed to decrease MS salivary levels, however this decrease did not persist after the interruption of the consumption. In addition, the usage of xilitol was not a determinant to the emergence of a specific X R genotype in vivo

O objetivo do presente estudo foi avaliar, em aparelhos disjuntores de Haas, a contaminação in situ por estreptococos do grupo mutans, sob a forma de colônias/biofilmes, nas diferentes superfícies (acrílico, fios, bandas e parafusos), com e sem o uso de bochechos com gluconato de clorexidina a 0,12%, por meio de Cultura Microbiana e Microscopia Eletrônica de Varredura (MEV). Adicionalmente, a corrosão na área da união entre o fio, a solda de prata e a banda dos aparelhos foi avaliada por meio de Estereomicroscopia Ótica, MEV e análise em Espectrometria de Energia Dispersiva (EDS). Foram selecionados 34 pacientes de 7 a 12 anos de idade, que compareceram à clínica de Ortodontia Preventiva da FORP/USP, com necessidade de correção com aparelho disjuntor de Haas por problemas transversais da maxila (mordida cruzada posterior). Em seguida, utilizando uma tabela de números randômicos, os pacientes foram aleatoriamente divididos em dois grupos de 17 indivíduos cada (Grupos I e II). Durante todo o período de permanência dos aparelhos na cavidade bucal, no Grupo I (controle; n=17) os pacientes foram orientados a utilizar dentifrício fluoretado para escovação diária e não empregar bochechos com soluções antimicrobianas. Por outro lado, no Grupo II (experimental; n=17) os pacientes foram orientados a realizar, além da escovação diária com o uso de dentifrício fluoretado, 2 bochechos por semana com solução de gluconato de Clorexidina a 0,12% (Periogard®). Decorridos cerca de 4 meses da permanência na cavidade bucal, os aparelhos foram removidos. Sendo seccionadas, aleatoriamente, partes dos aparelhos constituídas de uma banda com fio soldado, para análise em estereomicroscopia ótica, MEV e EDS. Os resultados em estereomicroscopia foram submetidos à análise estatística por meio do teste de Fisher, com nível de significância de 5%. Em seguida, os aparelhos foram submetidos ao processamento microbiológico, em meio de cultura CaSa B, para contagem das colônias/biofilmes de estreptococos do grupo mutans. O teste estatístico não-paramétrico de Mann-Whitney foi aplicado para verificação de possíveis diferenças entre os grupos, com relação à formação de colônias/biofilmes sobre a superfície das diferentes áreas (parafuso, resina acrílica, bandas, fio vestibular e fio palatino), como também para verificar se havia diferença entre a formação de colônias/biofilmes sobre as superfícies livres (voltadas para a cavidade bucal) e as superfícies não-livres (em contato direto com a mucosa palatina e sulco gengival). O nível de significância adotado foi de 5%. Após cultura microbiana, partes dos aparelhos representativas de cada grupo foram submetidas ao processamento e análise em MEV. Os resultados obtidos evidenciaram que, com relação às colônias/biofilmes das superfícies nãolivres dos Grupos I e II, não houve diferença entre os grupos (p=0,009). No entanto, quando as superfícies livres dos Grupos I e II foram comparadas, evidenciou-se diferença significante entre os 2 grupos, em todas as áreas analisadas (parafuso, resina acrílica e bandas) (p<0,001). Os resultados da cultura microbiana foram confirmados em MEV. A análise em estereomicroscopia ótica evidenciou a presença de áreas de alteração de coloração sugestivas de corrosão na região de solda em contato com a banda e com o fio, em ambos os grupos (p=1). Os picos dos elementos químicos observados em EDS também foram semelhantes em ambos os grupos. Pôde-se concluir que o uso do gluconato de clorexidina a 0,12% (Periogard®), sob a forma de bochecho, apresentou eficácia na redução da formação de colônias/biofilmes nas superfícies livres dos aparelhos disjuntores de Haas, in situ, sem elevação nos níveis de corrosão. / The purpose of this study was to evaluate, in situ, in Haas expanders, the contamination of different surfaces (acrylic resin, wires, bands and screws) by mutans group streptococci colonies/biofilms with and without prescription of 0.12% chlorhexidine gluconate mouthwashes, by means of microbial culture and scanning electron microscopy (SEM). Additionally, the corrosion in the area of union between the appliances wire, silver soldering and band was assessed by optical stereomicroscopy, SEM and energy dispersive spectroscopy (EDS). Thirty-four children aged 7 to 12 years were selected among the patients attending the Preventive Orthodontic Clinic at FORP/USP with need of corrective orthodontics with Haas expander due to transversal problems of the maxilla (posterior crossbite). Thereafter, using a table of random numbers, the patients were randomly assigned to two groups of 17 individuals each (Groups I and II). Throughout the time that the appliances remained in the oral cavity, the patients in Group I (control; n=17) were instructed to use a fluoridated dentifrice for daily toothbrushing and not to use antimicrobial mouthwash solutions. On the other hand, in Group II (experimental; n=17), in addition to daily toothbrushing with a fluoridated dentifrice, mouthwashes with an antimicrobial agent (0.12% chlorhexidine gluconate - Periogard®) were prescribed to the patients. After approximately 4 months of maintenance in the patients mouth, the appliances were retrieved. Thereafter, components of the appliances (consisting of a band with soldered wire) were sectioned at random for analysis under optical stereomicroscopy, SEM and EDS. The results of the optical stereomicroscopy were submitted to statistical analysis using Fisher\'s test at 5% significance level. The appliances were sent to microbiology processing, in CaSa B culture medium, for counting of the number of mutans streptococci colonies/biofilms. Mann-Whitney non-parametric statistical test was applied to verify possible differences between the groups with respect to the formation of colonies/biofilms on the surface of different areas (screw, acrylic resin, bands, buccal wire and palatal wire), as well as to determine whether there were differences between the formation of colonies/biofilms on free surfaces (facing the oral cavity) and non-free surfaces (in direct contact with the palatal mucosa and gingival sulcus). The level of significance was set at 5%. After microbial culture, components of the appliances that were representative of each group were submitted technical processing and SEM analysis. The obtained results showed that there was no statistically significant difference between Groups I and II (p=0.009) regarding the formation of colonies/biofilms on the non-free surfaces. However, when the free surfaces of Groups I and II were compared, statistically significant difference was observed between these groups in all analyzed areas (screw, acrylic resin and bands) (p<0.001). The results of the microbial culture were confirmed by the SEM analysis. The optical stereomicroscopic analysis showed the existence of color alteration suggestive of corrosion in the solder region in contact with the band and with the wire in both groups (p=1). The peaks of chemical elements observed in EDS were also similar in both groups. It may concluded that the use of 0.12% chlorhexidine gluconate mouthwashes (Periogard®) showed efficacy in reducing the formation of colonies/biofilms on the free surfaces of Haas expanders, in situ, without increasing the corrosion levels.

O objetivo deste estudo foi avaliar, em crianças de creches públicas e filantrópicas do município de São Paulo, a prevalência de cárie dental e os fatores de risco relacionados à presença de cárie e presença de altos níveis de estreptococos mutans (SM). Foram incluídas variáveis denominadas sociodemográficas, condições de gestação e nascimento, variáveis fisiológicas do estado nutricional, aleitamento materno e alimentação complementar, hábitos alimentares, variáveis comportamentais e odontológicas. O estudo foi desenvolvido em parceria entre a Disciplina de Odontopediatria da Faculdade de Odontologia da Universidade de São Paulo (FOUSP) e a Disciplina de Nutrologia do Departamento de Pediatria da Universidade Federal de São Paulo (UNIFESP EPM). É parte integrante do Projeto Crecheficiente - Ações de Segurança e Educação Alimentar em Creches Públicas e Filantrópicas do Município de São Paulo. A população deste estudo foi constituída de 150 e 144 crianças, que participaram da avaliação odontológica e microbiológica respectivamente, entre 12 e 24 meses, de ambos os gêneros, regularmente matriculadas em cinco creches, sendo duas públicas e três filantrópicas. A coleta dos dados foi obtida por meio de entrevistas com as mães ou responsáveis, antropometria, coleta de sangue para dosagem de hemoglobina, avaliação odontológica (índices ceo-d e ceo-d modificado) e coleta salivar das mães e das crianças para teste de estreptococos mutans. Os dados obtidos foram analisados estatisticamente utilizando análise de regressão logística univariada, considerando o nível de significância a 5%. Foram utilizadas as mesmas variáveis de risco e seus respectivos pontos de corte nas análises univariadas da avaliação odontológica e microbiológica. Das crianças examinadas 7,3% apresentaram experiência de cárie, sendo encontrada uma média do índice ceo-d igual a 0,09 e ceo-d mod de 3,37, sem diferença entre gêneros e tipo de creche. Em relação à presença de cárie, a análise de regressão univariada indicou significância dos seguintes fatores: o tempo de matrícula na creche, aleitamento materno exclusivo menor de 60 dias, idade do primeiro contato com açúcar, e níveis altos de SM que favoreceram a ocorrência da doença, o número maior de irmãos e a presença de biofilme visível foram significantes e associados com proteção. Dentre os alimentos citados encontrados na dieta dos lactentes, observa-se que o consumo de mel e o consumo de refrigerantes favoreceram a ocorrência da cárie dentária. Em relação à presença de SM, 72,9% das crianças tinham este microrganismo presente na cavidade bucal sendo que 9,5% apresentavam altos níveis de SM. As variáveis que influenciaram significantemente os altos níveis de SM foram: idade da mãe, idade de introdução do açúcar e presença de cárie dentária na criança. Em relação aos hábitos alimentares apenas o consumo de petit suisse favoreceu a quantidade elevada de SM. A contagem dos níveis salivares de SM da mãe não foi significante. / The aim of this study was to assess in children of public and philanthropic day care centers of São Paulo, the prevalence of dental carie and the risk factors associated to dental carie and high levels of mutans streptococci (MS). Were included variables denominated socio-demographic, pregnancy and birth conditions, physiological variables of nutrition status, breast feeding and complementary diet, dietary habits, behavior and dental variables. This study was the result of a partnership between the Pediatrics Department of the University of São Paulo and the Nutrology Department of Federal University of São Paulo, UNIFESP EPM. It is part of a larger study called Crecheficiente Project Security and Dietary Education actions of public and philanthropic day care centers of São Paulo city. The population of this study were consisted of 150 and 144 children that participated of the dental and microbiological evaluation respectively, between 12 and 24 years old, both gender, regularly registered in five day care centers, two publics and three philanthropic. The data collection were done by interviews with mothers or childrens responsible, anthropometry, bloody collection for hemoglobin, dental test (Knutson, dmf and modified dmf) and salivary collection from mothers and children for estreptococos mutans test analysis. Data analysis involved univariate logistic regression, The level of significance was set at 5%. The same variables presented at univariate analysis of the dental test and break point, were described into microbiological test. 7,3% of the children had dental carie. A mean of dmft index were 0,09 and modified dmft 3,37. No difference was found between gender and type of day care center. The univariate analyses indicated significance for caries experience: time of day care center admission, exclusive breast feeding least than 60 days, age of the first sugar contact, high levels of MS benefit caries, the major number of brothers and sisters, presence of dental plaque were associated to protection. The foods found in the dietary habits of the infants, honey and soft drinks exposed the occurrence of dental caries. For the streptococci mutans experience, 72,9% of the children had the microorganism present and 9,5% were high levels of MS. The variables that influence significantly this group were: mothers age, age of the first sugar contact and childrens carie experience. In the dietary habits, only the pettit suisses consume exposed to high levels of SM. The mothers mutans streptococci levels were not significant.

Este trabalho teve o objetivo de avaliar o efeito do uso de goma de mascar contendo 15% de xilitol, por 30 dias, em 12 indivíduos com altos níveis salivares (= 10 5 u.f.c./ml de saliva) de Estreptococos do grupo mutans (SM) sobre os níveis salivares de SM, os genótipos de S. mutans e a ocorrência de amostras xilitol-tolerantes (X R ) na saliva. Os voluntários foram instruídos a mascarem a goma por 5 minutos, 5 vezes ao dia, durante 30 dias, sem realizarem nenhuma alteração nos hábitos de higiene ou dieta. Amostras de saliva foram coletadas no início do experimento (antes do consumo da goma de mascar), 30 dias após a utilização da goma de mascar e 30 dias após a interrupção do uso da goma. Dois voluntários foram excluídos da última coleta devido ao uso de antibióticos. Os resultados mostraram que ocorreu redução acentuada nos níveis salivares de SM. A concentração média de SM no exame inicial foi de 9,8 x 10 5 ± 2,61 x 10 5 UFC/ml de saliva, enquanto este valor foi de 2,17 x 10 5 ± 1,94 x 10 5 após o consumo da goma, sendo a diferença estatisticamente significante (p < 0,05). Porém este efeito não foi duradouro, não apresentando diferença estatística 30 dias após a interrupção do consumo (3,5 x 10 5 ± 2,71 x 10 5 ), em relação aos valores iniciais. Foram identificados 17 genótipos distintos de S. mutans nas amostras salivares de 10 voluntários, que completaram as 3 etapas do estudo, pela técnica de RAPD-PCR, com o iniciador OPA-2. O ensaio de tolerância de S. mutans ao xylitol in vitro, em meio BHI e BHI /xilitol 1%, permitiu a identificação de 12 amostras X R entre 124 amostras estudadas, representantes dos 17 genótipos distintos. Amostras de S. mutans (X R ) foram identificadas nas 3 coletas, inclusive antes do consumo da goma. Foram identificadas amostras X R e X S com o mesmo genótipo por RAPD-PCR. A prevalência de amostras X R não apresentou relação direta com o uso de xilitol ou com os tipos genotípicos. Baseados nas condições do presente estudo, os resultados sugerem que o uso regular de gomas de mascar contendo 15 % de xilitol por 30 dias contribui para a redução dos níveis salivares de SM durante o período de consumo, porém esta redução não persiste após a interrupção do uso. Além disso, foi observado que o uso de xilitol não foi fator determinante na seleção in vivo de determinado genótipo X R / The aim of this study was to evaluate the effect of xylitol (15%) containing chewing gum, for 30 days , in 12 subjects with high mutans Streptococci (MS) salivary levels (= 10 5 c.f.u./ml) on Mutans streptococci (MS) salivary levels, on genotypes and detection of xilitol tolerant (X R ) strains of S. mutans . Volunteers were asked to chew one lozangle, for 5 minutes, five times per day, for 30 days with no change in their habitual diet or oral hygiene. Saliva samples were collected at baseline, 30 days after gum usage and 30 days after the interruption of the consumption. Two subjects were excluded from the last examination due to antibiotic use. The results showed a marked reduction in MS salivary levels. The average levels of MS in saliva in baseline was 9.8 x 10 5 ± 2.61 x 10 5 c.f.u./ml saliva and decreased to 2.17 x 10 5 ± 1.94 x 10 5 after 30 days of consumption. This difference was statistically significant (p< 0.05). However this effect was not long lasting, and 30 days after the interruption of consumption (3.5 x 10 5 ± 2.71 x 10 5 ) there was no statistically difference in relation to baseline data. Seventeen different genotypes were identified in MS salivary samples of 10 volunteers that completed the 3 sampling procedures, by RAPD-PCR using OPA-2 primer. The assay of xylitol tolerance of S. mutans in vitro, in BHI and BHI/1% xylitol allowed the identification of 12 X R isolates, among 124 tested strains representing all detected genotypes. X R strains were detected in all sampling steps, including at baseline, before chewing gum consumption. There were X R and X S isolates with the same genotypes by RAPD-PCR. The prevalence of X R strains did not show direct relation to consumption of xylitol neither to genotypes. Based on the conditions of the present study, the data suggested that the regular use of a chewing gum containing 15% xylitol for 30 days contributed to decrease MS salivary levels, however this decrease did not persist after the interruption of the consumption. In addition, the usage of xilitol was not a determinant to the emergence of a specific X R genotype in vivo

OBJECTIVE: The objective was to assess the role of socio-demographic, dietary, behavioral, and environmental factors in Mutans Streptococci (MS) colonization in young children from low socio-income families. METHODS: This study involves secondary analyses of data collected from 6 to 24 months old children (N=129) enrolled in the Supplemental Nutrition Program at WIC, Iowa. They were followed for 18 months assessing different socio-demographic, dietary, behavioral, and environmental factors at 5 time-points. Total 3 clinical examinations were conducted at baseline, 9 months, and 18 months. Salivary samples collected during the examinations by semi-quantitative method, serve to determine the subjects' MS levels, who were grouped into either 1) No MS at any time during the study (n=58); 2) MS at baseline (n=35); or 3) Acquired MS during the study period (n=36). Prediction of the group membership (1or3) over the three time points is used as outcome for this study. This paper reports important findings from pairwise comparison of the three groups at baseline, 9 months and 18 months. RESULTS: Consumption of sugar-rich beverages and tooth-related factors like plaque and number of teeth were significantly (p<0.05) higher in Group 2 children, than the others at baseline. Group 2 also had the oldest children (mean age at baseline - 16.7 months) than Group 1 (10.8 months) and Group 3 (12.6 months). The effect of age was reflected in longitudinal comparison of group1 and 3 as well. Different behavioral and dietary factors were significant at different time-points, specific to that age-group under observation. Higher maternal education was found to be a protective factor, whereas tooth-related factors such as, history of caries and number of teeth, were risk factor in longitudinal analyses. CONCLUSION: Time measured as age of the child is the key factor in MS infection in youg children. Dietary, behavioral, environmental, and tooth related factors acquire importance depending on age of the child. Future longitudinal analyses will further explore these relationships.

Background: The World Health Organization defines probiotics as ‘live microorganisms which, when administered in adequate amounts, confer a health benefit to the host’. Traditionally, probiotic microorganisms have been used to prevent or treat gastrointestinal tract diseases. In the last 15 years, there has been increasing interest of a possible probiotic impact on the oral microbiota and dental caries. Dental caries is a multifactorial disease, and the causative factor in the oral microbiota includes a shift from a balanced microflora to a microflora that includes more aciduric species such as mutans streptococci (MS), non-mutans streptococci, and Actinomyces. MS is considered an opportunistic pathogen although several other bacteria also contribute to the disease. Early acquisition of MS is associated with early development of caries; therefore a desirable complement to other prophylactic measures would be a MS colonization inhibitor. Objective: To better understand how selected strains of probiotic lactobacilli interact with MS in vitro and in vivo and to study the impact of probiotic lactobacilli on caries development during childhood. Material and methods: The in vitro properties of probiotic lactobacilli were studied with regard to (i) acid production from sugars and sugar alcohols, (ii) growth inhibition capacity on clinical isolates and reference strains of MS as well as Candida albicans and (iii) the capacity to co-aggregate with MS. A randomized controlled trial (RCT) tested the short-term effect of intervention with two Lactobacillus reuteri strains on MS, which was evaluated after treatment with chlorhexidine. The re-growth patterns of MS and 19 other selected strains were also evaluated. In the second clinical study  we investigated the long-term effect on MS prevalence and dental caries after an intervention with Lactobacillus paracasei ssp. paracasei F19 (LF19) between 4 and 13 months of age. Results: The results from the in vitro testing showed that strains of probiotic lactobacilli differed in their fermentation patterns, inhibition capacity and their capacity to co-aggregate, which should be kept in mind in the translation to clinical research. The clinical study on short-term effects of two L. reuteri strains on MS and other oral strains showed no effect on re-growth patterns after intervention. The clinical study on long-term effects of LF19 showed no effect on the prevalence of MS. Furthermore, the clinical follow-up at 9 years of age showed no differences in either decayed, missing, and filled surface (dmfs) or DMFS between the probiotic and placebo groups. Evaluation of saliva samples showed no signs of oral colonization with LF19 in the study group. Conclusion: The in vitro testing showed potentials of the selected probiotic Lactobacillus strains for interference with MS and C. albicans. The results from the clinical studies showed no such effect on MS or dental caries. Evidence regarding the effectiveness of specific probiotic applications in the prevention of dental caries is limited and does not allow for conclusions concerning the use of probiotic bacteria as a preventive measure.