Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Os Enterococcus sÃo cocos Gram-positivos catalase-negativos, habitantes do trato gastrintestinal e geniturinÃrio. SÃo identificados por meios manuais baseados no clÃssico de Facklam, por sistemas semi-automatizados, automatizados e por Biologia Molecular. Em vista da diversidade de mÃtodos de identificaÃÃo e com o intuito de se verificar a concordÃncia entre alguns mÃtodos, foi realizada identificaÃÃo de 62 cepas bacterianas, advindas da coleÃÃo do Centro Especializado em Micologia MÃdica (CEMM) da Universidade Federal do CearÃ, Brasil, presumidamente identificadas como Enterococcus sp. e Enterococcus faecalis pelo esquema de Facklam. Este estudo iniciou-se com a identificaÃÃo por trÃs mÃtodos manuais: esquema de facklam e dois modificados deste - Modificado 1 e Modificado 2 - e semi-automatizado â API 20 Strep. Para a definiÃÃo de resultados discordantes, recorreu-se ao semi-automatizado â BBL â e à Biologia Molecular â PCR. O esquema de Facklam identificou 16% (10) como cepas do gÃnero Enterococcus sp. e 84% (52) como E. faecalis. O esquema Modificado 1 apresentou a seguinte identificaÃÃo: 82,2% (51) E. faecalis, 9,7% (6) E. mundtii, e 8,1% (5) E. gallinarum. O Modificado 2 identificou 98,4% (61) E. faecalis e 1,6% (1) E. gallinarum. O API 20 Strep identificou 51,6% (32) E. faecalis, 19,4% (12) A viridans, 13,0% (8) E. avium, 4,8% (3) S. agalactiae, 3,2% (2) E. faecium, 1,6% (1) S. acidominimus, 1,6% (1) Leuconoctocc sp., 1,6% (1) S. uberis, 1,6% (1) A. adiacens e 1,6% (1) InaceitÃvel. Foram selecionadas 10 amostras (cepas nÂs 05, 13, 15, 20, 27, 31, 32, 33, 50 e 60), que apresentaram resultados discordantes entre os sistemas Modificado 1, Modificado 2 e API 20 Strep para serem identificadas pelo BBL Crystal e por PCR. O BBL identificou 6 amostras como E. faecium, inclusive a cepa-controle E. faecalis ATCC 29212 e nÃo identificou 4 amostras. Na PCR, uma amostra nÃo amplificou e 9 foram identificadas como Streptococcus spp.. Uma amostra apresentou-se positiva para o gÃnero Enterococcus, mas nÃo para a espÃcie E. faecalis e 1 (uma) amplificou para a espÃcie S. agalactiae. Nenhuma das amostras se apresentou positiva para a espÃcie E. gallinarum. A amostra-controle â E. faecalis ATCC 29212 - foi amplificada corretamente. Com a anÃlise dos resultados, verificou-se que houve concordÃncia de identificaÃÃo das 62 cepas em 84% das amostras entre os sistemas manuais (Facklam, Modificado 1 e Modificado 2) e em 52% das amostras entre os sistemas manuais e semi-automatizado (API 20 Strep). Nas 10 amostras com resultados discrepantes, nÃo houve concordÃncia de identificaÃÃo entre os sistemas manuais, API 20 Strep e o BBL. A PCR concordou com os sistemas manuais e o BBL e foi discordante do API 20 Strep, em gÃnero, em 01 amostra. Correlacionando a PCR com o API 20 Strep, houve concordÃncia, em gÃnero, em 01 amostra e foi discordante dos demais sistemas. Para aumentar a sensibilidade de identificaÃÃo de Enterococcus spp. devem ser utilizados pelo menos dois mÃtodos com testes fenotÃpicos. Amostras com identificaÃÃes discordantes devem ser reidentificadas por PCR / The Enterococcus are Gram-positive cocci catalase negative - which inhabit the gastrointestinal, and genitourinary tract. They are identified manually and the procedure is based on the classic of Facklam, by semi-authomatized and automatized systems as well as by Molecular Biology. Due to the diversity of identification methods and aiming to verify the concordance among some methods, it was accomplished the identification of 62 bacteria strains proceeding from the collection of the Centro Especializado em Micologia MÃdica (CEMM) of the Federal University of CearÃ, in Brazil. They were presumably identified as Enterococcus sp. e Enterococcus faecalis by the Facklam scheme. This study was begun with the identification for two manual methods, modified from the Facklam scheme â Modified 1 and Modified 2- and semi-automatized â API 20 Strep. For the inconsistent results definition we used the semi-automatized â BBL and the Molecular Biology- PRC. The Franklin scheme identified 16% (10) as strains of the genre Enterococcus sp. and 52% (84) as E. faecalis. The Modified Scheme 1 presented the following identification: 82, 2% (51) E. faecalis, 9, 7% (6) E. mundtii and 8, 1% (5) E. gallinarum. The Modified 2 identified 98, 4% (61) E. faecalis and 1, 6% (1) E. gallinarum. The API 20 Strep identified 51,6% (32) E. faecalis, 19,4% (12) A viridans, 13,0% (8) E. avium, 4,8% (3) S. agalactiae, 3,2% (2) E. faecium, 1,6% (1) S. acidominimus, 1,6% (1) Leuconoctocc sp., 1,6% (1) S. uberis, 1,6% (1) A. adiacens and1,6% (1) unacceptable. It was made the selection of 10 sample (strains), among them, the ones numbered (05, 13, 15, 20, 27, 31, 32, 33, 50 and 60), which presented discordant results among the systems Modified 1, Modified 2 and API 20 Strep to be identified by BBL Crystal and by PCR. The BBL identified 6 samples as E. faecium, including the control- strain E. faecalis ATCC 29212 and did not identify 4 samples. In the PCR, one sample did not amplify and 9 were identified as Streptococcus spp. One sample was identified as positive for the genre Enterococcus, but it did not for the species E. faecalis and 1 (one) was amplified for the species S. agalactiae. None of the samples presented were positive for the species E. gallinarum. The control-sample â E. faecalis ATCC 29212 â was correctly amplified. With the analysis of the results, it was noticed that there was identification concordance of the 62 strains in 84% of the samples among the manual systems (Facklam, Modified 1 and Modified 2) and in 52% of the samples among the manual and semi-automatized systems (API 20 Strep). In 10 samples with disagreeing results there was no identification concordance among the manual systems, API 20 Strep and the BBL. A PCR agreed with the manual systems and the BBL and did not agree with the API 20 step, in genre, in one sample. Correlating the PCR with the API 20 Strep, there was agreement, in genre, in 01 sample and disagreement with the other systems

Swine influenza viruses (SIV) are a common and an important cause of respiratory disease in pigs. Pigs can serve as mixing vessels for the evolution of reassortment viruses containing both avian and human signatures, which have the potential to cause pandemics. NS1 protein of influenza A viruses is a major antagonist of host defence and it regulates multiple functions during infection by interacting with a variety of host proteins. Therefore, it is important to study swine viruses and NS1-interacting host factors in order to understand the mechanisms by which NS1 regulates virus replication and exerts its host defense functions. Influenza A viruses enter the host through the respiratory tract and infect epithelial cells in the respiratory tract, which form the primary sites of virus replication in the host. Thus, studying SIV infection in primary swine respiratory epithelial cells (SRECs) would resemble conditions similar to natural infection. The objectives of this study were to identify NS1-interacting host factors in the virus-infected SRECs and to understand the physiological role of at least one of the factors in influenza virus infection. The approaches to meet this objective were to generate a recombinant SIV carrying a Strep-tag in the NS1 protein, infect SRECs with the Strep-tag virus, purify NS1-interacting host protein complex from the infected cells by pull-down using strep-tactin resin and then study the physiological role of one of the NS1-interacting partners during influenza infection. Using a reverse-genetics strategy, a recombinant virus carrying the Strep-tag NS1 was successfully rescued and the SRECs were infected with this recombinant virus. The Strep-tag in the NS1 protein facilitated the isolation of an intact NS1-interacting protein complex and the proteins present in the complex were identified by liquid chromatography-tandem mass spectrometry. The identified proteins were grouped to enrich for different functions using bioinformatics. This gave an insight into the different functions that NS1 may regulate during infection and the potential host partners involved in these functions. Among the host proteins identified as potential interaction partners, RNA helicases were particularly of interest to study. Influenza being an RNA virus, RNA helicases could have important functions in the virus life cycle. Among the identified RNA helicases, DDX3 has been shown to regulate IFNβ induction and affect the life cycle of a number of viruses. However, its function in influenza A virus life cycle has not been studied. Hence, this study explored whether DDX3 has any role in the influenza A virus life cycle. Immunoprecipitation studies revealed viral proteins NP and NS1 as direct interaction partners with DDX3. DDX3 is a known component of stress granules (SGs) and influenza A virus lacking the NS1 gene is reported to induce SG formation. Therefore, the role of DDX3 in SG formation, induced by PR8 influenza A virus lacking NS1 (PR8 del NS1) was explored. The results from this study showed that DDX3 co-localized with NP in SGs indicating that DDX3 may interact with NP in the SGs. NS1 protein was found to inhibit virus-induced SGs and DDX3 downregulation impaired virus-induced SG formation. The contribution of the different domains of DDX3 to viral protein interaction and virus-induced SG formation was also studied. While DDX3 helicase domain did not interact with NS1 and NP, it was essential for DDX3 localization in virus induced SGs. Moreover, DDX3 downregulation resulted in the increased replication of PR8 del NS1virus, accompanied by an impairment of SG induction in infected cells. Since DDX3 is reported to regulate IFNβ induction, the role of DDX3 in influenza A virus induced IFNβ induction was also examined. Using small molecule inhibitors and siRNA-mediated gene knockdown, the RIG-I pathway was identified as the major contributor of influenza induced IFNβ induction in newborn porcine tracheal epithelial (NPTr) cells. DDX3 downregulation and overexpression also showed that DDX3 has an inhibitory effect on IFNβ expression induced by both influenza infection and low molecular weight (LMW) poly I:C treatment, which is also a RIG-I ligand. RNA competition assay to identify the mechanism of DDX3-mediated inhibition, showed that RIG-I binding affinity to its ligands LMW poly I:C and influenza viral RNA (vRNA) is much higher than that of DDX3. Furthermore, DDX3 downregulation enhanced titers of the PR8 del NS1 virus, while it did not affect the titers of the wild-type strains of PR8 and SIV/SK viruses. Overall, the results show that DDX3 has an antiviral role and the SG regulatory function of DDX3 has a profound effect on virus replication than the IFNβ regulatory function.

Purpose: The purpose was to examine the maternal influences on the development of infant oral biofim and dominant bacterial strains of at risk populations. Methods: The study used a cross-sectional design to examine factors influencing biofilm colonization and the identification of bacterial strains transmitted from mother to child. Participants were enrolled in Children’s Health Involving Parents of Greater Richmond (CHIP). Plaque and saliva samples were collected from mothers and their children ages 6-36 months. The colonized oral bacteria strains of the mother infant dyads were then compared. Oral bacterial strain identification was completed using the HOMIM Forsythe microbe identification array. Examination for concordant strains was done using the statistical boot strap shuffle in Excel. Results: Forty-one CHIP families were involved in the pilot study. Participants were predominantly non-white , less than high school education 46.3%, and their average age was 29.1 years. Mothers had a caries prevalence of 87.8% and the infant’s caries rate was 26.7%. To date n=14 pairs of the n=41 samples have been processed and analyzed using the HOMIM microarray. Twelve paired samples were not processed due to non-detectable levels of bacterial DNA. Fifteen samples are currently being processed by HOMIM Forsyth. Predominate species transferred from mother to child include S. Oralis, S. parasanguinous, S. mitis, Slakia, and S. anginosis. 425 unique strains of bacteria were analyzed on the array with a maternal concurrence rate of 33%. Conclusion: When comparing total bacterial populations in the oral environment a concurrence of transmission from mother to child was 33%. Higher rates of vertical transmission were observed in S. Oralis, S. sanguinous, and Slakia.

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.

Background and aims: Most respiratory tract infections (RTI) are self-limiting. Despite this, they are associated with high antibiotic prescription rates in general practice in Sweden. The aim of this thesis was to evaluate the management of respiratory tract infections (RTIs) with particular emphasis on acute otitis media (AOM). Methods: Paper I: A prospective, open, randomized study of 179 children presenting with AOM and performed in primary care. Paper II & III: Study of 6 years data from primary care in Kalmar County on visits for RTI, retrieved from electronic patient records. Paper IV: Observational, clinical study of 71 children presenting with AOM complicated by perforation, without initial use of antibiotics. Results: Children with AOM who received PcV had some less pain, used fewer analgesics and consulted less, but the PcV treatment did not affect the recovery time or complication rate (I). Between 1999 and 2005, 240 445 visits for RTI were analyzed (II & III). Antibiotics were prescribed in 45% of visits, mostly PcV (60%) and doxycycline (18%). Visiting rates for AOM and tonsillitis declined by >10%/year, but prescription rates of antibiotics remained unchanged. For sore throat, 65% received antibiotics. Patients tested but without presence of S.pyogenes received antibiotics in 40% of cases. CRP was analyzed in 36% of consultations for RTI. At CRP<50mg/l antibiotics, mostly doxycycline, were prescribed in 54% of visits for bronchitis. Roughly 50% of patients not tested received antibiotics over the years.Twelve of 71 children with AOM and spontaneous perforation completing the trial received antibiotics during the first nine days due to lack of improvement, one child after 16 days due to recurrent AOM and six had new incidents of AOM after 30 days (IV). Antibiotics were used more frequently when the eardrum appeared pulsating and secretion was purulent and abundant. All patients with presence of S.pyogenes received antibiotics. Results: Children with AOM who received PcV had some less pain, used fewer analgesics and consulted less, but the PcV treatment did not affect the recovery time or complication rate (I). Between 1999 and 2005, 240 445 visits for RTI were analyzed (II & III). Antibiotics were prescribed in 45% of visits, mostly PcV (60%) and doxycycline (18%). Visiting rates for AOM and tonsillitis declined by >10%/year, but prescription rates of antibiotics remained unchanged. For sore throat, 65% received antibiotics. Patients tested but without presence of S.pyogenes received antibiotics in 40% of cases. CRP was analyzed in 36% of consultations for RTI. At CRP<50mg/l antibiotics, mostly doxycycline, were prescribed in 54% of visits for bronchitis. Roughly 50% of patients not tested received antibiotics over the years.Twelve of 71 children with AOM and spontaneous perforation completing the trial received antibiotics during the first nine days due to lack of improvement, one child after 16 days due to recurrent AOM and six had new incidents of AOM after 30 days (IV). Antibiotics were used more frequently when the eardrum appeared pulsating and secretion was purulent and abundant. All patients with presence of S.pyogenes received antibiotics.

Les vésicules géantes sont souvent utilisées comme modèles des membranes cellulaires. On a ici examiné :<br />1. leur adhésion spécifique via deux « colles moléculaires » :<br />– colle faible : fragments terminaux EC12 de E-cadhréine. Cette protéine<br />est impliquée dans la formation de tissus et dans de nombreux phénomènes<br />cancéreux. L'analyse statistique de l'adhésion bicouche supportée/vésicule<br />décorées par EC12 montre qu'existe une adhésion faible sensible au calcium et à une mutation des fragments ;<br />– colle forte : couple streptavidine/biotine. Lors de l'adhésion sur une bi-couche supportée, la tension de la vésicule joue fortement sur la morphologie du contact et sa cinétique.<br />2. la dynamique de pores transitoires (dus à une mise sous tension par éclairement<br />intense), en présence de tensioactif capable de s'insérer à leur bord. La tension de ligne en fonction de la concentration en tensioactif est un isotherme 1D, analysé en termes d'excès de ligne et d'énergie d'adsorption du tensioactif.

No description available.

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.