Resumo: Sabe-se que uma variedade de microrganismos produzem um ou mais tipos de amilases para degradar os amiláceos presentes no ambiente e que as amilases produzidas por microrganismos termofílicos apresentam características mais termoestáveis do que aquelas produzidas por mesófilos. Essas amilases termoestáveis são de grande interesse na indústria de processamento de amido, uma vez a temperatura do gelatinização do mesmo, etapa importante do processo, fica em torno de 70ºC. Além disso, os processos que ocorrem em altas temperaturas, têm menor risco de contaminação por mesófilos e a diminuição da viscosidade do meio permite trabalhar com elevadas concentrações de substrato. No presente trabalho foram isoladas, a partir de amostras de solos, compostagens e resíduos agro-industriais, 339 linhagens microbianas termofílicas (326 bacterianas e 13 fúngicas) capazes de crescer a 55ºC em meio contendo amido como única fonte de carbono. As 163 linhagens (162 bactérias e 1 fungo) que se destacaram quanto à produção de amilases e à velocidade de crescimento em meio sólido, foram selecionadas e submetidas às fermentações submersa (meio nutriente) e semi-sólida (farelo de trigo). As atividades enzimáticas foram determinadas pelo método dextrinizante (a- amilase), pela quantificação da glicose (glucoamilase) e de açúcares redutores liberados a partir da hidrólise do amido. Das linhagens testadas, nove mostraram-se boas produtoras de a-amilase em fermentação submersa e/ou semi-sólida, fato pelo qual foram selecionadas para produção de amilases em meios formulados a partir de resíduos líquidos da extração de mandioca e milho (fermentação submersa) e sólidos, constituídos de farelos de mandioca, milho e trigo (fermentação semi-sólida). Quatro linhagens se destacaram, sendo suas enzimas brutas caracterizadas quanto às propriedades físico-químicas...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: It is known that some microorganisms produce one or more amylase types that degrade organic compound present in the environment. The amylases produced by thermophilic microorganisms present characteristics more thermostable than those produced by mesophilics. Since starch gelatinization temperature is around 70ºC, the thermostable amylases are very important to starch processing industry. Besides, the processes that happen in high temperatures have less risk of contamination for the mesophilic microorganisms, the decrease of the viscosity of the media and allows to work with high substrate concentrations. In the present work thermophilic microorganisms (326 bacteria and 13 fungi) were isolated from soil samples, decaying agricultural waste and agriculture-industrial residues. They are able to grow at 55ºC in media containing starch as only carbon source. 163 strains (162 bacteria and 1 fungus) that showed high amylase production and fast growth in solid media were selected and submitted to the submerged (nutrient media) and semi-solid (wheat bran) fermentations. The enzymatic activities were determined by dextrinogenic method (a-amylase), by the quantification of the glucose (glucoamylase) and of the reducing sugars released from the starch hydrolysis. Among the tested strains, nine have shown high a-amylase production in submerged and/or semi-solid fermentations and because of this they were selected for amylase production in medium formulated with liquid wastes of extraction starch from the cassava and corn (submerged fermentation), and solid wastes constituted of cassava, corn, and wheat brans (semi-solid fermentation). Among these strains, four were selected and their crude enzymes were characterized regarding to their physicochemical properties. The strains Bacillus sp A13-22 and Mucor sp A13-36 produced amylases with optimum temperature of 70 and 80 ºC...(Complete abstract click electronic access below) / Orientador: Eleni Gomes / Coorientador: Célia M. L. Franco / Banca: Cláudio Cabello / Banca: Gustavo Orlando Bonilla Rodriguez / Mestre

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-09Bitstream added on 2014-06-13T20:35:51Z : No. of bitstreams: 1 moretti_mms_me_rcla.pdf: 1537204 bytes, checksum: ee5403abcf2b166e590ebe82f51303d0 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Dos 27 microrganismos recém isolados, A. fumigatus M.7.1 e Myceliophthora sp M.7.7 foram os melhores produtores de FPase (0,8 e 2,0 U/g de substrato) e xilanase (1040 e 1292 U/g de substrato), quando cultivados por fermentação em estado sólido (FES) em mistura de bagaço de cana e farelo de trigo (1:1). As produções máximas de xilanase (7237,9 U/g de substrato) e endoglucanase (46,2 U/g de substrato) por A. fumigatus M.7.1 foram observadas pelo cultivo do fungo em palha de milho e farelo de trigo (9:1 p/p) e bagaço de cana e farelo de trigo (9:1 p/p), respectivamente. Em relação ao isolado Myceliophthora sp M.7.7, os picos de produção de ambas as enzimas (xilanase: 1044,6 U/g de substrato; endoglucanase 53,7 U/g de substrato) foram obtidos quando este foi cultivado em mistura de bagaço de cana e farelo de trigo (9:1 p/p). A endoglucanase e xilanase produzida por A. fumigatus M.7.1 apresentaram atividade ótima em pH 4,5, a 70 e 60 ºC, respectivamente. Nos ensaios com a linhagem Myceliophthora sp M.7.7, ambas as enzimas apresentaram maior atividade em pH 5,0 a 65-70 ºC. Ambas as enzimas de Myceliophthora sp M.7.7 e a endoglucanase de A. fumigatus M.7.1 mantiveram aproximadamente 70-100% da atividade inicial na faixa de pH entre 3,5 a 9,0 e nas temperaturas de 35 a 65 ºC. A xilanase de A. fumigatus M.7.1 manteve-se estável em pH entre 5,5 e 10,5 e 40 e 50 ºC, respectivamente. Dos tratamentos ao qual o bagaço foi submetido, o que mostrou maior eficiência na liberação de açúcares redutores (0,09%) e compostos fenólicos (0,74%), foi a solução de glicerol em microondas por 5 min. O bagaço de cana pré tratado com glicerol foi incubado com o preparado enzimático de A. fumigatus M.7.1 a 55 ºC. Após 24 h de incubação, foram liberados 0,7 mg/mL de açúcar redutor. As mesmas condições foram utilizadas na sacarificação do bagaço utilizando o extrato enzimático... / The microorganisms A. fumigatus M.7.1 and Myceliophthora SP M.7.7 were the best producers of FPase (0,8 and 2,0 U/g of substrate) and xylanase (1040 and 1292 U/g of substrate) of the 27 isolated microorganisms, when cultivated by solid state fermentation (SSF) in a mixture of cane bagasse and wheat bran (1:1). The greatest production of xylanase (7237,9 U/g of substrate) and endoglucanase (46,2 U/g of substrate) by A. fumigatus M.7.1 were noted by the fungi cultivation in corn straw and wheat bran (9:1 p/p) and cane bagasse and wheat bran (9:1 p/p), respectively. In the isolated Myceliophthora sp M.7.7, the peak production of the both enzymes (xylanase: 1044,6 U/g of substrate; endoglucanase 53,7 U/g of substrate) were obtained when the microorganism were cultivated in a mixture of cane bagasse and wheat bran (9:1 p/p). The endoglucanase and the xylanase produced by A. fumigatus M.7.1 showed higher activity in pH 4,5, at 70 and 60 oC, respectively. In the essays with the Myceliophthora sp M.7.7 strains, the both enzymes showed higher activity in pH 5,0, at 65-70 oC. The both enzymes of Myceliophthora sp M.7.7 and the endoglucanase of A. fumigatus M.7.1 maintained approximately 70-100% of the initial activity between pH 3,5 and 9,0 and between 35 and 65 oC. The xylanase of A. fumigatus M.7.1 was stable between the pH 5,5 and 10,5 and the temperature 40 and 50 oC. About the treatments of the bagasse, the most efficient in the liberation of reducing sugars (0,09%) and phenolic compounds (0,74%) was the glycerol solution in microwave for 5 minutes. The cane bagasse pretreated with glycerol was incubated with the enzymatic solution of A. fumigatus M.7.1 at 55 oC. After 24 hours of incubation, 0,7mg/mL of reducing sugar was liberated. The same conditions were used in the saccharification of the bagasse using the enzymatic extract produced by Myceliophthora sp. M.7.7, with a reducing sugar... (Complete abstract click electronic access below)

Submitted by TÁSSIO BRITO DE OLIVEIRA null (tassio88@hotmail.com) on 2016-12-14T01:49:17Z No. of bitstreams: 1 Oliveira 2016 Tese.pdf: 5009678 bytes, checksum: 584b0994b57a0b3ea482753790174b74 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-20T13:43:51Z (GMT) No. of bitstreams: 1 oliveira_tb_dr_rcla.pdf: 5009678 bytes, checksum: 584b0994b57a0b3ea482753790174b74 (MD5) / Made available in DSpace on 2016-12-20T13:43:51Z (GMT). No. of bitstreams: 1 oliveira_tb_dr_rcla.pdf: 5009678 bytes, checksum: 584b0994b57a0b3ea482753790174b74 (MD5) Previous issue date: 2016-11-18 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A torta de filtro é gerada após o processo de filtração do caldo de cana (cerca de 26 a 40 kg por t de cana) e geralmente é utilizada como fertilizante nas lavouras sem qualquer tratamento prévio. A diversidade de fungos presentes na torta de filtro fresca e no processo de compostagem desse substrato foi acessada utilizando sequenciamento em larga escala. Além disso, fungos tolerantes ao calor foram isolados e avaliados quanto à capacidade de produzir enzimas de degradação da biomassa (celulase, xilanase, lacase e poligalacturonase). Considerando que esse grupo de fungos carece de uma revisão taxonômica atual, aproveitamos as recentes mudanças proporcionadas pelo Código de Nomenclatura para Algas, Fungos e Plantas para gerar uma revisão taxonômica do grupo. Uma gama de patógenos oportunistas foi encontrada entre os taxa mais abundantes na torta de filtro fresca, como Lomentospora prolificans (43,13%), Trichosporon sp. (10,07%), Candida tropicalis (7,91%) e Hormographiella aspergillata (8,19%). Isso indica que a torta de filtro pode ser uma potencial fonte de fungos patogênicos, apresentando riscos para a saúde humana se aplicado como fertilizante sem qualquer tratamento. No entanto, o processo de compostagem reduz efetivamente a carga desses fungos. Além disso, cria um ambiente interessante para fungos capazes de produzir enzimas com potencial aplicação biotecnológica, uma vez que todos os 110 isolados avaliados foram capazes de produzir, pelo menos, uma das enzimas avaliadas. Além disso, a análise comparativa de genes codificantes para peptidases presentes nos genomas de fungos termofílicos (encontrados em sistemas de compostagem) e mesofílicos mostrou que a termofilia levou à várias adaptações para a termoestablidade enzimática. / Pressmud is derived from sugarcane juice filtrate (around 26 to 40 kg per ton of sugarcane) and it is mainly used as fertilizer in crops without prior treatment. Here, the fungal diversity present in both fresh and composting pressmud was revealed by 454 pyrosequencing. In addition, heat-tolerant fungi were isolated and surveyed for their repertoire of biomass-degrading enzymes (cellulase, xylanase, laccase and polygalacturonase). The fact that the taxonomy of such organisms is still obscure, we revised their taxonomy in the light of the recent changes adopted in the Code of Nomenclature for Algae, Fungi and Plants. A wide range of opportunistic pathogens was found among the most abundant taxa in fresh pressmud, such as Lomentospora prolificans (43.13%), Trichosporon sp. (10.07%), Candida tropicalis (7.91%), and Hormographiella aspergillata (8.19%). This indicates that fresh pressmud may be a source of human pathogenic fungi, presenting a potential threat to human health if applied as fertilizer without treatment. Composting of the pressmud effectively reduces the load of such fungi. Furthermore, the composting system creates an interesting environment for fungi able to produce enzymes with biotechnological applications, since all the 110 isolates screened were able to produce at least one of the tested enzymes.Furthermore, comparative analysis of peptidases genes encoded by thermophilic (generally found in composting systems) and mesophilic fungi showed that thermophyly selected for thermostable enzymes. / FAPESP: 2012/14594-7 / FAPESP: 2015/25252-8

Resumo: Dos 27 microrganismos recém isolados, A. fumigatus M.7.1 e Myceliophthora sp M.7.7 foram os melhores produtores de FPase (0,8 e 2,0 U/g de substrato) e xilanase (1040 e 1292 U/g de substrato), quando cultivados por fermentação em estado sólido (FES) em mistura de bagaço de cana e farelo de trigo (1:1). As produções máximas de xilanase (7237,9 U/g de substrato) e endoglucanase (46,2 U/g de substrato) por A. fumigatus M.7.1 foram observadas pelo cultivo do fungo em palha de milho e farelo de trigo (9:1 p/p) e bagaço de cana e farelo de trigo (9:1 p/p), respectivamente. Em relação ao isolado Myceliophthora sp M.7.7, os picos de produção de ambas as enzimas (xilanase: 1044,6 U/g de substrato; endoglucanase 53,7 U/g de substrato) foram obtidos quando este foi cultivado em mistura de bagaço de cana e farelo de trigo (9:1 p/p). A endoglucanase e xilanase produzida por A. fumigatus M.7.1 apresentaram atividade ótima em pH 4,5, a 70 e 60 ºC, respectivamente. Nos ensaios com a linhagem Myceliophthora sp M.7.7, ambas as enzimas apresentaram maior atividade em pH 5,0 a 65-70 ºC. Ambas as enzimas de Myceliophthora sp M.7.7 e a endoglucanase de A. fumigatus M.7.1 mantiveram aproximadamente 70-100% da atividade inicial na faixa de pH entre 3,5 a 9,0 e nas temperaturas de 35 a 65 ºC. A xilanase de A. fumigatus M.7.1 manteve-se estável em pH entre 5,5 e 10,5 e 40 e 50 ºC, respectivamente. Dos tratamentos ao qual o bagaço foi submetido, o que mostrou maior eficiência na liberação de açúcares redutores (0,09%) e compostos fenólicos (0,74%), foi a solução de glicerol em microondas por 5 min. O bagaço de cana pré tratado com glicerol foi incubado com o preparado enzimático de A. fumigatus M.7.1 a 55 ºC. Após 24 h de incubação, foram liberados 0,7 mg/mL de açúcar redutor. As mesmas condições foram utilizadas na sacarificação do bagaço utilizando o extrato enzimático... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The microorganisms A. fumigatus M.7.1 and Myceliophthora SP M.7.7 were the best producers of FPase (0,8 and 2,0 U/g of substrate) and xylanase (1040 and 1292 U/g of substrate) of the 27 isolated microorganisms, when cultivated by solid state fermentation (SSF) in a mixture of cane bagasse and wheat bran (1:1). The greatest production of xylanase (7237,9 U/g of substrate) and endoglucanase (46,2 U/g of substrate) by A. fumigatus M.7.1 were noted by the fungi cultivation in corn straw and wheat bran (9:1 p/p) and cane bagasse and wheat bran (9:1 p/p), respectively. In the isolated Myceliophthora sp M.7.7, the peak production of the both enzymes (xylanase: 1044,6 U/g of substrate; endoglucanase 53,7 U/g of substrate) were obtained when the microorganism were cultivated in a mixture of cane bagasse and wheat bran (9:1 p/p). The endoglucanase and the xylanase produced by A. fumigatus M.7.1 showed higher activity in pH 4,5, at 70 and 60 oC, respectively. In the essays with the Myceliophthora sp M.7.7 strains, the both enzymes showed higher activity in pH 5,0, at 65-70 oC. The both enzymes of Myceliophthora sp M.7.7 and the endoglucanase of A. fumigatus M.7.1 maintained approximately 70-100% of the initial activity between pH 3,5 and 9,0 and between 35 and 65 oC. The xylanase of A. fumigatus M.7.1 was stable between the pH 5,5 and 10,5 and the temperature 40 and 50 oC. About the treatments of the bagasse, the most efficient in the liberation of reducing sugars (0,09%) and phenolic compounds (0,74%) was the glycerol solution in microwave for 5 minutes. The cane bagasse pretreated with glycerol was incubated with the enzymatic solution of A. fumigatus M.7.1 at 55 oC. After 24 hours of incubation, 0,7mg/mL of reducing sugar was liberated. The same conditions were used in the saccharification of the bagasse using the enzymatic extract produced by Myceliophthora sp. M.7.7, with a reducing sugar... (Complete abstract click electronic access below) / Orientador: Eleni Gomes / Coorientador: Daniela A. Bocchini-Martins / Banca: Beatriz Vahan Kilikian / Banca: Mauricio Boscolo / Mestre

Um sistema combinado anaeróbio-aeróbio composto de um filtro anaeróbio ascendente seguido de reator aeróbio de lodos ativados em batelada, foi continuamente alimentado com água residuária sintética da indústria de polpa celulósica não branqueada, durante o período de trezentos e sessenta e três dias, com o objetivo de verificar o desempenho dos reatores operados sob condições termofílicas. Os reatores foram inoculados com lodo mesofílico com gradual adaptação para a condição termofílica e operados em duas fases distintas. A primeira e segunda fases contemplam os períodos em que os reatores foram operados em temperatura termofílica de 55 e 65 graus Celsius, respectivamente. O filtro anaeróbio trabalhou com tempo de detenção hidráulica de 48 horas em fluxo contínuo e o reator aeróbio operado em bateladas seqüenciais (RBS) com tempos de ciclo de 12, 18 e 24 horas. A melhor eficiênica de remoção de DQO do sistema combinado anaeróbio-aeróbio foi de 65%, devida, em maior parte ao reator anaeróbio. O filtro anaeróbio operado em temperatura de 55 e 65 graus demonstrou eficiência na capacidade de retenção de biomassa termofílica, evitando a perda no efluente. O RBS, quando operado em temperatura de 55 graus e tempo de ciclo de 18 horas, demonstrou melhor eficiência para pós-tratamento de água residuária sintética. Os resultados gerais mostram uma melhor eficiência dos reatores operados em temperatura de 55 graus Celsius. / A combined anaerobic-aerobic system, composed of an upflow anaerobic filter followed by an aerobic reactor of activated sludge in batch, was continuously feed with synthetic wastewater from unbleached cellulose pulp plant during a period of three hundred and sixty three days, aiming to verify the performance of the reactors operating under thermophilic conditions. The reactors were inoculated with mesophilic sludge having a gradual adaptation to the thermophilic condition and they were operated in two distinct phases. The first and second phases contemplate the periods in which the reactors were operated in a thermophilic temperature of 55 and 65 Celsius degrees, respectively. The anaerobic filter worked with hydraulic detention time of 48 hours in a continuous flow, and the aerobic reactor was operated in a sequencing batch (SBR) with a cycle time of 12, 18 and 24 hours. The best efficiency of the combined anaerobic-aerobic system to remove DQO was 65% due to, in most part, the anaerobic reactor. The anaerobic filter, operating in temperatures of 55 and 65 degrees, has demonstrated efficiency in the capacity to retain thermophilic biomass, avoiding effluent lost. The SBR reactor, when operated in temperature of 55 degrees and a cycle time of 18 hours, has demonstrated better efficiency in the post-treatment of synthetic wastewater. The general results have showed a better efficiency of the reactors operating in a thermophilic temperature of 55 Celsius degrees.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-10-10T15:52:13Z No. of bitstreams: 2 Dissertação - Julierme José de Oliveira - 2014.pdf: 1186561 bytes, checksum: ee682becf7a3d3796f34ba3239320262 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-11T10:53:29Z (GMT) No. of bitstreams: 2 Dissertação - Julierme José de Oliveira - 2014.pdf: 1186561 bytes, checksum: ee682becf7a3d3796f34ba3239320262 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-10-11T10:53:29Z (GMT). No. of bitstreams: 2 Dissertação - Julierme José de Oliveira - 2014.pdf: 1186561 bytes, checksum: ee682becf7a3d3796f34ba3239320262 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Thermophilic species of Campylobacter spp. are often cited as the most common etiologic agent in cases of foodborne illnesses in several countries. When ingested even in small quantities (400 to 500 cells) this organism, through the mechanisms of pathogenicity, the host develops infectious gastroenteritis known as campylobacteriosis. This disease is closely related to the intake and improper handling of contaminated food, especially poultry, and birds the main reservoir of the microorganism. Poor knowledge climbs the role of eggs in the transmission of campylobacteriosis, based on the annual consumption of the food matrix, research clarifying all variables involving this food are essential. This study aimed to verify the occurrence of Campylobacter spp., identify thermophilic species involved and assess the level of antimicrobial susceptibility of isolates from chicken carcasses and eggs obtained from commercial establishments in the city of Goiânia. A total of 100 chicken carcasses and 100 dozens of eggs were evaluated using enzyme- linked immunosorbent assay for screening miniVIDAS ® with subsequent bacterial isolation and identification of samples by biotyping . By means of the methods employed observed that Campylobacter spp. was identified in 7% (7 /100) of chicken carcasses in 1% (1/100 ) of samples of eggs, 100 % of these were confirmed as belonging to the bacterial genus . The species with the highest number of isolates were Campylobacter jejuni (50 %), followed by Campylobacter coli ( 25 % ) and Campylobacter lari ( 25 % ). For antimicrobial resistance results were that 100% of the isolates were resistant to erythromycin, as well as ciprofloxacin and tetracyclines. For ceftriaxone and nalidixic acid resistance values were 91.7 % resistant. Chloramphenicol was 50% of resistant isolates, showed 33.3% sensitivity and 16.7% intermediate resistance. It was also observed that 100% of the isolates showed multidrug profile. Through the results obtained it is concluded that thermophilic Campylobacter are present in chicken carcasses and marketed in Goiânia eggs, whose distribution becomes relevant from the point of view of exposure to consumers. Campylobacter jejuni was the most frequent species. The six districts surveyed had bacteria of the genus in at least one category. Isolates when screened for response to antimicrobial proved multiresistant, denoting the importance of research as a way to support the election of bases and principles for the antibiotic treatment prescribed treatments. Poultry foods surveyed in this study and considered positive for the pathogen constitute risk to the consumer. / As espécies termotolerantes de Campylobacter spp. são frequentemente apontadas como o agente etiológico mais comum nos casos de doenças veiculadas por alimentos em diversos países. Ao ser ingerido mesmo em pequenas quantidades (de 400 a 500 células), esse microrganismo, por meio dos mecanismos de patogenicidade, desenvolve gastroenterite infecciosa no hospedeiro, conhecida como campilobacteriose. Esta doença está intimamente relacionada à ingestão e manipulação incorreta de alimentos contaminados, especialmente carne de frango, sendo as aves o principal reservatório. Pouco se sabe sobre o papel desempenhado pelos ovos na transmissão de campilobacteriose, tendo como base o consumo anual dessa matriz alimentar, pesquisas que esclareçam todas as variáveis envolvendo esse alimento são de fundamental importância. Pelo exposto, este estudo teve como objetivo verificar a ocorrência de Campylobacter spp., identificar as espécies termotolerantes circulantes na cidade de Goiânia, vinculadas aos estabelecimentos comercializadores de alimentos, relacionados à alta densidade populacional, localizados em distritos comerciais e avaliar o nível de suscetibilidade a antimicrobianos dos isolados de carcaças de frango e ovos provenientes destes estabelecimentos. Um total de 100 carcaças de frango e 100 dúzias de ovos foram avaliados utilizando como triagem o ensaio imunoenzimatico miniVIDAS® com posterior isolamento bacteriano das amostras e identificação por meio da biotipificação. Por meio dos métodos empregados observou-se que Campylobacter termotolerantes foi identificada em 7% (7/100) das carcaças de frango e em 1% (1/100) das amostras de ovos. A espécie com maior número de isolados foi Campylobacter jejuni (50%), seguidos por Campylobacter coli (25%) e Campylobacter lari (25%). Para a resistência à antimicrobianos os resultados reportaram que 100% dos isolados são resistentes à eritromicina, assim como à ciprofloxacina e tetraciclina. Para ácido nalidixico e ceftriaxona, os valores de resistência foram da ordem de 91,7%. Cloranfenicol foi resistente em 50% dos isolados, apresentou sensibilidade a 33,3% e resistência intermediária a 16,7%. Observou-se ainda que 100% dos isolados apresentaram perfil de multirresistência. Pelos resultados obtidos conclui-se que Campylobacter termotolerantes estão presentes em carcaças de frango e ovos comercializados na cidade de Goiânia, cuja distribuição torna-se relevante do ponto de vista da exposição aos consumidores. Campylobacter jejuni foi a espécie mais frequente. Os seis distritos pesquisados apresentaram bactérias do gênero pelo menos em um estabelecimento. Os isolados quando pesquisados quanto a resposta a antimicrobianos mostraram-se multirresistentes, denotando a importância desta investigação como forma de respaldar a eleição de bases e princípios para a antibioticoterapia para tratamentos prescritos. Os alimentos avícolas pesquisados neste estudo e considerados positivos para o patógeno constituem risco ao consumidor.

Les amibes sont des organismes unicellulaires eucaryotes, de 10 à 300 µm, pouvant vivre sous forme parasite ou évoluer librement dans les sols et les milieux aquatiques ou encore adopter l’une ou l’autre forme en fonction de l’environnement. Certaines amibes libres sont hautement pathogènes pour les animaux et l’homme, en particulier Naegleria fowleri qui se développe dans les eaux naturellement chaudes à des températures comprises entre 27 et 45°C. Cette espèce est responsable d'une encéphalite généralement mortelle, la méningo-encéphalite amibienne primitive (MEAP), dont un cas a été recensé en Guadeloupe en 2008. Un enfant de 9 ans ayant contracté la maladie est décédé quelques jours après un bain dans les eaux chaudes de Dolé, sur la commune de Gourbeyre. Notre travail de Thèse à l’Institut Pasteur, en collaboration avec l’ARS, a permis d’initier un programme de recherche sur ces amibes pathogènes très peu connues sur notre territoire, afin de mieux évaluer le risque lié aux baignades dans les sources géothermales de la Guadeloupe. Nous avons développé et mis au point une méthode de détection et de dénombrement des amibes faisant appel à la biologie moléculaire qui est aujourd’hui appliquée en routine au sein de notre laboratoire. Depuis Janvier 2011, un suivi mensuel a révélé la présence des amibes thermophiles et de l’amibe pathogène dans presque tous les sites analysés (Ravine Chaude, Bains de La Lise et Bain du Curé à Pigeon, Bains Jaunes, Bain de Dolé, Bain de Capes, Bain des Amours, Bain de la rivière Grosse-Corde, Chute du Carbet, Bain de Morphy et Bain chaud de Matouba), à l’exception des eaux chaudes soufrées et/ou salées de Sofaïa et de l’Anse Thomas à Bouillante. Le séquençage des produits de PCR obtenus et déposés dans Genbank nous a permis de confirmer l’appartenance de cette souche de N. fowleri, au type 3 Euro-américain, identique à celui détecté lors de l’accident de 2008 (Moussa et al. 2013). Le nombre d’amibes pathogènes retrouvées varie de 2 à 30 amibes/litre selon les bains, sans dépasser la limite de 100 amibes/litre recommandée par les autorités sanitaires. En dépit de ces faibles concentrations, une surveillance régulière des bassins est nécessaire. Sur l’ensemble de l’année 2011-2012, l’espèce pathogène N. fowleri a été la plus fréquemment rencontrée par rapport aux espèces non pathogènes N. lovaniensis et Hartmanella sp. Ces données de surveillance ont amené l’ARS et les communes concernées à organiser une campagne de sensibilisation à destination des populations locales et touristiques, reposant sur l’installation de panneaux de prévention à proximité des principaux sites où N. fowleri a été retrouvée.La présence régulière de N. fowleri tout au long de l’année dans la plupart des bains chauds et notamment dans les bains les plus fréquentés, a orienté notre travail sur la recherche de l’origine de leur contamination. Nous avons découvert que les sources géothermales ne sont pas contaminées à leur émergence, mais que les amibes proviennent de la terre en amont des bassins. Ce résultat qui montre que le sol est le réservoir naturel des amibes, nous a permis de proposer aux collectivités un moyen de lutte efficace en canalisant l’eau depuis l’émergence jusqu’au bassin, sans passer par le sol. / Amoebas are eukaryotic unicellular organisms, measuring from 10 to 300 µm, being able to live under a parasite form or to evolve freely in soils and aquatic media. They can even adopt either form depending of the environment. Some free living amoebas are highly pathogenic for animals and humans, especially Naegleria fowleri which develops in naturally hot waters at temperatures between 27 and 45°C. This species is responsible for an encephalitis generally lethal, the primary amoebic meningoencephalitis (PAM), a case of which occured in Guadeloupe in 2008. A 9-year-old child having contracted the disease, died a few days after a bath in the hot waters of Dolé, in Gourbeyre. Our Thesis at the Institut Pasteur of Guadeloupe, in association with the ARS, initiated a research program on these pathogenic amoebas poorly known in our territory, to better estimate the risk to contract the disease while bathing in geothermal recreational waters of Guadeloupe. We developed and worked out a method of detection and enumeration of amoebas using the molecular biology, which is currently applied in routine in our laboratory. Since January, 2011, a monthly monitoring of the baths revealed the presence of thermophilic free living amoebas and pathogenic amoeba in almost all the analyzed sites (Ravine Chaude, La Lise, Bain du Curé à Pigeon, Bains Jaunes, Bain de Dolé, Bain de Capès, Bain des Amours, Bain de la rivière Grosse-Corde, Chutes du Carbet, Morphy and the hot bath of Matouba), with the exception of sulphurated and/or salty hot waters of Sofaïa and the Anse-Thomas in Bouillante. The sequencing of the PCR products obtained were deposited in Genbank and confirmed that the Guadeloupean N. fowleri belonged to the Type-3 Euro-American, identical to that detected in the patient in 2008 (Moussa et al. 2013). The number of pathogenic amoebas varied from 2 to 30 amoebas / liter according to the baths, without exceeding the limit of 100 amoebas / liter recommended by the health authorities. In spite of this rather low concentration the baths require a regular surveillance. On the full year 2011-2012, the pathogenic species N. fowleri was the most frequently encountered species followed by N. lovaniensis and Hartmanella sp. These data of the surveillance led the ARS and the municipalities concerned to organize an awareness campaign destinated to the local and tourist population, based on the installation of prevention panels near the main sites where N. fowleri was found. The regular presence of N. fowleri during the year in most of the hot baths, especially the most frequented ones, leads us to look for the origin of their contamination. We discovered that geothermal springs are not contaminated at their emergence, but that amoebas come from the soil upstream the baths. This result which shows that soil is the natural reservoir of amoebas, allowed us to propose to the authorities effective means of prevention by installing pipes to carry the water from the emergence to the bath, without touching the soil.

This diploma thesis deals with selected thermophilic representatives of genera Geobacillus, Saccharococcus and Bacillus, taking screening of its biotechnological potential into account. Bacteria from the first two genera came from Czech and German collection of microorganisms, while bacteria of genus Bacillus were natural isolates. Researched strains were examined from a viewpoint of carbon source utilization and furthermore, production of biosurfactants, extracellular hydrolytic enzymes (protease, amylase, lipase, cellulase, xylanase), organic acids, antimicrobial agents and microbial plastics – polyhydroxyalkanoates was also tested. Bacteria S. thermophilus, G. uzenensis and G. zalihae evinced a substantial ability of biosurfactant production. Strains G. jurassicus, G. uzenensis, G. gargensis and G. lituanicus were capable of intensive production of all tested, technologically significant enzymes. Highest antimicrobial effects were reached with bacteria G. stearothermophilus and G. thermocatenulatus. Largest production of acetic acid was achieved with G. jurassicus and lactic acid with G. thermodenitrificans. Ability to produce polyhydroxyalkanoates was proved at genotype level by some cultures only, however at fenotype level, response was negative. On the contrary, bacteria genus Bacillus were able to produce polyhydroxyalkanoates, although in small amounts under given circumstances. With remaining researched metabolites, production ability was considerably lower, compared to genera Geobacillus and Saccharococcus.

Viruses are the most abundant organisms on Earth, yet their collective evolutionary history, biodiversity and functional capacity is not well understood. Viral metagenomics offers a potential means of establishing a more comprehensive view of virus diversity and evolution, as vast amounts of new sequence data becomes available for comparative analysis.Metagenomic DNA from virus-sized particles (smaller than 0.2 microns in diameter) was isolated from approximately 20 liters of sediment obtained from Boiling Springs Lake (BSL) and sequenced. BSL is a large, acidic hot-spring (with a pH of 2.2, and temperatures ranging from 50°C to 96°C) located in Lassen Volcanic National Park, USA. BSL supports a purely microbial ecosystem comprised largely of Archaea and Bacteria, however, the lower temperature regions permit the growth of acid- and thermo-tolerant Eukarya. This distinctive feature of the BSL microbial ecosystem ensures that virus types infecting all domains of life will be present. The metagenomic sequence data was used to characterize the types of viruses present within the microbial ecosystem, to ascertain the extent of genetic diversity and novelty comprising the BSL virus assemblage, and to explore the genomic and structural modalities of virus evolution.Metagenomic surveys of natural virus assemblages, including the survey of BSL, have revealed that the diversity within the virosphere far exceeds what has currently been determined through the detailed study of viruses that are relevant to human health and agriculture. The number of as-yet-uncharacterized virus protein families present in the BSL assemblage was estimated by clustering analysis. Genomic context analysis of the predicted viral protein sequences in the BSL dataset indicates that most of the putative uncharacterized proteins are endemic or unique to BSL, and are largely harbored by known virus types. A comparative metagenomic analysis approach identified a set of conserved, yet uncharacterized BSL protein sequences that are commonly found in other similar and dissimilar environments.New sequence data from metagenomic surveys of natural virus assemblages was also used to better characterize and define known virus protein families, as some of the viruses found in the BSL environment represent distant relatives of well-characterized isolates. By comparing viral genes and protein sequences from these highly divergent species, it is possible to better understand the dynamics of adaptation and evolution in the virosphere. Additionally, as structures of virus proteins continue to be experimentally determined by X-ray crystallography and cryo-electron microscopy, a merger of structural and metagenomic sequence data allows the opportunity to observe the structural dynamics underlying virus protein evolution.Capsid (structural) proteins from two distinct Microviridae strains; a globally ubiquitous and highly sequence-diverse virus family, were identified in, and isolated from the BSL metagenomic DNA sample. These BSL capsid protein sequences, along with several other homologous sequences derived from metagenomic surveys and laboratory isolates, were mapped to the solved structure of a closely related capsid protein from the Spiroplasma phage-4 microvirus. Patterns of amino acid sequence conservation, unveiled by structure-based homology modeling analysis, revealed that the protein sequences within this family exhibit a remarkable level of plasticity, while remaining structurally and functionally congruent.Lateral gene transfer is thought to have had a significant impact on the genomic evolution and adaptation of virus families. Genomic context analysis was also utilized to identify interviral gene transfer within the BSL virus assemblage. An ostensibly rare interviral gene transfer event, having transpired between single-stranded RNA and DNA virus types, was detected in the BSL metagenome. Similar genomes were subsequently detected in other ecosystems around the globe. The discovery of this new virus genome dramatically underscores the scope and importance of genetic mobility and genomic mosaicism as major forces driving the evolution of viruses.The analyses conducted herein demonstrate the many ways in which viral metagenomic sequence data may be utilized to not only evaluate the composition of a natural virus assemblage, but to discover new viral genes, and to better understand the dynamics of both genomic and structural evolution within the virosphere.

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