La technique d’empreinte génétique par rep-PCR, qui utilise des séquences d’ADN répétitives, a été utilisée pour mettre en évidence la présence de groupes d’Escherichia coli signatures pour divers poulaillers et d’évaluer leur évolution suite au détassement. L’amorce (GTG)5 a été utilisée pour générer des empreintes d’ADN de 522 isolats provenant de 7 poulaillers échantillonnés deux fois : juste avant et 5 jours après le détassement. Les empreintes d’ADN ont été analysées selon l’algorithme de correspondance de bandes de Jaccard. Les analyses de Jackknife des coefficients de similitude ont révélé qu’entre 73% et 93% des isolats ont pu être correctement regroupés selon leur poulailler d’origine. Un dendrogramme construit à partir des coefficients de similitude de Jaccard a groupé les isolats dans 42 grappes avec près de la moitié dans une seule grappe. Environ 80% des isolats ont été groupés dans les 6 plus grosses grappes. Quatre de ces grappes été constituées majoritairement d’isolats provenant d’un seul site. Ces grappes pourraient être des grappes signatures qui permettraient d’identifier des poulaillers en particulier. La comparaison des nombres de grappes présentes avant et après le détassement a révélé une variabilité de l’impact du détassement sur les populations fécales d’E. coli. Pour certains sites, il y avait peu d’agrégats présents tant avant qu’après le détassement alors que pour d’autres sites c’était le contraire. Quoique plus de recherches soient nécessaires afin de valider les conclusions, nos résultats suggèrent la présence de sous-populations signatures d’E. coli pour certains poulaillers et une réponse variable à l’effet du détassement. / Rep-PCR genomic fingerprinting, which uses repetitive intergenic DNA sequences, was investigated as a mean to identify signature pattern of chicken fecal Escherichia coli populations and evaluate their changes over time. The (GTG)5 primer was used to generate DNA fingerprints from 522 isolates originating from 7 chicken houses just prior to, and five days after, thinning. The DNA fingerprints were analysed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that between 73% and 93% of the isolates could correctly be grouped in their house of origin. A dendrogram constructed by using Jaccard similarity coefficients grouped the isolates in 42 clusters with approximately half of them in the same cluster. Out of the 6 largest clusters, containing 80% of all isolates, 4 consisted mostly of isolates coming from only 1 house. These clusters could represent signature clusters identifying specific houses. A comparison of the number of clusters present before and after thinning for each house revealed a substantial difference in the behaviour of the fecal E. coli. For some houses, there were few clusters represented both before and after thinning, with a high number of new clusters appearing after thinning whereas in other houses, the contrary was observed. Our results suggest the presence of a signature subpopulation in some chicken houses and a variable response of the E. coli population to the effect of thinning.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-17T14:39:22Z No. of bitstreams: 1 Janaina Cortez de Oliveira.pdf: 616667 bytes, checksum: 9d56953508b1cd4518224b8966b15c92 (MD5) / Made available in DSpace on 2017-03-17T14:39:22Z (GMT). No. of bitstreams: 1 Janaina Cortez de Oliveira.pdf: 616667 bytes, checksum: 9d56953508b1cd4518224b8966b15c92 (MD5) Previous issue date: 2010-10-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The bacterial blight caused by Xanthomonas citri subsp. malvacearum (Xcm) is the most important bacterial disease of cotton in the Middle-West and Northeast Regions of Brazil. The variability of a population formed by 100 Xcm strains obtained from commercial fields in the states of Goiás, Mato Grosso, Mato Grosso do Sul and Bahia, Brazil, was analyzed based upon genetic and phenotypic characteristics. The enzymes amylase, lipase, cellulase and the polysaccharide levan were produced by all Xcm isolates while the caseinase was only produced by 20 strains. All strains induced hipersensitivity reaction in tomato leaves. The 100 strains were sensitive to copper oxychloride (1500 mg L-1), copper hidroxide (1614 mg L-1), oxytetracicline (600 mg L-1), oxytetracicline (90 mg L-1) + tribasic copper sulfate (1500 mg L-1) and oxytetracicline (76.6 mg L-1) + streptomycin sulfate (367.2 mg L-1), but resistant to kasugamycin (60 mg L-1). Among the eighteen tested antibiotics the strains presented variable reaction in relation to: nalidixic acid, amoxicillin, azithromycin, bacitracin, eritromicin, kanamycin, pefloxacin, tobramycin e vancomycin. Rep-PCR (REP, ERIC and BOX) showed the formation of five groups at 70% similarity level. Group I contained 98 strains, including the reference strain Xcm IBSBF1733, group II contained only two strains from Bahia and the other three groups included strains of Xanthomonas citri pv. anacardii, X. citri pv. mangiferaeindicae e X. axonopodis pv. spondiae used for comparison. This work also evaluated the effect of silicon (Si) on the bacterial blight of cotton control and the putative mechanisms involved in the resistance potentialized by this element were evaluated. Calcium silicate (CaSiO3) was incorporated into the soil at concentrations of 0.00, 0.25, 0.50, 1.50 and 3.00 g of SiO2 kg-1 soil 25 days before planting. Leaves of 33-day-old-plants were inoculated by infiltration with 0.5 mL suspension of Xcm (108 CFU ml-1). Components of resistance were evaluated every two days until 10 days after inoculation, at which point plant development and the accumulation of Si and Calcium (Ca) were also determined. H2O2 production and the activity of enzymes related to plant defenses were analyzed at 6, 12 and 24 hours after inoculation in plants +/-Si. In vitro inhibition of pathogen growth due to Si was also assessed. No significant difference was seen among the treatments regarding incubation period, disease incidence or inhibition of bacterial growth. However, with the application of 1.50 g of SiO2 kg-1 of soil, a reduction in severity (54.9%) was observed along with a reduction in the area under the disease progression curve (35.76%) and an increase in plant height (7.04%). There was no observable accumulation of Si in the cotton leaves. Levels of soluble proteins and H2O2 and activity of the enzymes SOD, APX, guaiacol peroxidase, PAL and b Glu were altered in the presence of Si (1.80 g SiO2 kg-1). Thus based on the genetic and phenotypic characteristics studied we conclude that the population of 100 Xcm strains presents low variability, and that the reduction of cotton bacterial blight severity mediated by Si is probably associated to the characteristic events of induced resistance observed. / A mancha-angular, causada por Xanthomonas citri subsp. malvacearum (Xcm), é a principal doença bacteriana do algodoeiro no Centro-Oeste e Nordeste do Brasil. A variabilidade de uma população de 100 isolados de Xcm, oriundos de plantas com sintomas típicos de mancha-angular em áreas produtoras de algodoeiro dos estados de Goiás, Mato Grosso, Mato Grosso do Sul e Bahia, Brasil, foi analisada com base em características fenotípicas e genéticas. As enzimas amilase, lipase e celulase e o polissacarídeo levana foram produzidos por todos os isolados de Xcm, enquanto a caseinase foi produzida apenas por 20 isolados. Todos os isolados induziram reação de hipersensibilidade em folhas de tomateiro. Os 100 isolados foram sensíveis a oxicloreto de cobre (1500 mg l-1), hidróxido de cobre (1614 mg l-1), oxitetraciclina (600 mg l-1), oxitetraciclina (90 mg l-1) + sulfato tribásico de cobre (1500 mg l-1) e oxitetraciclina (76,6 mg l-1) + sulfato de estreptomicina (367,2 mg l-1), e resistentes a casugamicina (60 mg l-1). Dentre os 18 antibióticos testados, os isolados bacterianos apresentaram reação variável a nove: ácido nalidíxico, amoxicilina, azitromicina, bacitracina, eritromicina, kanamicina, pefloxacina, tobramicina e vancomicina. Rep-PCR (REP, ERIC e BOX) revelou a formação de cinco grupos ao nível de 70% de similaridade, sendo o grupo I constituído de 98 isolados, incluindo o isolado referência de Xcm IBSBF1733, o grupo II, formado apenas por dois isolados oriundos da Bahia e os três outros grupos por isolados de X. citri pv. anacardii, X. citri pv. mangiferaeindicae e X. axonopodis pv. spondiae utilizados para comparação. Neste trabalho também foi avaliado o efeito do silício (Si) no controle da mancha-angular do algodoeiro, e os possíveis mecanismos envolvidos na resistência potencializada por este elemento. O silicato de cálcio (CaSiO3) foi incorporado ao solo nas doses de 0,00; 0,25; 0,50; 1,50 e 3,00 g de SiO2 kg-1 de solo 25 dias antes do plantio. Plantas com 33 dias tiveram as folhas inoculadas por infiltração com 0,5 mL de suspensão de Xcm (108 UFC ml-1). Os componentes da resistência foram avaliados a cada dois dias até 10 dias após a inoculação, quando o desenvolvimento da planta e o acúmulo de Si e Ca foram também determinados. Foram ainda analisadas a atividade de enzimas relacionadas à defesa vegetal e a produção de H2O2 às 6, 12 e 24 h após a inoculação em plantas +/-Si e a inibição in vitro do crescimento do patógeno. Não foi verificada diferença significativa entre os tratamentos quanto ao período de incubação, incidência da mancha-angular e inibição do crescimento da bactéria. Com a aplicação da dose 1,50 g SiO2 kg-1 de solo, observou-se redução da severidade (54,9%), da área abaixo da curva de progresso da doença (35,76%) e aumento na altura das plantas (7,04%). Não foi verificado acúmulo de Si nas folhas de algodoeiro. Níveis de proteínas solúveis e H2O2 e atividade das enzimas SOD, APX, POX, PAL e GLU foram alterados na presença do Si (1,80 g SiO2 kg-1). Com base nas características fenotípicas e genéticas analisadas, conclui-se que a população de 100 isolados de Xcm estudada apresenta baixa variabilidade e que a redução da severidade da mancha-angular do algodoeiro mediada por Si está provavelmente associada aos eventos característicos de indução de resistência observados.

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-16T13:41:30Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-16T20:21:01Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-16T20:21:17Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) / Made available in DSpace on 2018-05-16T20:21:17Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Aline_de_Oliveira_Rodrigues.pdf: 787004 bytes, checksum: 42043083bf6f4a2ee9080a2164a23e88 (MD5) Previous issue date: 2015-04-30 / Sem bolsa / O setor hoteleiro vem se expandindo mundialmente a cada ano e dentre os serviços oferecidos, a gastronomia é um dos mais requisitados, através da qual busca-se a satisfação do cliente, oferecendo produtos seguros e de qualidade. Paralelamente ao incremento do turismo, a incidência de doenças de origem alimentar está crescendo em todo mundo. Acredita-se que o consumo de alimentos contaminados seja a principal causa de doenças em turistas. Neste contexto o objetivo deste estudo foi avaliar a aplicação das boas práticas em restaurantes hoteleiros da cidade de Pelotas/RS e Rio Grande/RS e relacioná-las com a qualidade microbiológica dos alimentos oferecidos, além de identificar fontes de contaminação. O setor de alimentos e bebidas (A&B) de quatro hotéis foi avaliado quanto às condições higiênicossanitárias em duas visitas, com intervalo mínimo de 30 dias, através de aplicação de check list fundamentado na norma da RDC 216/2004, e de análises microbiológicas de amostras coletadas no local. Foram realizadas coletas de amostras de superfícies de utensílios, equipamentos e mãos de manipuladores para contagem de Staphylococcus coagulase positiva e de coliformes termotolerantes. Isolados obtidos das culturas dessas contagens foram comparados através de perfis de genéticos obtidos por rep-PCR. Considerando as não conformidades observadas no check list, na primeira visita apenas um hotel foi classificado como ótimo, dois foram classificados como bons e um recebeu a classificação ruim. Entretanto, na segunda visita o hotel classificado como ruim passou para bom, devido à realização de melhorias no restaurante. Alguns resultados das análises microbiológicas apresentaram limite acima do permitido para a contagem dos micro-organismos analisados, identificando falha na aplicação das boas práticas. Através da técnica de rep-PCR, foi identificada uma mesma cepa em amostras de queijo e mãos, manteiga e tábua de corte, queijo e tábua de corte e salada de frutas e mãos, evidenciando contaminação cruzada devido falhas no processo de higiene e manipulação dos alimentos. / The hotel industry has been expanding every year and among the services offered, the cuisine is one of the most requested, which seeks customer satisfaction by offering safe and quality products. Parallel to the tourism development, the incidence of foodborne diseases is growing worldwide. It is believed that the consumption of contaminated food is the major cause of disease in tourists. This study goal was to evaluate the application of best practices in hotel restaurants in the cities of Pelotas/RS and Rio Grande/RS and relate it to the microbiological quality of the food offered, besides to identify sources of contamination. The sector of food and beverages (F&B) of four hotels was evaluated in two visits with an interval of 30 days regarding hygiene-sanitary conditions, by application of check list and microbiological analyzes of samples collected on site. Surface, utensils, equipment and hands handlers samples have been collected for counting coagulase positive Staphylococcus and thermotolerant coliform. Isolates obtained from these counting cultures were compared by bands profiles obtained by rep-PCR. According to the non-conformity observed in the check list on the first visit, only one hotel was rated as great, two were classified as good and one received a bad rating. However, on the second visit, the one considered bad got a good rate due to improvements in its unit. Some microbiological analysis showed a limit above the allowed for the count of analyzed microorganisms, identifying failure in the hygiene-sanitary process. By rep-PCR, was identified the same strain in samples of cheese and hands, butter and cutting board, cutting board and cheese and fruit salad and hands, showing cross-contamination due to flaws in the process of care and handling of food.

Dans la mucoviscidose, le tableau clinique est dominé par les infections respiratoires qui constituent la cause majeure de morbidité. Diverses espèces fongiques sont décrites dans ces infections, notamment les Scedosporium et le complexe Rasamsonia argillacea pour lesquels les connaissances restent limitées. Dans ce travail, nous avons évalué l’intérêt de l’amplification des séquences répétitives de l’ADN pour la différenciation spécifique et infraspécifique de ces champignons. La rep-PCR a d’abord été appliquée à l’analyse d'isolats du genre Scedosporium. La rep-PCR a produit des résultats concordants avec ceux obtenus par séquençage du gène de la bêta-tubuline, amplification aléatoire de fragments d’ADN polymorphes et séquençage multi-loci. Elle permet également l’identification précise des espèces au sein du complexe R. argillacea. L’analyse des résultats obtenus sur 116 isolats a montré la capacité de ces champignons à coloniser de manière chronique les voies respiratoires, un même profil électrophorétique étant observé généralement chez un même patient. Enfin, nous avons produit de nouvelles données sur l’écologie des Scedosporium en étudiant différents biotopes au Maroc. Comme précédemment rapporté,ces champignons préfèrent les environnements impactés parles activités humaines. On les rencontre dans des sols de pH neutre, riches en matières organiques et pauvres en sels. Enfin, si S. apiospermum était l’espèce la plus abondante, S. boydii, S. aurantiacum et S. dehoogii ont été rencontrés avec des fréquences comparables. En conclusion, les données générées renforcent nos connaissances sur l’épidémiologie des infections causées par ces pathogènes émergents. / In cystic fibrosis (CF), respiratory infections are the leading cause of morbidity and mortality. Bacteria are the most common causative agents of these infections, but an increasing number of fungal species may also be found, including Scedosporium species and species of the Rasamsonia argillacea complex. In order to improve our knowledge on the epidemiology of these infections, here we evaluated the interest of PCR amplification of repetitive DNA sequences (rep-PCR) for species identification and strain delineation. rep-PCR was applied to the retrospective analysis of a panel of 63 isolates belonging to the Scedosporiumgenus. Results were consistent with those obtained by beta-tubulingene sequencing, random amplification of polymorphic DNA and Multilocus Sequence Typing. rep-PCR also permitted precise species identification in the R. argillacea complex. Analysis of the data obtained on 116 isolates revealed the capacity of these fungi to chronically colonize the airways of patients with CF, a unique electrophoretic profile being observed usually for each patient. Finally, we produced in this thesis new data about the ecology of Scedosporium species by studying different biotopes in Morocco. As previously reported, these fungi are mainly found in human-impacted areas. Also they prefer soils with a neutral pH, a high organic matter content and a very low mineral salts content. In addition, if S. apiospermum was by far the predominant species, S. boydii, S. aurantiacum and S. dehoogii were encountered with similar frequencies. In conclusion, data provided here reinforce our knowledge on the epidemiology of the infections caused by these emerging pathogens.