Sepsis is one of the leading causes for fatalities in the intensive care unit, and also one of the biggest health problems worldwide. It is a disease caused primarily by bacterial infections but can also be caused by viral or fungal infections. Since it is such a big health problem being associated with increased risk of sepsis, coupled with longer stays in the intensive care unit, the need for fast diagnosis and treatment is very important. Currently, culture is the leading diagnostic method for identification of bacteria, although other methods are currently being tested to improve identification time and decrease cost and workload. Next generation sequencing (NGS) has the capacity to output several million reads in a single experiment, making it very fast and relatively cheap compared to other older sequencing methods such as Sanger sequencing. The ability to analyze genes and even whole genomes, opens the possibilities to identify factors such as bacterial species, virulence genes and antibiotic resistance genes. The aim of this study was to find any possible correlations between 16 species of streptococci and clinical data in patients with suspected sepsis. Initial species identification was performed using MALDI-TOF before the samples were sequenced using NGS. Sequence files were then quality controlled and trimmed before being assembled. Following assembly, coverage was controlled for all assembled genomes before the downstream analysis started. Different tools such as 16S RNA species identification, multi locus sequence typing and antibiotic resistance finder were used, among other tools. The results were extremely mixed, with the overall quality of the data being of good quality, but the assembly and downstream analysis being worse. The most consistent species was S. pyogenes. No correlation between sepsis patients and relevant clinical data was found. The mixed quality of results from assembly and downstream analysis were most likely contributed to difficulties in culturing and sequencing of the streptococci. Finding ways to circumvent these problems would most likely aid in general sequencing of streptococcal species, and hopefully in clinical applications as well.

Made available in DSpace on 2014-06-11T19:27:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-03-08Bitstream added on 2014-06-13T20:17:10Z : No. of bitstreams: 1 jesus_cm_me_arafo.pdf: 199618 bytes, checksum: d0ce2867f80614585999c16e15edac52 (MD5) / Por meio de exames clínicos e análises laboratoriais, o presente trabalho avaliou a prevalência de cárie dentária e a relação entre os fatores presença de cárie dental, biofilme bacteriano visível e contagem de Streptococcus grupo mutans em crianças com idade entre 12 e 48 meses, sendo 26 portadoras (grupo teste) e 142 não portadoras da síndrome de Down (grupo controle). A prevalência de cárie dentária no grupo teste foi de 15,38% e no grupo controle de 31,69%. No grupo controle houve aumento estatisticamente significativo do índice ceo-d e dos níveis de Streptococcus grupo mutans a partir dos 36 meses de idade. Nos dois grupos, com o aumento da idade, aumentou o número de crianças colonizadas por Streptococcus grupo mutans. Foi observada dependência estatisticamente significativa entre a presença de lesão cárie dentária e altos níveis de Streptococcus do grupo mutans tanto no grupo teste quanto no grupo controle. Houve também correlação positiva estatisticamente significativa entre a presença de lesão de cárie dentária e a presença de biofilme bacteriano visível, e entre a presença de biofilme bacteriano visível e altos níveis de Streptococcus do grupo mutans apenas no grupo controle. / This study evaluated the dental caries prevalence and the relationship among dental caries, visible bacterial biofilm and mutans streptococci counts in children with Down syndrome (test-group=26) and in health children (control-group=142), aged from 12 to 48 months. The caries prevalence was 15.38% in the test group and 31.69% in the control-group. The dmf-t index and the mutans streptococci levels had a significant statistic increase after 36 months of age in the control group. The number of children who harbored mutans streptococci had a significant increased with age in all children (test and control groups). In both groups dental caries and high levels of mutans streptococci presented a strong positive correlation. High positive correlation was also observed between visible bacterial biofilm and dental caries lesions and between visible bacterial biofilm and high levels of mutans streptococci in the control-group, but not in the test-group. Keywords: Down syndrome, dental caries, mutans streptococci, biofilm.

Orientadores: Regianne Umeko Kamiya, Reginaldo Bruno Gonçalves / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-13T03:23:31Z (GMT). No. of bitstreams: 1 Lima_BrunadeAraujo_M.pdf: 718582 bytes, checksum: 4999de5a34cfebba63ad6e57d6621afa (MD5) Previous issue date: 2009 / Resumo: Streptococcus mutans é o principal agente etiológico da cárie dental. A busca por novos agentes antimicrobianos naturais capazes de inibir os principais mecanismos de virulência de S. mutans, como a síntese e tolerância a ácidos e a formação de biofilme dental pode representar uma importante estratégia de prevenção e tratamento desta infecção. Estudos recentes confirmaram que produtos naturais de origem marinha possuem significativa atividade antimicrobiana contra microrganismos patogênicos. Assim, o objetivo deste estudo foi analisar a atividade antimicrobiana de extratos ou frações purificadas obtidos a partir de invertebrados e microrganismos marinhos associados sobre S. mutans e outros estreptococos orais como S. sobrinus e S. sanguinis, pela determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM). Adicionalmente, avaliamos a influência de extratos e/ou frações ativas sobre a formação de biofilme e sobre a expressão dos genes associados aos principais fatores de virulência de S. mutans UA159 e um isolado clínico da espécie. Foram analisados genes associados com a capacidade de formação de biofilme (gtf B/C, gbpB), com a síntese de ácidos (ldh) e com tolerância ao estresse ácido (dnaK, groEL, ATPase). De 768 frações/extratos testados cerca de 10% inibiram de 50% a 100% do crescimento microbiano de estreptococos orais (com CIM e CBM inferiores a 500 ìg/mL). Dentre os extratos e frações utilizadas, foram selecionados um extrato (DR(M3)6) e duas frações purificadas (PcS2d e DR-MSP- 10d-P3) com atividade antimicrobiana e CIM/CBM inferiores a 125 µg/ml sobre S. mutans, para avaliação da atividade sobre fatores de virulência da espécie. Nas concentrações 7ìg/mL, 31ìg/mL e 62ìg/mL, as frações PcS2d, DR-MSP-10d-P3 e o extrato DR(M3)6, respectivamente, inibiram a formação do biofilme sem interferir no crescimento planctônico das duas cepas analisadas, em adição o tratamento com os respectivos antimicrobianos, nestas mesmas concentrações por 30 minutos, resultou principalmente na redução da expressão dos genes gtfB, gbpB, dnaK e ATPase em proporções diferentes para as cepas S. mutans UA 159 e o isolado clinico. Em conclusão, as substâncias avaliadas, principalmente as frações purificadas podem futuramente ser empregadas como potenciais anticariogênicos/cariostáticos, entretanto mais estudos serão necessários para tal aplicabilidade. / Abstract: Streptococcus mutans are the principal etiologic agent of dental caries. The search for new natural antimicrobial agents capable of inhibiting the mechanisms of virulence of S. mutans, as synthesis and tolerance to acid and dental biofilm formation may represent an important strategy for prevention and treatment of this infection. Recent studies have confirmed that natural products of marine origin have significant antimicrobial activity against pathogenic microorganisms. The aim of this study was to evaluate the antimicrobial activity of extracts or purified fractions obtained from marine invertebrates and microorganisms associated on S. mutans and other oral streptococci such as S. sobrinus and S. sanguinis by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Additionally, we evaluated the influence of extracts and / or active fractions on the formation of biofilm and the expression of genes associated with virulence factors of S. mutans UA159 and a S. mutans clinical isolate. We analyzed genes associated with the capacity of biofilm formation (gtfB/C, gbpB), acids synthesis (ldh) and acid stress tolerance (dnaK, GroEL, ATPase). 768 fractions / extracts was tested and about 10% inhibited by 50% to 100% of the microbial growth of oral streptococci (MIC and MBC with less than 500ìg/mL). Among the extracts and fractions used were selected an extract (DR (M3) 6) and two purified fractions (PcS2d and DR-MSP-10d-P3) with antimicrobial activity and MIC / MBC less than 125ìg/mL on S. mutans, to evaluate the activity of virulence factors. At concentrations 7ìg/mL, 31ìg/mL e 62ìg/mL, the fractions PcS2d, DRMSP- 10d-P3 and extract DR (M3) 6, respectively, inhibited the formation of biofilms without interfering with planktonic growth the two strains examined, in addition to treatment with the antimicrobial agents, these same concentrations for 30 minutes, resulted in reducing the expression of genes gtfB, gbpB, dnaK ATPase in different proportions for the strains S. mutans UA159 and clinical isolate. In conclusion, substances evaluated, especially the purified fractions can further be used as anticariogenic / cariostatic potential; however, more studies are needed for this application. / Mestrado / Microbiologia e Imunologia / Mestre em Biologia Buco-Dental

Orientador: Altair A. Del Bel Cury / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-07-25T04:05:54Z (GMT). No. of bitstreams: 1 Rocha_EduardoPassos_M.pdf: 8338827 bytes, checksum: a8aba43a0026699db752f3c1473b688b (MD5) Previous issue date: 1999 / Resumo: Parte da literatura relaciona o uso da prótese parcial removível (PPR) com níveis diferenciados de estreptococos do grupo mutans (EGM). No entanto, os relatos são inconclusivos e os trabalhos que associam medidas preventivas de controle químico são poucos e inexistentes a longo prazo. Dessa forma, o objetivo do trabalho foi avaliar a influência da PPR no índice de EGM na saliva, e o seu controle químico com o uso do gel de digluconato de clorexidina a 1 % (CHX). Para tanto, amostras de saliva foram coletadas imediatamente antes da instalação da PPR (tempo zero=T0), 8 dias após (T8), 48 dias após (T 48), 92 dias após (T92), 140 dias após (T140) e 189 dias após a instalação da PPR (T189). Estas foram homogeneizadas em agitador de tubos (Vortex) por 1 min. Diluições seriadas de '10 POT. ¿1' a '10 POT. ¿6' foram realizadas em solução estéril de tampão fosfato, 0,05 M, pH 7,0. Em duplicata, alíquotas de 25 'muL' foram semeadas em placas de petri contendo ágar MSB. A seguir, as placas foram incubadas em estufa de C02 (5%), a 37°C, por 72 horas. Os resultados apresentaram diferença estatística (Test t, p<0,05) entre os valores de T0 e T48, respectivamente: (média/desvio-padrão em UFC de EGM/mL de saliva) 2,26 x '10 POT. 6'/4,43 x '10 POT. 6' e 4,79 x '10 POT. 7'/1,48 x '10 POT. 8'. Decorrido esses períodos, a CHX foi aplicada em 29 voluntários, não sendo eficaz na redução dos níveis de EGM (Test t, p>0,05). Novas amostras de CHX foram formuladas e avaliadas quanto à efetividade. A nova formulação foi aplicada em 15 voluntários. Amostras de saliva foram coletadas nos períodos: antes da aplicação da clorexidina (tempo zero=T0), 24 horas após (T24h) e 82 dias após (T82). Os resultados apresentaram diferença estatística (Sign Rank, p<0,05) entre os valores de T0 e T24h, respectivamente: (média/desvio-padrão em UFC de EGM/mL de saliva) 6,64 x '10 POT. 6'/8,47 x '10 POT. 6' e 3,20 x '10 POT.5'/4,27 x '10 POT. 5'. O índice de EGM na saliva retomou aos valores iniciais num período de 82 dias (T82). Concluiu-se Que o uso da PPR promoveu aumento significativo do índice de EGM após 48 dias de instalação da prótese e Que o gel de clorexidina a 1 % foi eficaz na redução do índice de EGM num intervalo de tempo entre 24h e 82 dias após a segunda aplicação / Abstract: Many studies have reported an evaluating of the salivary levels of MS in patients submitted to oral rehabilitation with RPD. Thus, after subscription of consent term and approval of ethic committee, saliva samples (2.0 mL) were obtained from 32 patients in 6 periods: (T0): immediately before installation of RPD; (T8): 8 days after T0; (T48): 48 days after T0; (T92): 92 days after T0; (T140): 140 days after T0 and (T189): 189 days after T0. The samples were vortexed and serially diluted from '10 POT. ¿1' to '10 POT. ¿6' in 0.05 M phosphate buffer in the number of MS in saliva between the periods T0 and T48 (mean/SD, CFU/mL saliva): T0: 1.90/4.02 x '10 POT. 6' and T48: 0.38/1.33 x '10 POT. 8'. After this, intensive treatment with CHX was (pH 7.4). From each dilution, 0.025 mL was plated on MSB. The plates were incubated in 5% COz at 37°C for 72 hours. There was a statistically significant increase (Test t, p<0.05) accomplished in 29 patients. Saliva samples (2.0 mL) were obtained after treatment in 4 periods: (T24h): 24 hours after T24h; (T14): 14 days after T24h; (T28): 28 days after T24h and (T63): 63 days after T24h. After treatment with CHX, the number of MS in saliva did not decrease (Test t, p>0.05). A new CHX formulation was applied in 15 patients. Saliva samples (2.0 mL) were obtained in periods: (T0): before CHX; (T24h): 24 hours after T0 and (T82): 82 days after T0. The new CHX reduced the MS in periods T0 and T24h: (mean/SD, CFU/mL saliva): T0: 6.64/8.47 x '10 POT. 6' and T24h: 3.2/4.27 x '10 POT. 5' (Sign Rank, p<0.05). In conclusion, there was a significant increase in the number of MS in saliva after installation of RPD and that the intensive treatment with a properly formulated 1 % chlorhexidine gel was effective in the reduction of MS between 24h and 82 days after new appliance of CHX / Mestrado / Protese Dental / Mestre em Clínica Odontológica

Orientadores: Monica Campos Serra, Leonor de Castro Monteiro Loffredo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-07-25T16:16:10Z (GMT). No. of bitstreams: 1 Pinelli_Camila_M.pdf: 4056278 bytes, checksum: 4fff171bfc31ff5c42bfd597a8e4421e (MD5) Previous issue date: 1999 / Resumo: O objetivo deste trabalho foi verificar a eficácia de um teste microbiológico para estreptococos do grupo mutans na avaliação do risco de cárie. Oitenta e um alunos de 6a a 8a séries da rede pública escolar de Piracicaba foram selecionados e se submeteram a um exame clínico odontológico - feito por um pesquisador clínico com experiência no diagnóstico e tratamento de pacientes de alta atividade de cárie - e a um exame salivar, feito com o produto CARITEST SM®, a partir de um mesmo lote de fabricação. Utilizando-se a estatística kappa, a reprodutibilidade do teste microbiológico foi verificada através da concordância interexaminador, na avaliação dos resultados microbiológicos de maneira independente. A validade foi verificada, através da sensibilidade e especificidade, comparando-se os resultados microbiológicos com o critério de validação ("gold standard") da pesquisa. Os valores de kappa, k=0,55 e k=0,78, representaram, respectivamente, uma concordância interexaminador regular considerando a avaliação segundo 6 escores propostos pelo fabricante, e uma concordância boa para a classificação segundo alta e baixa contagem microbiológica,- dada pelo agrupamento dos escores. O valor para a sensibilidade foi de 0,59 e para a especificidade foi de 0,85, mostrando que o teste foi mais específico do que sensível, identificando melhor os indivíduos de baixo risco de cárie. Esse teste microbiológico pode ser considerado um bom auxiliar na avaliação do risco de cárie, porém não é o único fator preditivo a ser avaliado, frente ao caráter multifatorial da doença / Abstract: The aim of this study was to evaluate the efficacy of a microbiological test for estimation of mutans streptococci levels in caries risk assessment. Eighty-one volunteers from 6th up to 8th grades from Piracicaba's public schools were assessed for dental caries. A researcher with great skills in examining and treating patients with high caries activity performed the examinations. The students were then submitted to a salivary test, CARITEST SM® (from the same batch number). Kappa statistics (K) was applied to verify the repeatibility of the simplified test checked through interexaminer agreement, in the classification of the microbiological tests, independently and blind. The validity of the test was verified according to sensibility and specificity, comparing the microbiological results with the gold standard for caries risk. A fair agreement was verified when the examiners classified according to 6 scores (K=0.55) and a good agreement when they classified according to high or low density of unit colonies formation (K=0.78). The sensibility was 0.59 and the specificity was 0.85, showing that there was a moderate ability of the test to identify the subjects with high caries risk and a substantial ability to identify the group of low caries risk. The microbiological test represents an aid for cariesrisk assessment, but it is not the only criteria to be used for caries risk diagnosis, considering the multifactorial character of the disease / Mestrado / Dentística / Mestre em Clínica Odontológica

Streptococcus pyogenes is an important human pathogen. One of the most prominent virulence factors produced by S. pyogenes is SfbI, a surface adhesin composed of three domains: thioester domain (TED), proline-rich repeat domain (PRR) and fibronectin-binding repeat domain (FnBD). The structures and functions of TED and PRR and their contributions to the pathogenesis of streptococcal diseases are unknown. The interaction between PRR and its putative target, the intracellular actin cytoskeleton regulator Arp2/3, was investigated by both in vitro and in vivo studies. PRR was shown to inhibit Arp2/3-dependent actin polymerisation. The expression of PRR in HeLa cells caused disruption to the cytoskeleton of the cells. All data point towards a role of PRR in inhibiting the Arp2/3 complex but more evidence is needed to support this. The N-terminal domain of SfbI (TED) and four homologous domains from S. pyogenes, group G streptococci and Streptococcus pneumoniae were characterised by mass spectrometry, NMR spectroscopy and biochemical assays. All were shown to possess intramolecular thioester bonds, spontaneously formed between sides chains of Cys and Gln residues. Fibrinogen (Fg) was identified as the first binding target of bacterial TEDs with direct evidence that the thioester bond was involved in the interaction with Fg. A pull-down experiment using human plasma showed Fg is a specific binding partner of SfbI-TED. The binding sites were narrowed down to the thioester-forming Gln of SfbI-TED and Lys residues in the Fg-Aα chain, and binding potentially occurred via covalent isopeptide linkage. The data presented here suggest two new roles for SfbI, previously unknown in bacterial pathogenesis. The PRR may be the first bacterial inhibitor of the actin cytoskeleton acting by inhibiting the Arp2/3 complex. Thioester domains appear to be a shared common feature of surface proteins of many Gram-positive pathogens. They may form covalent crosslinks between bacteria and host tissue.

This study evaluated four lactic streptococcal pairs which had been used routinely in cheese plants to determine (1) if strain growth interactions would produce stimulated growth and acid production; (2) final percent strain ratios and generation times after 1/1 initial inocula volumes and (3) whether final strain ratios could be controlled through adjustment of initial inocula volumes. Strains were propagated in fortified and unmodified whey substrate under pH control at 27C for 12 hours. Cell number and acid activity were evaluated on single and paired strains. Phage typing was used to determine generation times and the percent of each strain in a pair. Cell number increase was found in two of the four pairs grown in the unmodified whey substrate but in only one of the four pairs grown in the fortified medium. Acid production increase was observed in one pair when grown in the unmodified whey substrate, and in one other pair when grown in the fortified medium. Final strain percent ratios of the two of the four pairs (with initial inocula of a 1/1, v/v, ratio), demonstrated unbalanced growth, with one strain accounting for 76 to 92% of the final flora. Generation times of the dominant strain varied significantly with incocula ratios. Increasing the inocula volume ratio of the dominated strain of one pair from 1/1 to 19/1 dropped the final percent of the dominant strain from an average value of 83 to 66%. The dominant organism of this pair increased its doubling time when the inocula ratio was varied from 3/1 to 19/1 (dominated/dominant).

Im Rahmen der Untersuchung wurde bei 27 Patienten, die klinische Zeichen der Karies, aber keine pulpitischen Symptome zeigten, kariöses Dentin mit Hilfe eines sterilen Exkavators entnommen. Das durchschnittliche Alter der Patienten beträgt 41 Jahre und der Durchschnittswert des DMF-T-Index 12,5. Die Untersuchungsgruppe bestand zu 55,5 % aus männlichen Probanden sowie zu 44 % aus Rauchern. Nach Isolierung von 107 Reinkulturen aus den Patientenproben erfolgte die Identifizierung der oralen Streptokokken mittels eines mikrobiologischen Standardtests (RapidID-32Strep der Firma BioMérieux) und MALDI-TOF-MS-Analyse. Parallel wurden speziesspezifische PCRs der Dentinproben für S. sanguinis, S. constellatus, S. intermedius, S. anginosus, S. mutans, S. salivarius, S. oralis, S. mitis, S. gordonii und S. parasanguinis durchgeführt. Mittels MALDI-TOF-MS-Analyse konnten insgesamt sechs verschiedene Spezies oraler Streptokokken in den Dentinproben nachgewiesen werden. Am häufigsten kamen Vertreter der Mitis-Gruppe vor (in 89 % der Dentinproben), gefolgt von S. gordonii und S. sanguinis (zu 52 % und 26 % vertreten). Die MALDI-TOF-MS-Methode erwies sich als geeignetere der mit Kultivierung verbundenen Nachweismethoden. Ihre Ergebnisse wurden durch selektive PCRs einzelner Subkulturen und DNA-Sequenzierung bestätigt. Mittels der speziesspezifischen PCRs der Dentinspäne wurden zehn verschiedene Spezies oraler Streptokokken identifiziert. Vertreter der Mutans-Gruppe wurden so zu durchschnittlich 44 %, S. salivarius zu 37 % nachgewiesen. Es zeigte sich ein signifikantes Vorkommen von S. anginosus in Proben, die ebenfalls S. mutans enthielten (p= 0,00213). Alle drei Verfahren sind zur Untersuchung klinischer Proben geeignet, wobei die MALDI-TOF-MS-Analyse die genaueste Differenzierung auf Speziesebene ermöglicht. Die Non-Mutans-Streptokokken S. oralis, S. gordonii und S. anginosus scheinen die Mikroflora von kariösem Dentin zu dominieren. Sie übertrafen in der vorliegenden Arbeit in ihrem Vorkommen S. mutans in mehr als der Hälfte der untersuchten Proben. Diese Beobachtung stützt die erweiterte ökologische Plaquehypothese.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal. / Group B streptococcus (GBS) is the leading cause of neonatal sepsis and meningitis and has recently been recognized as an increasingly common cause of invasive disease in immunocompromised adults (elderly or chronic diseases). GBS produces a number of extracellular enzymes, several of which interact with the host immune system and are important for the GBS- host interaction and for the development of disease. Previous studies showed that metalloproteases are involved in several metabolic pathways in different cellular types. For this reason, we decided to investigate the possible involvement of GBS metalloproteases during cell interaction and apoptosis/necrosis induced by microorganism in human umbilical vein endothelial cells (HUVEC) and epithelial respiratory cells line (A549). Treatment of GBS with metalloproteases inhibitors (EDTA, EGTA and PHEN) did not induce alteration on bacterial growth, but promoted changes in the expression of surface proteins, adhesive capacity and profile of intracellular survival of the pathogen. The GBS and supernatant of bacterial growth medium (conditioned medium; MC) promoted the death of HUVEC and A549 cells. However, the metalloproteases inhibitors treatment restored the cellular viability induced by GBS and MC, suggesting that GBS metalloproteases are involved in the disruption of cell barrier, promoting bacterial dissemination. This study describes for the first time apoptosis and necrosis induced by GBS and MC in HUVEC and A549 cells after 24h incubation, respectively. We also observe reduction of pro-caspase-3 after infection of HUVEC with GBS and MC, suggesting activation of caspase-3. Moreover, the over-expression of pro -apoptotic protein Bax and decrease of anti-apoptotic protein Bcl-2 levels in HUVEC show the involvement of mitochondrial apoptotic mechanism (intrinsic via). Enhanced understanding of the molecular basis of GBS pathogenesis may pinpoint novel bacterial and host molecules that can represent novel therapeutic or immunoprophylactic targets against disease caused by this foremost of neonatal pathogens.