Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-10T17:58:31Z No. of bitstreams: 1 Marques, Leila Márcia Peres [Dissertação, 2014].pdf: 1219156 bytes, checksum: dbca893aaf472356f21637055bd0d192 (MD5) / Made available in DSpace on 2017-04-10T17:58:31Z (GMT). No. of bitstreams: 1 Marques, Leila Márcia Peres [Dissertação, 2014].pdf: 1219156 bytes, checksum: dbca893aaf472356f21637055bd0d192 (MD5) / O presente trabalho tem por objetivo caracterizar fenotípica e genotipicamente cepas de S. aureus isoladas de queijo Minas frescal (QMF), quanto a qualidade microbiológica, perfil de resistência a diferentes antimicrobianos, presença do gene de resistência a meticilina (mecA), presença de genes que codificam para enterotoxinas estafilocócicas (SE) clássicas; e para cepas resistentes a meticilina, pesquisa dos genes que codificam para a citotoxina Panton Valentine Leukocidin (PVL) e o perfil de diversidade genética, através da tipificação dos tipos SCCmec, Eletroforese em gel de campo pulsado (PFGE) e do sequenciamento da proteína estafilococócica A (SpA). Estafilococos coagulase-positivos (CoPS) foram isolados a partir de 4 amostras (13,3%) de QMF, sendo que 3 (10%) amostras estavam impróprias para consumo segundo a legislação (acima de 5,0.10² UFC/g). As 31 cepas de CoPS isoladas apresentaram a sequência gênica 16S rRNA e o gene nuc, sendo confirmadas como S. aureus. As 31 cepas S. aureus apresentaram resistência a penicilina, 21 (67,74%) a eritromicina, 12 (38,71 %) a ciprofloxacina, 4 (12,9%) a clindamicina, 4 (12,9%) a oxacilina e cefoxitina, 2 (6,45%) a rifampicina, 1 (3,23%) ao cloranfenicol e a tetraciclina. Todas as 31 cepas foram sensíveis ao trimetoprim-sulfametoxazole, gentamicina e a linezolida. Sete cepas (22,58%) carreavam o gene mecA, destas, 4 apresentaram fenótipo de resistência a meticilina, sendo classificadas como S. aureus resistentes a meticilina (MRSA), sendo todos sensíveis a vancomicina e com resistência constitutiva a clindamicina. Cinco cepas (16,1%) apresentaram resistência induzida a clindamicina. Os genes que codificam para as SE clássicas (sea/seb e seb/sec) foram encontrados em 2 isolados (6,45%) MRSA. Através da PFGE e da tipificação SpA, as cepas MRSA isoladas, apresentaram 3 diferentes perfis genotípicos. Essas cepas MRSA não apresentaram os tipos SCCmec I a V, nem os genes que codificam para PVL. Os resultados obtidos neste trabalho indicam que cepas MRSA estão sendo veiculadas através do QMF, provavelmente por manipulação humana inadequada e/ou condições higiênico-sanitárias precárias. O potencial enterotoxigênico destas bactérias indica que o QMF pode causar intoxicação alimentar, sendo um risco para a saúde pública / This study aimed to characterize genotypic and phenotypically S. aureus strains isolated from Minas frescal cheese (MFC) to evaluate the microbiological quality, resistance profile to diferent antimicrobials, the presence of methicillin resistance (mecA) gene and the presence of genes encoding classical staphylococcal enterotoxins (SE). Only for methicillin resistant S. aureus (MRSA) strains were performed the gene encoding Panton Valentine Leukocidin cytotoxin (PVL) and the genetic diversity profile through Pulsed-field gel electrophoresis (PFGE), typing of SCCmec and SpA typing. Coagulase-positive staphylococci (CoPS) were identified in four MFC samples (13.3%), and three (10%) samples showed the number of colony forming units (CFU) higher than allowed to MFC by Brazilian legislation (5,0x10² UFC/g). All the 31 CoPS strains carried the gene sequence 16S rRNA and nuc gene, and were confirmed as S. aureus. All 31 strains of S. aureus were resistant to penicillin, 21 (67.74%) to erythromycin, 12 (38.71%) to ciprofloxacin, 4 (12.9%) to clindamycin, 4 (12.9%) to oxacillin and cefoxitin, 2 (6.45%) to rifampicin, 1 (3.23%) to chloramphenicol and tetracycline. All the 31 CoPS strains were susceptible to trimethoprim-sulfamethoxazole, gentamicin and linezolid. Seven strains (22.58%) encoded mecA gene, four of them showed the methicillin resistance phenotypic, been classified as methycilin resistant S. aureus (MRSA). All MRSA isolates were susceptible to vancomycin and showed constitutive clindamycin resistance. Five strains (16.1%) showed induced clindamycin resistance. The gene encoding the classical SE (sea/seb and seb/sec) were detected in two isolates (6.45%) MRSA. Genetic analysis of MRSA strains was performed by PFGE and SpA typing showed three different profiles. The strains that showed mecA gene, did not show PVL genes coding, neither the types of SCCmec typing. The results obtained in this study showed that MRSA strains are being transmitted by MFC samples, probably due to inadequate human manipulation and/or poor and inefficient hygienic-sanitary conditions. The enterotoxigenic potential of these strains is a concern for public health due to the risk of food poisoning among the MFC consumers

Bacillus cereus est un pathogène opportuniste à l'origine de deux types de toxi-infections alimentaires classées en syndrome émétique ou diarrhéique. Le syndrome diarrhéique résulte de la production d'entérotoxines (Hbl, Nhe et CytK) au niveau de l'intestin grêle de l'hôte, caractérisé par une atmosphère anaérobie et un faible potentiel d'oxydo-réduction (POR). La capacité de B. cereus à se développer et à produire des entérotoxines dans ces conditions est sous le contrôle de deux systèmes qui agissent, en partie, indépendamment du régulateur pléiotrope connu, PlcR (Phospholipase C Regulator). Il s'agit du système à deux composants ResDE et de la protéine Fnr (Fumarate Nitrate Reductase). Le but de cette étude a été de caractériser d'un point de vue fonctionnel l'implication du régulateur Fnr et du système ResDE dans la toxinogenèse de B. cereus. Les résultats ont montré que la régulation de la transcription de hbl et nhe était sous le contrôle direct et indirect de Fnr et de ResD. En aérobiose, la fixation de Fnr (forme Apo) sur les régions promotrices des gènes de structure des entérotoxines (pnhe et phbl) et des gènes de régulation (presDE, pfnr et pplcR) dépend des conditions redox. L'affinité de ResD pour pnhe, phbl, presDE, pfnr et pplcR dépend des séquences de ces régions promotrices et son affinité pour les régions promotrices presDE et pfnr dépend de son état de phosphorylation. ResD et ApoFnr sont capables de se fixer simultanément sur les régions promotrices étudiées et sont également capables d'interagir physiquement en l'absence d'ADN. Nous avons proposé un modèle de régulation de la toxinogenèse dans lequel ResDE et Fnr pourraient agir en synergie. Enfin des expériences de double hybride ont permis de mettre en évidence que la protéine PlcR pourrait interagir in vivo avec les régulateurs ResD et Fnr. La régulation de la toxinogenèse impliquerait donc la formation d'un complexe multi-moléculaire / Bacillus cereus is an opportunistic pathogen responsible of two types of food-borne diseases, classified as emetic and diarrhoeal syndromes. The diarrhoeal syndrome results from the production of enterotoxins (Hbl, Nhe and CytK) in the host small intestine, which constitutes a high reducing anoxic environment. The ability of B. cereus to produce enterotoxins and grow well in such environment is controlled by two global regulators that may function independently of the pleiotropic virulence regulator PlcR (Phospholipase C Regulator). These two regulators are the two-component system ResDE and the redox regulator Fnr (Fumarate Nitrate Reductase). The aim of this study was to establish the role of Fnr and ResDE in the virulence regulatory pathway of B. cereus. The results showed that transcriptional regulation of hbl and nhe was directly and indirectly controlled by Fnr and ResD. In aerobiosis, Fnr interaction (apo form) with the promoter regions of the enterotoxin structural genes (pnhe and phbl) and the enterotoxin regulator genes (presDE, pfnr and pplcR) depends on its oligomeric state. DNA binding affinity of ResD for pnhe, phbl, presDE, pfnr and pplcR depends on the promoter sequences and affinity for presDE and pfnr depends on its phosphorylation state. ResD and Fnr were found to physically interact and simultaneously bind their target DNAs. We proposed a model for regulation of enterotoxin genes expression in which ResD and Fnr could act synergically. Finally, yeast two-hybride experiments showed that PlcR could physically interact in vivo with Fnr and ResD. Enterotoxin genes expression of B. cereus could thus be controlled through a mechanism including a ternary complex

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-09T15:48:50Z No. of bitstreams: 1 Milena da Silva Albuquerque.pdf: 915379 bytes, checksum: dd414d9f832cb823db0efc59b06ce1fb (MD5) / Made available in DSpace on 2017-02-09T15:48:50Z (GMT). No. of bitstreams: 1 Milena da Silva Albuquerque.pdf: 915379 bytes, checksum: dd414d9f832cb823db0efc59b06ce1fb (MD5) Previous issue date: 2014-12-17 / The objective of this work was to investigate the occurrence of encoding staphylococcal enterotoxin (sea, seb, sec and seg) genes and the toxin gene 1 of Toxic Shock Syndrome (tst) from Staphylococcus aureus, coming from mastitis cases bovine in the micro region of Garanhuns, State of Pernambuco. 93 isolates from Staphylococcus aureus were analyzed, which were obtained from milk samples from cows with clinical mastitis and subclinical from 17 properties in 11 municipalities of the Micro Region of Garanhuns, State of Pernambuco. For the molecular characterization of the species Staphylococcus aureus, one of the Polymerase Chain Reaction (PCR) was performed in order to identify the presence of the nuc gene, and to the molecular characterization of enterotoxins, and Staphylococcal toxin Toxic Shock Syndrome. Specific genes were identified: sea, seb, sec, seg and tst. 93 genes were analyzed and we observed the presence of enterotoxigenic gene in 20 (21.6%) samples, of which 11 (55.0%) were positive for tst gene, seven (35.0%) for the sec gene two (10.0%) for the seg gene. 20 isolates amplified segments to the presence of the sec, seg and tst genes. 16 of these (80.0%) were positive for only one gene, and four (20.0%) were positive for both genes (tst and sec). 17 properties were studied, of which seven (41.2%) had positive cultures for at least one of the genes sec, seg and tst. This was the first hit record of encoding gene of the toxin of toxic shock syndrome in mastitis cows milk samples in the state of Pernambuco. Since there was a variation in the distribution of sec, seg and tst genes in strains from different property, it can be inferred that there is genotypic variation in S. aureus strains that cause bovine mastitis. / Objetivou-se com este trabalho pesquisar a ocorrência de genes codificadores de enterotoxinas estafilocócicas (sea, seb, sec e seg) e do gene da toxina 1 da síndrome do choque tóxico (tst) a partir de isolados de Staphylococcus aureus procedentes de casos de mastite bovina na microrregião Garanhuns, estado de Pernambuco. Foram analisados 93 isolados de Staphylococcus aureus obtidos a partir de amostras de leite de vacas com mastite clínica e subclínica, provenientes de 17 propriedades localizadas em 11 municípios da microrregião Garanhuns, no estado de Pernambuco. Para a caracterização molecular da espécie Staphylococcus aureus, foi realizada uma Reação em Cadeia da Polimerase (PCR) visando a identificação pela presença do gene nuc, assim como para a caracterização molecular das Enterotoxinas Estafilocócicas e da toxina da Síndrome do Choque Tóxico. Foram identificados os genes específicos sea, seb, sec, seg e tst. Dos 93 isolados analisados, observou-se a presença de genes enterotoxigênicos em 20 (21,6%) amostras, das quais 11 (55,0%) foram positivas para o gene tst, sete (35,0%) para o gene sec, duas (10,0%) para o gene seg. Dentre os 20 isolados que amplificaram segmentos para a presença dos genes sec, seg e tst, 16 (80,0%) foram positivos apenas para um gene e quatro (20,0%) foram positivos para dois genes (sec e tst). Das 17 propriedades estudadas, sete (41,2%) apresentaram amostras positivas para pelo menos um dos genes sec, seg e tst. Este foi o primeiro registro de ocorrência do gene codificador da toxina da síndrome do choque tóxico em amostras de leite de vacas com mastite no estado de Pernambuco. Como houve uma variação na distribuição dos genes sec, seg e tst nas cepas procedentes de diferentes propriedades, pode-se inferir que há uma variação genotípica nas cepas de S. aureus que causam mastite bovina.

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-08Bitstream added on 2014-06-13T21:05:07Z : No. of bitstreams: 1 ferreira_lm_dr_jabo.pdf: 458853 bytes, checksum: 0406f864aa66f98b313ad2405eae5c45 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Entre agosto de 2005 e dezembro de 2006, foram obtidas 245 estirpes de Staphylococcus aureus isoladas de amostras de leite de vacas com mastite, de óstios papilares da glândula mamária e de insufladores da ordenhadeira, procedentes de um rebanho bovino produtor de leite tipo B. Com a finalidade de monitorar as estirpes de S. aureus envolvidas em casos de mastite bovina por meio da verificação da relação epidemiológica existente entre as estirpes isoladas, especialmente com vistas aos sítios de localização e vias de transmissão, as estirpes foram submetidas à Eletroforese de Campo Pulsado (PFGE), à amplificação das seqüências codificadoras (sea, seb, sec, sed e tst), por intermédio da Reação em Cadeia da Polimerase (PCR), e suas sensibilidades in vitro a 12 antimicrobianos foram determinadas. Os resultados obtidos revelaram 51 diferentes perfis, sendo que a resistência à penicilina foi a predominante entre as 179 (73,1%) estirpes de S. aureus, quando considerada de forma particular (54,8%) ou em conjunto (29,4%). As 66 (26,9%) estirpes restantes foram sensíveis aos 12 antimicrobianos testados e a vancomicina foi o único princípio ativo que se mostrou eficiente a 100% das estirpes testadas. A PFGE revelou 39 pulsotipos distintos, dos quais 25 (64,1%) encontraram-se distribuídos nas 137 (55,9%) estirpes obtidas do leite. Dentre estas, 92 (67,1%) estirpes apresentaram resistência a um ou mais antimicrobianos e foram agrupadas em 22 (88,0%) pulsotipos distintos. Foi observado, também, por meio da PFGE, que nenhum pulsotipo foi isolado por mais de três colheitas consecutivas e que somente o pulsotipo 29 foi identificado em 5 (31,2%) colheitas... / Two hundred and forty five Staphylococcus aureus strains were isolated between August of 2005 and December of 2006 from samples collected from a type-B milk-producing herd. Samples encompassed milk collected directly from mastitic cows, papillas osteuns of mammary glands, and milking machine cups. Samples were subjected to Pulsed Field Gel Electrophoresis (PFGE) to monitor for the strains of S. aureus and to determine epidemiologic relationships between the isolated strains, taking into account the different precedence of strains. PFGE was used to monitor the presence of specific coding sequences (i.e., sea, seb, sec, sed, and tst) in the milk samples using polymerase chain reaction (PCR) amplification. Moreover, sensitivity of strains to 12 different antibiotics was determined in vitro. Results revealed the presence of 51 different PFGE profiles of S. aureus in the samples, and highlighted 179 (73.1%) penicillin resistant strains. Nonetheless, the 66 (26.9%) other strains were sensitive to all 12 antibiotics tested, and vancomycin was the only antibiotic effective against all tested strains. PFGE also revealed 39 distinct peaks with 25 of them (64.1%) being present in 137 (55.9%) strains obtained from milk. Of these strains, 92 (67.1%) were resistant to one or more antibiotics, and were further grouped in 22 (88.0%) distinct peaks. Additionally, PFGE revealed the presence of no distinct peaks in samples from three consecutive collection times, and that peak 29 was the only one identified in 5 collection times (31.2%). Importantly, PGFE also indicated that in the periods A and H, which corresponded to 12.5% of the total time points, the isolated strains from one collection day (samples from all 3 sources) all had the peaks 2 and 12, respectively...(Complete abstract, click electronic access below)

Made available in DSpace on 2015-03-26T13:47:11Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1370501 bytes, checksum: 64432b0656ba736f2b945a205fa9a8c4 (MD5) Previous issue date: 2012-11-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The genus Staphylococcus is constituted of numerous pathogenic species, including S. aureus, which is often related to food poisoning cases and outbreaks, especially in dairy products. Its pathogenic activity is due to the ability of some strains to produce thermostable enterotoxins (SE). S. aureus detection in foods is often performed using conventional methods, which requires additional tests, such as biochemical and molecular characterization with the purpose of estimating its enterotoxigenic potential. The aim of this study was to characterize the enterotoxigenic potential of Staphylococcus spp. isolates and determine their genetic variability. In a previous study, a Staphylococcus spp. culture collection was obtained, from which 89 isolates were selected and subjected to phenotypical (coagulase and thermonuclease production, biochemical profile and SE production) and molecular analysis (SmaI macrorestriction, SE gene detection by PCR and DNA sequencing). PFGE analysis obtained by SmaI macrorestriction patterns revealed a highly heterogeneous population. Of the 89 isolates, 15.7% were capable of producing classical enterotoxins (SEA-SEE). 21.4% of isolates showed matching results between production of enterotoxins and detection of classical SE genes. 62.9% of isolates showed at least one of the classical SE genes, in association with other non-classical SE genes. SE genes were observed in all isolates and in different combinations, which revealed 59 distinct genotypes. sek was the least frequent observed SE gene, while sei was present in 98.9% of isolates. Partial sequencing of agr locus in 41 isolates showed the ocurrence of agr groups I (68.3%) and II (31.7%). No significant associations were found between agr groups, enterotoxin genes profiles, occurrence of egc cluster, PFGE profiles and/or SE production. Our findings suggest the absence of phenotypic or genotypic markers ideally correlated with the enterotoxigenic production of staphylococci of food origin, which demands further studies for better understanding the occurrence of these associations. / O gênero Staphylococcus é constituído de inúmeras espécies patôgenicas, especialmente S. aureus, que estão frequentemente relacionados a surtos de intoxicação alimentar, principalmente em leite e derivados. Essa atividade patogênica se deve à capacidade de algumas cepas em produzir enterotoxinas termoestáveis. A detecção de S. aureus em alimentos é realizada através de métodos clássicos de cultivo, porém requer testes complementares, que visam a caracterização adequada do potencial patogênico dos isolados. O objetivo deste trabalho foi caracterizar o potencial enterotoxigênico e determinar o grau de variabilidade genética de isolados de Staphylococcus spp.. A partir de uma coleção de microorganismos obtidos de leite cru e queijo frescal em um estudo preliminar, 89 isolados foram selecionados e submetidos à análises fenotípicas (produção de coagulase e termonuclease, perfil bioquímico e produção de enterotoxinas) e moleculares (macrorrestrição por SmaI, PCR para genes de enterotoxinas e sequenciamento de DNA). As análises de PFGE dos perfis de macrorrestrição por SmaI revelaram uma população altamente heterogênea. Dos 89 isolados, 15,7% dos isolados foram produtores de enterotoxinas clássicas. 21,4% dos isolados apresentaram resultados correspondentes entre a presença de genes e a detecção de enterotoxinas. 62,9% dos isolados apresentaram ao menos um dos genes de enterotoxinas clássicas, sempre em associações com os demais genes de enterotoxinas pesquisados. Todos os isolados apresentaram ao menos um dos genes de enterotoxinas pesquisados, em diversas combinações, com a ocorrência de 59 genótipos diferentes. sek foi o gene menos observado, enquanto sei esteve presente em 98,9% dos isolados. O locus egc foi detectado em 5 isolados, porém sua presença foi observada de forma incompleta em diversos isolados. O sequenciamento parcial do locus agr em 41 isolados revelou a ocorrência dos grupos agr I (68,3%) e II (31,7%). Não foram encontradas associações significativas entre grupos agr, perfis de genes de enterotoxinas, ocorrência do egc, perfis obtidos na PFGE e produção de enterotoxinas. As observações do presente estudo sugerem a ausência de marcadores idealmente correlacionados com a capacidade enterotoxigênica de isolados de estafilococos provenientes de amostras de alimentos, o que demanda a realização de outros estudos para compreender melhor a ocorrência dessas associações.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2016-09-15T16:50:29Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_mario_coppola.pdf: 733232 bytes, checksum: aefd4ed8b53d5285cb8c54b6894c3fc1 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2016-09-15T19:51:47Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_mario_coppola.pdf: 733232 bytes, checksum: aefd4ed8b53d5285cb8c54b6894c3fc1 (MD5) / Made available in DSpace on 2016-09-15T19:51:47Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_mario_coppola.pdf: 733232 bytes, checksum: aefd4ed8b53d5285cb8c54b6894c3fc1 (MD5) Previous issue date: 2014-04-04 / Sem bolsa / A mastite bovina é uma doença importante dos rebanhos leiteiros por trazer prejuízos à sanidade e produção. Os objetivos desse estudo foram isolar Staphylococcus de amostras de leite de casos de mastite bovina clínica e subclínica no Rio Grande do Sul, confirmar a presença de S. aureus através da amplificação do gene nuc por PCR, identificar os genes das EEs A, B e D e os grupos agr I-IV e a relação entre os genes dos grupos agr e de EEs. Duzentas e sessenta e seis amostras de leite foram analisadas, sendo 218 provenientes de vacas com mastite subclínica e 48 de casos clínicos. Bactérias do gênero Staphylococcus foram isoladas em 88 amostras. A identificação por PCR confirmou 58 amostras como S. aureus, o que correspondeu a 21,7% do total de amostras, 25 foram classificadas como Staphylococcus coagulase negativa (SCN) e 5 como coagulase positiva. Nas amostras de leite originárias de vacas com mastite subclínica e nos casos clínicos o isolamento de S. aureus foi superior aos SCN e Staphylococcus coagulase positiva. S. aureus foi isolado em maior número de rebanhos, comparado aos SCN e Staphylococcus coagulase positiva. Esses 58 isolados de S. aureus juntamente com outros 32 isolados de casos de mastite bovina no Rio Grande do Sul pertencentes ao banco de isolados do Laboratório de Bacteriologia do IPVDF, num total de 90 isolados foram testados por PCR para a presença de genes dos grupos agr I-IV e das EEs A, B e D. O gene agr mais frequente foi agr-III, seguido por agr-I. Quanto à distribuição por rebanhos, foi identificada maior ocorrência do gene agr-III. O gene de EEs mais encontrado foi sea e quanto à relação entre gene de EEs e agr, foi observada associação significativa entre a presença de sea e agr-I. / Bovine mastitis is an important disease of dairy cattle, which affects sanity and production. The objectives of this study were to isolate Staphylococcus on milk samples from subclinical and clinical cases in the Rio Grande do Sul state, to confirm the presence of S. aureus through amplification of the nuc gene by PCR, to identify the occurrence of enterotoxin (SE) genes A, B e D and agr I-IV groups in S. aureus and the relationship among agr genes and SEs. Two hundred sixty six milk samples were analyzed, 218 from cows with subclinical mastitis and 48 from clinical cases. The Staphylococcus genus was identified in 88 samples. The PCR identification confirmed 58 samples as S. aureus which corresponded to 21,7% of all samples, 25 were classified as CNS and 5 as coagulase positive In milk samples from cows with subclinical mastitis and clinical cases, the isolation of S. aureus was superior to CNS and coagulase positive Staphylococcus. S. aureus was isolated on most herds compared to CNS and coagulase positive Staphylococcus. This 58 isolates of S. aureus with others 32 isolates of bovine mastitis cases from Bacteriology Laboratory of IPVDF, were tested through PCR to presence of agr-I-IV groups and SEs A, B e D genes. The most frequent agr gene was agr-III, followed by agr-I. On herds, the most occurrence gene was agr-III. The most frequent SEs gene found was sea and as for the relationship among SEs genes and agr genes, occurred association for sea and agr-I.

Du fait de leurs propriétés immunostimulantes, les entérotoxines de Staphylococcus aureus (SEs) sont aussi considérées comme des outils thérapeutiques anticancéreux potentiels. Cependant, leurs implications dans de nombreuses pathologies humaines limitent leurs utilisations. Récemment, un opéron dénommé enterotoxin gene cluster (egc) codant pour cinq entérotoxines (SEG, SEI, SElM, SElN et SElO) supposées être de moindre virulence pour l’organisme, a été mis en évidence. En 2004, des patients atteints de carcinome broncho-pulmonaire ont été traités par l’administration d’un surnageant de culture d’une souche de S. aureus, contenant l’opéron egc. Ce traitement a permis d’allonger la durée de survie, et n’a eu aucun effet secondaire. Dans ce cadre, l’objectif de cette thèse a été d’étudier l’activité anti-tumorale des toxines de l’egc. Nos travaux ont mis en évidence l’activité tumoricide de ces toxines, induite par l’activation du système immunitaire. Cette toxicité est médiée par la sécrétion de nombreux médiateurs solubles comme le TNF-α et le NO. Nous avons confirmé le caractère pro-inflammatoire de type Th1 des toxines de l’egc. Nos travaux ont également montré qu’hormis SEI, les toxines de l’egc induisent des sécrétions de cytokines, chimiokines, protéases matricielles (MMPs) et facteurs de croissances nettement inférieures à celle induites par le reste des SEs. Ces résultats pourraient expliquer la faible toxicité associée aux toxines de l’egc. Enfin, nous avons montré que SElO possèdent une toxicité intrinsèque vis-à-vis des lignées tumorales. Cette étude plaide en faveur de l'intérêt des toxines de l’egc dans le développement de nouvelles approches en thérapie anti-tumorale / The use of classical superantigens (e.g. SEA, SEB and SEC) for treatment of cancer has resulted in a low response rates due to serious toxicity in humans. However, in a recent clinical study, remarkable results in treating lung cancer were obtained using superantigens encoded by the enterotoxin gene cluster (egc) without causing any significant toxicity. The current study was performed to investigate how egc superantigens (i.e. SEG, SEI, SElM, SElN and SElO) have tumoricidal activity with low toxicity. Indeed, we first demonstrated that tumoricidal activity of egc-SEs is mediated by immune cell activation, in particular, by secretion of soluble mediators such as nitric oxide and TNF-α. Thus, the proteomic analysis of the PBMC supernatants, showed that SEs-egc enhance the expression of pro-inflammatory cytokines, chimokines and many other biomarkers. Interestingly, levels were significantly higher in supernatants of SEA-stimulated PBMC than those with egc superantigens suggesting that staphylococcal superantigens differs in their inflammatory proprerties. Our results suggest that the relative lower pro-inflammatory activity of egc toxins may explain the low toxicity of these toxins observed during the clinical trial. Finally, we showed that SElO have a direct cytostatic activity against tumor cells. These findings suggest that egc-SEs seems to be good candidates for the development of new drugs in cancer therapy

Orientadores: Tomomasa Yano, Maria Celia S. Lanna / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T10:36:24Z (GMT). No. of bitstreams: 1 Ribeiro_DanielaAlves_M.pdf: 9526817 bytes, checksum: 451baed1219c35350316a70f67d5b336 (MD5) Previous issue date: 2006 / Resumo: Estudos de infecções causadas pela ingestão de águas contaminadas, em especial por Escherichia coli, são importantes para definir o papel dessas bactérias em casos de gastroenterites. Devido à ocorrência freqüente da diarréia infantil na cidade de Ouro Preto-MG, análises microbiológicas da sua água de consumo determinaram a presença de E. coli. No presente trabalho, propriedades fenotípicas e genotípicas de virulência foram estudadas em 97 amostras de E. coli isoladas da água de consumo desse município. Os resultados obtidos para padrões de adesão mostraram que 79 (81,4%) amostras aderiram a células HEp-2 em diferentes padrões de adesão, sendo que 49 (62%) amostras aderiram de forma agregativa, 16 (20,3%) apresentaram o padrão difuso, 12 (15,2%) aderiram de forma não característica e 2 (2,5%) apresentaram o perfil de adesão localizada ¿like¿. Por ensaios de hemaglutinação verificou-se que 70 (72%) amostras hemaglutinaram hemácias de cobaia, 64 (65,9%) aglutinaram hemácias eqüinas, 5 (5,2%) foram positivas com hemácias humanas do grupo A e 10 (10,3%) aglutinaram hemácias bovinas. Todas essas amostras evidenciaram um perfil de hemaglutinação manose sensível, o que está associado à presença da fímbria tipo 1. Outro fator de virulência detectado foi a produção de aerobactina em 11 (11,3%) isolados. Em relação à expressão de hemolisinas em meio sólido, 50 amostras de E. coli foram positivas em placas de ágar sangue contendo hemácias de cavalo, 37 lisaram hemácias de carneiro, 28 lisaram eritrócitos de cobaia, nove lisaram eritrócitos humanos e seis foram positivas com hemácias bovinas. Entretanto, os sobrenadantes de cultura de todas essas bactérias não apresentaram atividade hemolítica ¿in vitro¿. Em relação à atividade citotóxica dos sobrenadantes de cultura das amostras, verificou-se que 89 deles provocaram diferentes alterações morfológicas em células Vero, HeLa e CHO. Deste total, 39 apresentaram efeito citotóxico caracterísitico de desarranjo do citoesqueleto celular, foram estáveis ao aquecimento por 100ºC e demonstraram atividade enterotóxica em camundongos recém nascidos. Estudos genotípicos pela PCR, identificaram nove amostras eltIA+, uma amostra stI+, 14 stII+, seis _ hLy+, três sat+, três astA+, duas pic+, 10 kps+, 11 iucD+, 88 fimH+, seis eae+, três papC+ e uma papG1+. Nenhuma amostra foi positiva para os genes stx1, stx2, stx2v, eltIIA, cnfs, cdt, enhly, pet, bfp, inv, K88 e 987p, aggA, aafA e papGII/GIII. Baseado na aderência em células, na produção de toxinas e na presença de genes de virulência, classificou-se 24 amostras como ETEC, 6 como EPEC atípicas , 36 EAEC e 13 como DAEC. Nenhuma amostra foi classificada como EHEC e EIEC. Estes resultados sugerem um possível envolvimento destas amostras de E. coli como um dos agentes enteropatogênicos envolvidos nas infecções entéricas ocorridas na cidade de Ouro Preto / Abstract: Infections studies caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the pathogenic role of these bacteria in gastroenteritis. Due to frequent infantile diarrhea in the city of Ouro Preto-MG, this city's water microbiologic analysis displayed the presence of E. coli. In the present study, genotypic and phenotypic virulence properties were studied among 97 E. coli strains isolated from the drinking water provided for this city. The obtained results for adhesion patterns showed that 81,4% of the samples adhere to HEp-2 cells in different adhesion patterns, wherein 49 strains adhere in a aggregative pattern, 16 displayed the diffuse pattern, 12 adhered in a non-characteristic pattern and 2 showed the localized adhesion profile ¿like¿. By means of hemagglutination tests we verified that 72 % of the strains hemagglutinated guinea pig erythrocytes, 65,9% agglutinated horse erythrocytes, 5,2% were positives on group A human erythrocytes and 10,3% agglutinated bovine erythrocytes. All these strains made evident a manose sensible hemagglutination profile, which is associated with type 1 fimbriae. Another virulence factor detected was the production of aerobactin in 11,3% of the isolates. Regarding the hemolysin expression in solid medium, 50 E. coli strains were positive in blood agar plates containing horse erythrocytes, 37 lysated sheep erythrocytes, 28 lysated guinea pig erythrocytes, 9 lysated human erythrocytes and 6 were positives on bovine erythrocytes. However, the culture supernatants of all these bacterias didn't displayed any hemolytic activity ¿in vitro¿. This could be associated with a possible b-hemolysin production. In respect of the cytotoxic activity produced by the strains culture supernatants, we verified that 89 of them provoked different morphological alterations in Vero, HeLa and CHO cells. From these, 39 showed a characteristic cytotoxic effect of disarrangement of the cellular cytoskeleton, remained stable when heated by 100ºC and also showed enterotoxic activity in suckling mice. Genotipic studies performed by PCR identified nine eltIA+ strains, one stI+ strain, 14 stII+, six _ hLy+, three sat+, three astA+, two pic+, 10 kps+, 11 iucD+, 88 fimH+, six eae+, three papC+ and one papG1+. None of these results were positive for the following genes stx1, stx2, stx2v, eltIIA, cnfs, cdt, enhly, pet, bfp, inv, K88 e 987p, aggA, aafA e papGII/GIII. Based on the adherence to cells, toxin production and the presence of the virulence genes, 24 strains were classified as ETEC, 6 as atypical EPEC, 36 as EAEC and 13 as DAEC. Anyone strain were classified as EHEC and EIEC. These results suggest a possible involvement of these E.coli strains as one of the enterophatogenic agents related with the enteric infections occurred in the city of Ouro Preto / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Orientador: Jose Luiz Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-07T14:47:03Z (GMT). No. of bitstreams: 1 Pereira_KarenSignori_D.pdf: 795788 bytes, checksum: 3c56762962a9dfbb567a0cc78805f20c (MD5) Previous issue date: 2006 / Resumo: As bactérias pertencentes ao gênero Staphylococcus são bastante peculiares porque apesar de terem distribuição ubiqüitária e formarem a microbiota residente na pele e mucosa de humanos também podem ser causadoras de diversas enfermidades aos mesmos, tais como a intoxicação alimentar estafilocócica.Todavia, para a indústria de produtos cárneos a importância dos estafilococos está além do fato de serem microrganismos patogênicos. Algumas espécies do gênero são coadjuvantes de tecnologia para a fabricação de salames, estando presentes como componentes de culturas starter ou iniciadoras. Entre as características importantes das espécies utilizadas em culturas iniciadoras está a incapacidade enterotoxigênica, o que tem sido diretamente relacionado às espécies coagulase positiva. Diversos trabalhos, porém, têm demonstrado a capacidade de estafilococos coagulase negativa produzirem enterotoxina em meio de cultivo laboratorial e há, inclusive, registros de surtos de intoxicação estafilocócica associados a espécies não produtoras de coagulase. Assim, 90 amostras de salames industrializados, pertencentes a seis diferentes marcas, foram analisadas visando a enumeração, identificação e verificação do potencial enterotoxigênico das espécies de estafilococos coagulase negativa (ECN). Determinações de pH, Aw, análises de coliformes termotolerantes, Salmonella, Staphylococcus coagulase positiva e bactérias láticas também foram realizadas. Salmonella foi detectada em uma amostra. Entre os 266 isolados de ECN nenhum produziu enterotoxina, e dos 252 identificados cerca de 90% eram S. xylosus e S. carnosus / Abstract: Staphylococcus is ubiquitous distribution bacteria and present in skin of humans and other animals. However, staphylococci species can cause various diseases. Staphylococcal food poisoning is one of these diseases. But for meat industry staphylococci importance is not only because diseases. Some species are very important for the fermented sausage¿s manufacture like starter cultures. To be used, staphylococci have do not produce coagulase. However, many researches have demonstrated capacity of coagulase negative staphylococci (CNS) to produce enterotoxin and there are registers of staphylococcal food poisoning outbreaks linked to CNS. Ninety samples of industrialized salamis, for six different brands, had been studied. Enumeration, identification and potencial enterotoxigenic of CNS were analyzed. Additionally, termotolerants coliforms, staphylococci coagulase positive and lactic acid were counted; Salmonella isolated; pH and Aw measured. Salmonella was isolated of one sample. None of 266 CNS produced enterotoxins, 252 were identified and about 90% were S. xylosus and S. carnosus / Doutorado / Doutor em Ciência de Alimentos

Notre travail montre la prédominance du clone « Lyon » parmi les souches de Staphylococcus aureus responsables d’infections invasives (i.e. bactériémies). Il est caractérisé par la présence du gène codant la «staphylococcal enterotoxin » (SE) A. Afin de mieux comprendre le lien éventuel entre la prédominance de la SEA et la gravité des infections induites, nous avons orienté nos travaux sur le versant immuno‐inflammatoire des SE. L’étude des répertoires Vβ induits par l’ensemble des SE s’est révélée peu discriminante. A l’aide d’approches génomiques et protéiques réalisées sur des lymphocytes et monocytes humains, nous avons observé le puissant potentiel inflammatoire de la SEA en particulier par rapport à la SEG (SE appartenant à l’opéron egc retrouvé dans les infections de faible sévérité) et la prédominance de la réponse lymphocytaire T. Enfin, afin de mieux comprendre la spécificité des effets observés, nous avons conduit des expériences sur lymphocytes purifiés en ciblant les Ly T effecteurs (CD4+CD25‐) et Ly T régulateurs (CD4+CD25+FOXP3+). Nos travaux ont confirmé l’induction par la SEA d’une forte réponse inflammatoire, associant une synthèse de cytokines de type Th1 et Th17 dans les deux lignées lymphocytaires. De plus, nos résultats suggèrent que les SE provoquent une perte des fonctions suppressives des Ly T régulateurs ouvrant de nouvelles perspectives quant à l’implication des SAgs dans de nombreuses situations cliniques en particulier le choc septique, et les infections chroniques / Our work shows the prevalence of the “Lyon” clone among Staphylococcus aureus strains responsible for severe systemic infections (i.e. bacteremia). This clone is characterized by the presence of the staphylococcal enterotoxin (SE) A coding gene. To better understand the possible link between the prevalence of SEA and the severity of infections, we focused our work on the immuno inflammatory responses of SE. By using genomic and protein studies, in human lymphocytes and monocytes, we observed the potent inflammatory property of SEA (especially in comparison with SEG ‐ belonging to the egc cluster mainly found in less severe infections) mainly targeting T cell response over monocytes. Finally, to better understand the specificity of these effects, we performed experiments on purified lymphocytes and targeted effectors (CD4+CD25‐) and regulatory T lymphocytes (CD4+CD25+FOXP3+). Our works confirmed the induction by SEA of a strong inflammatory response, characterized by the release of Th1 and Th17 cytokines in both lymphocyte lineages. Furthermore, our results also showed the loss of the regulatory T cell suppressive functions after SE stimulation. Collectively, these results offer new perspectives for the implication of the SAgs in numerous clinical situations in particular toxic shock and the chronic infections.