Malassezia pachydermatis is an opportunistic fungus associated to dermatomycoses and otopathies in domestic and wild animals. The aim of this study was to compare the susceptibility profile of clinical isolates of M. pachydermatis against topic and systemic antifungals, through two standardized CLSI techniques: broth microdilution (M27-A3, 2008) and disk diffusion (M44-A, 2004). A standard Candida albicans strain (ATCC 28367) was used as quality control. M. pachydermatis isolates assayed through the broth microdilution method showed susceptibility to anfotericina-B (100%), fluconazole (97,83%), ketoconazole (95.66%), itraconazole (93,48%), followed by clotrimazole and miconazole (86.96%). The disk diffusion method showed susceptibility of 97,83% to nystatin, 95,66% to itraconazole and to amphotericin B, 91,32% to ketoconazole, 89,14% to fluconazole and of 86.96% to miconazole and clotrimazole. Through the in vitro induction of resistance to fluconazole (100%), cross-resistance was observed through the broth microdilution method among the 30 M. pachydermatis isolates against the azoles itraconazole (93%), ketoconazole (97%) and voriconazole (100%). The study of the activities of the essential oils obtained through the broth microdilution, based on geometric means of the minimum fungicidal concentration, showed that concentrations above 195.42 μg/mL, 332.49 μg/mL and 448.8 μg/mL of oregano, Mexican oregano and cinnamon essential oils, respectively, have fungicidal action against M. pachydermatis, independently of the sensitivity or resistance to fluconazole. The fungicidal activity of the essential oils against fluconazole-resistant M. pachydermatis strains is important for further therapeutic studies of this mycosis. Despite the susceptibility observed among sensitive M. pachydermatis strains suggesting fluconazole as a safe drug for the treatment of most cases of superficial and invasive malasseziosis, it is essential continuous surveillance studies to detect changes in the microbiological profile due to therapeutic practices. / Malassezia pachydermatis é um fungo oportunista, associado à dermatomicoses e otopatias em animais domésticos e selvagens. O objetivo deste estudo foi comparar o perfil de susceptibilidade de isolados clínicos de M. pachydermatis frente antifúngicos tópicos e sistêmicos, através de duas técnicas padronizadas pelo CLSI: microdiluição em caldo (M27- A3, 2008) e disco-difusão (M44-A, 2004). Para todos os testes foi utilizada uma cepa padrão de Candida albicans (ATCC 28367) como controle de qualidade. Isolados de M. pachydermatis testados através do método de microdiluição em caldo apresentaram em ordem decrescente de susceptibilidade: anfotericina-B (100%), fluconazol (97,83%), cetoconazol (95,66%), itraconazol (93.48%), seguido de clotrimazol e miconazol (86,96%). Pelo método de disco-difusão 97,83% dos isolados foram suscetíveis a nistatina, 95,66% ao itraconazol e a anfotericina-B; 91,32% ao cetoconazol, 89,14% ao fluconazol; e 86,96% ao miconazol e clotrimazol. Através da indução de resistência in vitro, de 30 isolados de M. pachydermatis, ao fluconazol (100%) foi possível evidenciar resistência cruzada pelo método de microdiluição em caldo frente aos antifúngicos azólicos, itraconazol (93%), cetoconazol (97%) e voriconazol (100%). O estudo da atividade de óleos essenciais gerados pelo método de microdiluição em caldo, baseado nas médias geométricas das CFMs, evidenciou-se que concentrações acima de 195.42 μg/mL de óleo essencial de orégano, 332.49 μg/mL de óleo essencial de orégano mexicano e 448.8 μg/mL de óleo essencial de canela, possuem ação fungicida in vitro sobre M. pachydermatis, independente da sensibilidade ou resistência ao fluconazol. A atividade de óleos essenciais frente M. pachydermatis fluconazol resistente, é um achado que consolida a importância destes para estudos futuros voltados a terapêutica desta micose. Apesar dos dados de susceptibilidade apresentados pelas cepas sensíveis de M. pachydermatis sugerirem que o fluconazol ainda é uma droga segura para terapêutica da maioria dos casos de malasseziose superficial ou invasiva, é fundamental que estudos de vigilância epidemiológica sejam realizados continuamente para evidenciar mudanças no perfil microbiológico em função de práticas terapêuticas.

Made available in DSpace on 2014-07-29T15:30:40Z (GMT). No. of bitstreams: 1 Dissertacao CrystianeAraujo.pdf: 881195 bytes, checksum: da026b75ed78f3c38d78f44f6a15271e (MD5) Previous issue date: 2007-05-31 / Dermatophytes are keratinophilic fungi that colonize and invade the stratum corneum of the skin, hair and nails causing the dermatophytosis. An increasing number of antifungal agents has become available for the treatment of dermatophytosis, however not all species have the same susceptibility pattern and may occur relative or absolute resistance of some dermatophytes. The document M38-A developed by Clinical and Laboratory Standards Institute (CLSI) for determining the minimal inhibitory concentration (MIC) of different antifungal agents against filamentous fungi, has not included the dermatophytes. The broth microdilution method has been evaluated by various researchers, and some parameters as inoculum size, temperature and duration of incubation and endpoint determination has been investigated. In this study, the in vitro activity of fluconazole, itraconazole, ketoconazole, griseofulvin and terbinafine against 60 dermatophyte isolates, belong to three species, using the broth microdilution technique, with modifications at temperature and incubation time was used. Additionally, the MIC values obtained by broth microdilution method were compared with those obtained by the agar dilution technique. The results obtained by broth microdilution method showed that all isolates produced clearly detectable growth at 28oC and the MIC values could be determined after 4 days of incubation for the isolates of Trichophyton mentagrophytes and 5 days for T. rubrum and Microsporum canis isolates. Itraconazole, ketoconazole and terbinafine had the lowest MIC values (0.03 μg/ml) for 33.3%, 31.6% and 15% of the isolates, respectively. A good concordance was observed between the agar dilution and broth microdilution methods. The levels of agreement were 91.6% with ketoconazole and griseofulvin, 83.3% with itraconazole, 81.6% with terbinafine and 73.3% with fluconazole for all the tested isolates. In summary, the results of this study suggest that an incubation time of 5 days and temperature at 28oC used in broth microdilution and agar dilution methods can contribute to define and to better interpret the MIC values. Beside, until a reference method for testing the susceptibilities of dermatophytes is standardized, the similar results with broth microdilution method become the agar dilution useful for testing the susceptibility of these fungi. / Os dermatófitos são fungos filamentosos queratinofílicos capazes de colonizar e invadir o extrato córneo da pele, pêlo e unhas produzindo as dermatofitoses. Há vários agentes antifúngicos disponíveis para o tratamento das dermatofitoses, entretanto as espécies de dermatófitos possuem perfis de suscetibilidade diferentes, podendo ocorrer resistência relativa ou absoluta. O documento M38-A desenvolvido pelo Clinical and Laboratory Standards Institute (CLSI) para determinar a concentração inibitória mínima (CIM) de diferentes agentes antifúngicos para fungos filamentosos, não inclui os dermatófitos. O método de microdiluição em caldo tem sido avaliado por vários pesquisadores, e alguns parâmetros como tamanho do inóculo, temperatura e tempo de incubação e determinação das leituras de CIM são investigados. Neste trabalho, foi avaliada a atividade in vitro de fluconazol, itraconazol, cetoconazol, griseofulvina e terbinafina para 60 isolados de dermatófitos, pertencentes a três espécies, através da técnica de microdiluição em caldo, com modificações na temperatura e tempo de incubação. Adicionalmente, os valores de CIM obtidos pelo método de microdiluição em caldo foram comparados com aqueles obtidos pela técnica de diluição em ágar. Os resultados encontrados através do método de microdiluição em caldo mostraram que todos os isolados produziram crescimento claramente detectável a temperatura de 28oC e os valores de CIMs foram determinados após 4 dias de incubação para os isolados de Trichophyton mentagrophytes e 5 dias para os isolados de T. rubrum e Microsporum canis. Itraconazol, cetoconazol e terbinafina mostraram os menores valores de CIM (0,03 μg/ml) para 33,3%, 31,6% e 15% dos isolados, respectivamente. Uma boa concordância foi observada entre os métodos de diluição em ágar e microdiluição em caldo. A concordância entre os dois métodos foi de 91,6% para cetoconazol e griseofulvina, 83,3% para itraconazol, 81,6% para terbinafina e 73,3% para fluconazol para todos os isolados analisados. Em conclusão, os resultados deste estudo sugerem que uma incubação de 5 dias e temperatura de 28oC, utilizados no método de microdiluição em caldo e diluição em ágar, podem contribuir para definir e melhor interpretar os valores de CIM. Além disso, até que um método de referência de teste de suscetibilidade para dermatófitos seja padronizado, os resultados similares entre os métodos de microdiluição em caldo e diluição em ágar tornam este último útil para testar a suscetibilidade in vitro destes fungos.

Hudens normalflora består av lågpatogena bakterier där stafylokockerna är de vanligaste förekommande bakterierna. Studier visar att antimikrobiella medel kan förändra hudbakteriepopulationer och att dessa förändringar kan leda till kritiska konsekvenser för hudens försvar. Propylenglykol är en substans som klassas som antimikrobiell och bakteriedödande. Propylenglykol har ett brett användningsområde och används ofta som hjälpmedel i en mängd olika läkemedel. Den finns bland annat i kosmetika såsom hudprodukter där den fungerar som fuktbindande och som konserveringsmedel. Vanliga koncentrationer av propylenglykol som fuktighetsbevarande ämne i topikaler är cirka 15 % och i kosmetika finns propylenglykol i koncentrationerna <0,1 % - >50 %. Det saknas i nuläget forskning om propylenglykols effekt på hudens bakterieflora. Syftet med studien var att med olika koncentrationer av propylenglykol bestämma MIC (minsta hämmande koncentration) och MBC (minsta baktericida koncentration) på vanliga stafylokocker (S. epidermidis, S. aureus, S. hominis och S. capitis) som ingår i hudens normalflora. För att bestämma MIC och MBC användes buljongspädningsmetoden där propylenglykol späddes ut i olika koncentrationer med buljong i en mikrotiterplatta. Sedan tillsattes valda testbakterier och OD600 mättes i 24 timmar. Resultatet visade att MIC och MBC för propylenglykol var 12,5 % respektive 25 % på vanliga stafylokocker som finns på huden. Vid koncentrationer av propylenglykol på 12,5 % hämmades synlig bakterietillväxt av S. epidermidis, S. aureus, S. hominis och S. capitis och vid 25 % uppstod en baktericid effekt på bakterierna. Mer forskning behövs dock för att få reda på hur hudens bakterier påverkas av propylenglykol och konsekvenserna av det. / The normal flora of the human skin is consisting of low pathogen bacteria, where the staphylococci are the most common bacteria. Studies show that antimicrobial substances can alter populations of skin bacteria and that these alterations can lead to critical consequences for the resistance of the skin. Propylene glycol is a substance that is classified as antimicrobial and bactericidal and the substance has a wide area of use and is frequently used as a supportive substance in various pharmaceuticals. Propylene glycol can be found in cosmetics and skincare products where it functions as moisture-binding and preservative. Normal concentrations of propylene glycol as moisture-binding substance in topicals is approximately 15 % and in cosmetics the concentration of propylene glycol is <0,1 % - >50 %. As of today, there are few scientific studies regarding the effects of propylene glycol to the bacterial flora of the human skin. The purpose of this study was to determine MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) for normal staphylococci (S. epidermidis, S. aureus, S. hominis och S. capitis) included in the normal flora of the skin using various concentrations of propylene glycol. To be able to determine MIC and MBC the broth dilution method was used, where propylene glycol was diluted in various concentrations with broth in a microtiter plate. Hereafter, selected test bacteria were added and OD600 was measured during 24 hours. The results implicated that MIC and MBC for propylene glycol were 12,5 % and 25 % for common staphylococci located on the skin. At concentrations of propylene glycol of 12,5 %, visible bacterial growth of S. epidermidis, S. aureus, S. hominis and S. capitis was inhibited and at 25 % a bactericidal effect occurred on the bacteria. It shall be noted that further research is needed to find out how the skin's bacteria are affected by propylene glycol and its consequences.

Resistensbestämningar som genererar ett kvantitativt MIC-värde är värdefullt för att kunna välja adekvat antibiotikabehandling. Referensmetoden för MIC-bestämning är mikrobuljongspädning där kommersiella antibiotikapaneler underlättar handhavandet av metoden på kliniska laboratorier. Syftet med studien var att verifiera mikrobuljongspädning med Sensititre™-panelerna SEMSE7 och SEMST7 avsedda för grampositiva kocker, genom att jämföra erhållet resultat med tidigare uppmätta referensvärden av MIC samt SIR-kategorier. Mikrobuljongspädning utfördes med SEMSE7-panel och MH-buljong för Staphylococcus spp. (n=21) och Enterococcus spp. (n=13) samt med SEMST7-panel och MH-F buljong för Streptococcus pneumoniae (n=20). Avläsning av MIC utfördes samt tolkades enligt SIR-systemet. Resultaten jämfördes mot kända referensvärden tidigare uppmätta med mikrobuljongspädning. Isolat analyserade med SEMSE7-panel och SEMST7-panel erhöll en överensstämmelse av MIC på 88,2% respektive 96,7%. Motsvarande kategorisk överensstämmelse av SIR var 92,5% respektive 92,6%. Resultatet visar att Sensititre™-panelen SEMST7 är godkänd i verifieringen av mikrobuljongspädning med god överensstämmelse med referensvärden av MIC samt SIR-kategorier. Vidare bedöms att fortsatt verifiering krävs av SEMSE7-panelen eftersom ej tillfredställande överenstämmelse med referensvärden av MIC erhölls, trots god överenstämmelse av SIR-kategorier. Studien visar att SEMST7-panelen kan implementeras för Streptococcus pneumoniae i den kliniska verksamheten på mikrobiologilaboratoriet, Jönköping. / Antimicrobial susceptibility testing methods generating a quantitative MIC are valuable for choosing adequate antibiotic treatment. Broth microdilution is the reference method for MIC determination and commercial panels with antibiotics facilitate the procedure in clinical laboratories. The aim of the study was to verify broth microdilution with the Sensititre™ panels SEMSE7 and SEMST7 designed for gram-positive cocci, by comparing results with previously measured reference values of MIC and SIR. Broth microdilution was performed using the SEMSE7 panel with MH broth for Staphylococcus spp. (n=21) and Enterococcus spp. (n=13), and using the SEMST7 panel with MH-F broth for Streptococcus pneumoniae (n=20). Reading MIC endpoints was performed and interpreted according to SIR system. Isolates analysed with the SEMSE7 and SEMST7 panel obtained essential agreement of 88,2% and 96,7%, and categorical agreement of 92,5% and 92,6% respectively. In conclusion, the Sensititre™ panel SEMST7 is approved in the verification of broth microdilution with satisfactory essential agreement and categorical agreement. Furthermore, it is considered that further verification is required for the SEMSE7 panel as unsatisfactory essential agreement was obtained, despite satisfactory categorical agreement. The study shows that the SEMST7 panel can be implemented for Streptococcus pneumoniae in the clinical practice at the microbiology laboratory, Jönköping.

Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Author: Bc. Lenka Králíková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma: Atypical mycobacteria as causative agents of human diseases The aim of this diploma thesis is to summarize the history of mycobacteria, their classification, epidemiology of nontuberculous infections, clinical significance, diagnosis, resistance, and treatment options. Within the epidemiology, the way of managing tuberculosis and non-tuberculous mycobacterioses is compared. Mycobacterioses are rare diseases, which incidence is gradually increasing with the increasing number of immunocompromised patients. Based on the spot of infection, we divide them into lymphadenitis, skin lesions, lung disease and disseminated infections. The most frequently isolated pathogens are Mycobacterium avium complex, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium marinum and Mycobacterium kansasii. The experimental part is devoted to the antimycobacterial activity of newly synthesized compounds, which could find application in the future in the treatment of tuberculosis and other mycobacterioses. Substance testing was performed on strains of the genus Mycobacterium...

Durant les cultures cellulaires en bioréacteur, la physiologie des micro-organismes et les paramètres physico-chimiques (alimentations en gaz et en substrat, agitation, température, pH, pression) interagissent très fortement. La spécificité des bioréactions microbiennes, en relation avec les couplages irréductibles entre les transferts de chaleur, de matière et de quantité de mouvement, réside dans la complexité (milieu triphasique) et la dynamique (bioréaction autocatalysé) de ces systèmes. L’objectif de ce travail est de progresser dans la compréhension et le contrôle dynamique des intéractions entre les aspects biologiques et les aspects physiques à différentes échelles (macro, micro et moléculaire) pour conduire la réaction biologique vers l’objectif défini (production de biomasse, de métabolites intra ou extra cellulaires, …) et l’optimiser. Les cellules (concentration, forme, dimension, physiologie, …) affectent fortement les propriétés physico-chimiques des moûts et par conséquent, les performances des bioprocédés (vitesses spécifiques, rendements, productivité). Le comportement rhéologique particulier du moût est souvent utilisé pour comprendre l’impact de la biomasse microbienne sur le rendement et les performances du bioprocédé.Dans ce travail, des cultures axéniques, définies comme des cultures pures de microorganismes unicellulaires procaryote et eucaryote, sont considérées. Notre approche s’appuie sur des mesures physiques et physico-chimiques en ligne et hors ligne réalisées sur un bioréacteur instrumenté, mesures qui sont mises en place de façon à respecter les conditions imposées par les contraintes biologiques propres aux microorganismes et à la stratégie de culture choisie. Des cultures d’Escherichia coli et d’Yarrowia. lipolytica, à taux de croissance controlé par l’apport de substrat, ont été réalisées dans une gamme de concentration allant de 0.1 à 100 g l-1. Le bilan qui peut être dressé pour ce travail, tant sur les aspects scientifiques que technologiques, est le suivant :- conception et réalisation d’un outil d’investigation original construit sur la base d’un bioréacteur (20 l) et pourvu d’une boucle de recirculation instrumentée pour la mesure,- identification hydrodynamique (courbes de frottement) de conduites calibrées permettant la viscosimétrie en ligne durant une culture cellulaire, - conception, développement et validation d’un code, LoCoPREL, permettant simultanément le contrôle de la culture cellulaire suivant une stratégie définie, la gestion de séquences de débit dans la boucle de dérivation et l’acquisition des données issues de l’instrumentation spécifique employée,- comparaison des mesures réalisées en ligne à débit constant ou selon des séquences de débit,- mise en évidence du comportement non newtonien des moûts et d’écarts entre les mesures en ligne et hors ligne,- analyse des mesures physiques réalisées en ligne et hors ligne, en lien avec les performances de la culture / During cell cultures in bioreactors, micro-organism physiology closely interacts with physico-chemical parameters such as gas and feed flowrates, mixing, temperature, pH, pressure. The specificity of microbial bioreactions in relation with irreductible couplings between heat and mass transfers and fluid mechanics, led into complex (three-phase medium) and dynamic (auto-biocatalytics reaction) systems. Our scientific approach aims to investigate, understand and control dynamic interactions between physical and biological systems at different scales (macro, micro and molecular) for molecules, strains and/or bioprocess innovation. Cells (concentration, shape, dimension, physiology…) strongly affect physico-chemical properties of broth. Then the modification of these characteristics interacts with bioprocess performances (specific rates, yields…) with an improvement or, more frequently, a decrease of yields. Among these properties, rheological behaviour is a strategy widely used to understand the impact of cells and the derivation of bioprocess performances.In this manuscript, axenic cultures, defined as cultures of a pure and unicellular Prokaryote and Eukaryote microorganisms in bioreactors, are considered. Our approach is based on physical and physico-chemical on-line and off-line measurements in respect with accurate and stringent conditions imposed by cell culture strategy. Escherichia coli and Yarrowia lipolytica cultures were investigated with a control of growth rate by carbon feed in the range from 0.1 up to 100 g l-1. Our scientific and technical actions and results led:- to design and realize an original pilot based on a bioreactor (20 l) with a derivation loop including a specific on-line rheometric device as well as additional physical and biological measurements,- to identify, from a hydrodynamic standpoint, the generalized friction curves of calibrated ducts enabling on-line viscosimetry during cell cultures,- to conceive and validate a homemade software, named LoCoPREL, enabling simultaneously to control cell cultures under defined strategy and to manage flow sequences within the derivation loop,- to discuss and compare on-line physical measurements under constant flow rate and various sequence strategy related to investigated shear-rates,- to highlight about the non-newtonian rheological behaviour of broths and the gap between on-line and off-line measurements,- to analyse on-line and off-line physical measurements in the light of biological performances during fed-batch cultures (mass balance, specific rate, yield).

Neste trabalho foi avaliada a possibilidade do emprego de caldo de cana-de-açúcar (Saccharum officinarum) como matéria-prima para obtenção de 2,3-butanodiol por fermentação, utilizando a bactéria Klebsiella pneumoniae NRRL B199. A pesquisa compreendeu o teste de diferentes nutrientes para a composição de um meio de fermentação eficiente com o caldo de cana e experimentos em que a influência da concentração inicial de sacarose (S0) sobre o processo em regime descontínuo foi estudada. Ensaios realizados em frascos agitados, com S0 entre 140 e 150 g/L, mostraram que a fermentação de caldo de cana clarificado não suplementado com nutrientes não é viável. Por outro lado, a adição de 5,0 a 10,0 g/L de fosfato de amônio ao caldo levou a concentrações finais de 2,3-butanodiol de até 64 g/L e rendimentos, em relação ao máximo teórico, da ordem de 81% em cerca de 38 horas. Estes resultados se aproximam daqueles alcançados na fermentação do caldo enriquecido com vários nutrientes, usado como referência, na qual foram produzidos 66,7 g/L de diol com um rendimento de 85,6%. A utilização de concentrações de fosfato de amônio de até 4,0 g/L resultou em fermentações incompletas. A cinética da fermentação foi estudada com o caldo diluído a, aproximadamente, 180 g/L de sacarose e suplementado com 8,0 g/L de fosfato de amônio e, também, com o meio rico, em regime descontínuo em fermentador de bancada. Foram obtidos, nestes ensaios, concentrações de butanodiol de 71,7 e 71,1 g/L, rendimentos de 78,0 e 74,1 %, produtividades de 2,1 e 2,0 g/Lh e tempos de fermentação de 34,5 e 35,7 horas, respectivamente. A principal diferença foi observada na concentração de biomassa que atingiu 9,1 g/L, no meio com fosfato, e 11,8 g/L no meio rico. As máximas velocidades específicas de crescimento (µx, m), 0,61 e 0,45 h-1, observadas com oxigênio dissolvido acima de zero, indicam que o crescimento celular, no meio rico, foi inibido pela alta concentração de nutrientes. Na seqüência das fermentações, com concentrações nulas de oxigênio dissolvido, a produção de diol foi iniciada e as máximas velocidades específicas de formação de produto (µP,m), 0,53 h-1 com fosfato de amônio e 0,45 h-1 com o meio rico, foram atingidas, sugerindo que altas concentrações de nutrientes afetam, também, a formação de produto. Nos dois ensaios foi observada a ocorrência de uma fase estacionária de crescimento que, no caso do meio contendo fosfato de amônio, teve uma duração 12 horas mais longa devido a um insuficiente suprimento de oxigênio ou do esgotamento de algum nutriente. Neste ponto, foi verificada uma queda das velocidades específicas de formação de produto (µP). Com o caldo diluído a cerca de 20 g/L de sacarose e 8,0 g/L de fosfato de amônio, a fermentação transcorreu em um tempo muito longo (23,5 horas) para a baixa concentração de sacarose empregada. Com S0 semelhante e o meio rico, o tempo de processo foi de apenas 6,5 h, o que demonstra que, no primeiro caso, ocorreu uma diluição excessiva dos nutrientes naturalmente presentes no caldo. O estudo do efeito da concentração inicial de sacarose sobre a fermentação, conduzido em fermentador de bancada e com o meio rico em nutrientes, mostrou que este parâmetro influencia fortemente o crescimento celular e a produção de 2,3-butanodiol. O aumento de S0 levou a valores decrescentes dos fatores de conversão de substrato em células, enquanto os rendimentos em diol aumentaram com S0 até 152 g/L. Valores crescentes de S0 provocaram uma inibição do crescimento celular, evidenciada pela diminuição de µx,m de 0,91 a 0,45 h-1, com S0 entre 24 e 181 g/L. Valores aproximadamente constantes de µP,m (0,62 a 0,68 h-1) foram observados com S0 até 152 g/L, enquanto uma queda acentuada foi verificada com S0=181 g/L (µP,m=0,45 h-1). As máximas produtividades (3,2 g/Lh) foram obtidas com valores de S0 de 105 e 152 g/L. Os dados gerados neste trabalho permitem concluir que caldo de cana-de-açúcar é uma matéria-prima adequada para a produção de 2,3-butanodiol por Klebsiella pneumoniae. / The use of sugar cane (Saccharum officinarum) juice, as raw material for the fermentative production of 2,3-butanediol by Klebsiella pneumoniae NRRL B199, was evaluated in this work. The research was focused on testing different nutrients to formulate an efficient fermentation medium with the juice and some experiments to study the effect of the initial sucrose concentration (S0) on the batch process. Shaker flasks experiments with S0 between 140 and 150 g/L have shown that the fermentation of sugar cane juice without added nutrients is not viable. On the other hand, the addition of 5.0 to 10.0 g/L ammonium phosphate to the juice led, after 38 hours, to final 2,3-butanediol concentrations of 64 g/L, with yields of 81% of the theoretical maximum. These results are similar to those found in the fermentation of a reference medium composed of juice enriched with several nutrients in which 66.7 g/L butanediol and a yield of 85.6% were obtained. Ammonium phosphate concentrations up to 4.0 g/L resulted in incomplete fermentations. The fermentation kinetics was studied in a laboratory scale fermentor, with the sugar cane juice diluted to ca. 180 g/L sucrose. Both ammonium phosphate (8.0 g/L) and reference media were tested. In these experiments, after process times close to 35 hours, butanediol concentrations of 71.7 and 71.1 g/L, yields of 78.0 and 74.1 % , and productivities of 2.1 and 2.0 g/Lh, respectively, were obtained. The most important difference was the final biomass concentration that reached 9.1 g/L with the ammonium phosphate medium and 11.8 g/L with the rich medium. The maximum specific growth rates (µx,m=0.61 and 0.45 h-1), measured with dissolved oxygen concentrations above zero, have indicated that cell growth in the rich medium was inhibited by high nutrient concentration. In the sequence, when the oxygen concentration was zero, butanediol production started and maximum specific production rates (µP,m of 0.53 h-1 with the ammonium phosphate medium and 0.45 h-1 with the rich medium were reached. These values suggest that nutrient concentration in the rich medium also affects product formation. ln both runs, a stationary growth phase was noticed. In the experiment with the ammonium phosphate medium, the stationary phase was 12 hours longer due to the insufficient oxygen supply rate or the shortage of some nutrient. At that moment, decreasing specific product formation rates (7#181;P) were observed. With the sugar cane juice diluted to approximately 20 g/L and 8.0 g/L ammonium phosphate, fermentation time was 23.5 hours, too long to such sucrose concentration. With similar S0 and the rich medium, fermentation time was reduced to 6.5 hours. That shows that, in the first case, natural nutrients of the juice were excessively diluted. The study on the effect of the initial sucrose concentration on the process, performed in a laboratory fermentor with the rich medium, has shown that this parameter strongly affects both cell growth and 2,3-butanediol production. Increasing S0 led to decreasing cell yields and, for S0 up to 152 g/L, diol yields increased. Cell growth inhibition was stronger as higher sucrose concentrations were used. This is evidenced by the values of µx,m that decreased from 0.91 to 0.45 h-1 as S0 was augmented from 24 to 181 g/L. Approximately constant µP,m (0.62 to 0.68 h-1) were observed with S0 up to 152 g/L. With S0=181 g/L, µP,m is reduced to 0.45 h-1 . The maximum productivities (3.2 g/Lh) were obtained with S0 between 105 and 152 g/L. From the results of this work, one can conclude that sugar cane juice is a suitable raw material for the production of 2,3-butanediol by Klebsiella pneumoniae.

Neste trabalho foi avaliada a possibilidade do emprego de caldo de cana-de-açúcar (Saccharum officinarum) como matéria-prima para obtenção de 2,3-butanodiol por fermentação, utilizando a bactéria Klebsiella pneumoniae NRRL B199. A pesquisa compreendeu o teste de diferentes nutrientes para a composição de um meio de fermentação eficiente com o caldo de cana e experimentos em que a influência da concentração inicial de sacarose (S0) sobre o processo em regime descontínuo foi estudada. Ensaios realizados em frascos agitados, com S0 entre 140 e 150 g/L, mostraram que a fermentação de caldo de cana clarificado não suplementado com nutrientes não é viável. Por outro lado, a adição de 5,0 a 10,0 g/L de fosfato de amônio ao caldo levou a concentrações finais de 2,3-butanodiol de até 64 g/L e rendimentos, em relação ao máximo teórico, da ordem de 81% em cerca de 38 horas. Estes resultados se aproximam daqueles alcançados na fermentação do caldo enriquecido com vários nutrientes, usado como referência, na qual foram produzidos 66,7 g/L de diol com um rendimento de 85,6%. A utilização de concentrações de fosfato de amônio de até 4,0 g/L resultou em fermentações incompletas. A cinética da fermentação foi estudada com o caldo diluído a, aproximadamente, 180 g/L de sacarose e suplementado com 8,0 g/L de fosfato de amônio e, também, com o meio rico, em regime descontínuo em fermentador de bancada. Foram obtidos, nestes ensaios, concentrações de butanodiol de 71,7 e 71,1 g/L, rendimentos de 78,0 e 74,1 %, produtividades de 2,1 e 2,0 g/Lh e tempos de fermentação de 34,5 e 35,7 horas, respectivamente. A principal diferença foi observada na concentração de biomassa que atingiu 9,1 g/L, no meio com fosfato, e 11,8 g/L no meio rico. As máximas velocidades específicas de crescimento (µx, m), 0,61 e 0,45 h-1, observadas com oxigênio dissolvido acima de zero, indicam que o crescimento celular, no meio rico, foi inibido pela alta concentração de nutrientes. Na seqüência das fermentações, com concentrações nulas de oxigênio dissolvido, a produção de diol foi iniciada e as máximas velocidades específicas de formação de produto (µP,m), 0,53 h-1 com fosfato de amônio e 0,45 h-1 com o meio rico, foram atingidas, sugerindo que altas concentrações de nutrientes afetam, também, a formação de produto. Nos dois ensaios foi observada a ocorrência de uma fase estacionária de crescimento que, no caso do meio contendo fosfato de amônio, teve uma duração 12 horas mais longa devido a um insuficiente suprimento de oxigênio ou do esgotamento de algum nutriente. Neste ponto, foi verificada uma queda das velocidades específicas de formação de produto (µP). Com o caldo diluído a cerca de 20 g/L de sacarose e 8,0 g/L de fosfato de amônio, a fermentação transcorreu em um tempo muito longo (23,5 horas) para a baixa concentração de sacarose empregada. Com S0 semelhante e o meio rico, o tempo de processo foi de apenas 6,5 h, o que demonstra que, no primeiro caso, ocorreu uma diluição excessiva dos nutrientes naturalmente presentes no caldo. O estudo do efeito da concentração inicial de sacarose sobre a fermentação, conduzido em fermentador de bancada e com o meio rico em nutrientes, mostrou que este parâmetro influencia fortemente o crescimento celular e a produção de 2,3-butanodiol. O aumento de S0 levou a valores decrescentes dos fatores de conversão de substrato em células, enquanto os rendimentos em diol aumentaram com S0 até 152 g/L. Valores crescentes de S0 provocaram uma inibição do crescimento celular, evidenciada pela diminuição de µx,m de 0,91 a 0,45 h-1, com S0 entre 24 e 181 g/L. Valores aproximadamente constantes de µP,m (0,62 a 0,68 h-1) foram observados com S0 até 152 g/L, enquanto uma queda acentuada foi verificada com S0=181 g/L (µP,m=0,45 h-1). As máximas produtividades (3,2 g/Lh) foram obtidas com valores de S0 de 105 e 152 g/L. Os dados gerados neste trabalho permitem concluir que caldo de cana-de-açúcar é uma matéria-prima adequada para a produção de 2,3-butanodiol por Klebsiella pneumoniae. / The use of sugar cane (Saccharum officinarum) juice, as raw material for the fermentative production of 2,3-butanediol by Klebsiella pneumoniae NRRL B199, was evaluated in this work. The research was focused on testing different nutrients to formulate an efficient fermentation medium with the juice and some experiments to study the effect of the initial sucrose concentration (S0) on the batch process. Shaker flasks experiments with S0 between 140 and 150 g/L have shown that the fermentation of sugar cane juice without added nutrients is not viable. On the other hand, the addition of 5.0 to 10.0 g/L ammonium phosphate to the juice led, after 38 hours, to final 2,3-butanediol concentrations of 64 g/L, with yields of 81% of the theoretical maximum. These results are similar to those found in the fermentation of a reference medium composed of juice enriched with several nutrients in which 66.7 g/L butanediol and a yield of 85.6% were obtained. Ammonium phosphate concentrations up to 4.0 g/L resulted in incomplete fermentations. The fermentation kinetics was studied in a laboratory scale fermentor, with the sugar cane juice diluted to ca. 180 g/L sucrose. Both ammonium phosphate (8.0 g/L) and reference media were tested. In these experiments, after process times close to 35 hours, butanediol concentrations of 71.7 and 71.1 g/L, yields of 78.0 and 74.1 % , and productivities of 2.1 and 2.0 g/Lh, respectively, were obtained. The most important difference was the final biomass concentration that reached 9.1 g/L with the ammonium phosphate medium and 11.8 g/L with the rich medium. The maximum specific growth rates (µx,m=0.61 and 0.45 h-1), measured with dissolved oxygen concentrations above zero, have indicated that cell growth in the rich medium was inhibited by high nutrient concentration. In the sequence, when the oxygen concentration was zero, butanediol production started and maximum specific production rates (µP,m of 0.53 h-1 with the ammonium phosphate medium and 0.45 h-1 with the rich medium were reached. These values suggest that nutrient concentration in the rich medium also affects product formation. ln both runs, a stationary growth phase was noticed. In the experiment with the ammonium phosphate medium, the stationary phase was 12 hours longer due to the insufficient oxygen supply rate or the shortage of some nutrient. At that moment, decreasing specific product formation rates (7#181;P) were observed. With the sugar cane juice diluted to approximately 20 g/L and 8.0 g/L ammonium phosphate, fermentation time was 23.5 hours, too long to such sucrose concentration. With similar S0 and the rich medium, fermentation time was reduced to 6.5 hours. That shows that, in the first case, natural nutrients of the juice were excessively diluted. The study on the effect of the initial sucrose concentration on the process, performed in a laboratory fermentor with the rich medium, has shown that this parameter strongly affects both cell growth and 2,3-butanediol production. Increasing S0 led to decreasing cell yields and, for S0 up to 152 g/L, diol yields increased. Cell growth inhibition was stronger as higher sucrose concentrations were used. This is evidenced by the values of µx,m that decreased from 0.91 to 0.45 h-1 as S0 was augmented from 24 to 181 g/L. Approximately constant µP,m (0.62 to 0.68 h-1) were observed with S0 up to 152 g/L. With S0=181 g/L, µP,m is reduced to 0.45 h-1 . The maximum productivities (3.2 g/Lh) were obtained with S0 between 105 and 152 g/L. From the results of this work, one can conclude that sugar cane juice is a suitable raw material for the production of 2,3-butanediol by Klebsiella pneumoniae.

Le couplage fonctionnel des opérations de séparation et de réaction ainsi que les bio-procédés sont deux axes de recherche largement explorés. Cependant, l’industrie du génie des procédés a du mal à se tourner vers des technologies de ce type car il demeure un réel manque de connaissances et d’outils de conception pour ce genre de procédés. Une méthodologie de conception générale pour l'extraction liquide-liquide réactive est introduite dans cette étude. Elle est composée de trois étapes différentes: l'analyse de faisabilité, la synthèse ou dimensionnement du procédé et la validation par simulation. Cette méthodologie conduit à des paramètres structuraux et opératoires de la colonne étudiée à partir seulement des informations concernant le comportement physico-chimique du système étudié, en exploitant les équations d’équilibre chimique et entre phase ainsi que les bilans matières. Les résultats de cette méthode sont un bon point de départ pour une étude d'optimisation ou d'un processus de calcul d'investissement. Cette méthodologie a été appliquée à différentes études de cas: regroupant deux stratégies différentes d'extraction avec plusieurs solvants pour récupérer l'acide succinique dans un milieu de fermentation / The functional coupling of separation and reaction operations and bioprocesses are two widely explored areas of research. However, process engineering industry is struggling to turn to these technologies because it remains a real lack of knowledge and design tools for this kind of processes. A general design methodology for reactive liquid-liquid extraction is introduced in this study. It is composed of three different steps: feasibility analysis, pre-design determination and simulation validation. This methodology leads to the design specifications of the units from the information concerning the physicochemical behaviour of the studied system, exploiting the equilibrium and material balance equations. The results of this methodology are a good starting point for an optimization study or for an investment calculation process. This methodology has been applied to different case studies: two different strategies of extraction and several solvents to recover succinic acid in fermentation broth.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The Mycobacterium genus includes M. tuberculosis complex (CMTB) species and others called nontuberculous mycobacteria (MNT). The CMTB bacilli cause tuberculosis (TB), a bacterial infectious disease, which commonly affects the lungs. Since the MNT cause other mycobacterial infections. The correct diagnosis of diseases caused by mycobacteria is essential for determining treatment. The tuberculosis treatment regimen in Brazil is recommended by the National Tuberculosis Control/Ministry of Health (PNCT/MS) and was recently modified. The main changes proposed by the Technical Advisory Committee of PNCT/MS were: to introduce a fourth drug, ethambutol, during the attack and take the combination of drugs in tablet form, with fixed-dose combinations (FDC) 4 in 1, for intensive treatment phase, and 2 in 1 for the maintenance phase. Due to the high incidence of tuberculosis in Brazil and worldwide, the emergence of resistant strains and the deployment of new therapies by the Ministry of Health, this study aimed to evaluate the susceptibility to antituberculosis or individually associated clinical isolates of Mycobacterium tuberculosis from the University Hospital Mary. The antimicrobial susceptibility alone or associated (rifampicin, isoniazid, pyrazinamide and ethambutol) was evaluated using the microdilution method (MMC) and compared to the proportion method (MP), gold standard method for susceptibility to mycobacteria. The MMC has proven to be a rapid, easily performed and well correlated with the MP. We have found various clinical isolates of M. tuberculosis resistant to one, two or three drug when tested against four drugs alone. However, when they were tested against four drugs associated with the FDC no strain was considered resistant. This fact is against the concept of FDC which aims to unite the four anti-TB drugs to combat the resistance of the bacilli. / O gênero Mycobacterium é constituído por espécies do Complexo M. tuberculosis (CMTB) e outras denominadas de micobactérias não tuberculosas (MNT). Os bacilos do CMTB causam a tuberculose (TB), uma doença infecciosa bacteriana, a qual comumente afeta os pulmões. Já as MNT causam outras micobacterioses. O diagnostico correto das doenças causadas pelas micobactérias é essencial para a definição do tratamento. O esquema de tratamento para a tuberculose no Brasil é preconizado pelo Programa Nacional de Controle da Tuberculose/Ministério da Saúde (PNCT/MS) e foi recentemente modificado. As principais mudanças propostas pelo Comitê Técnico Assessor do PNCT/MS foram: introduzir um quarto fármaco, o etambutol, na fase de ataque e adotar a associação dos fármacos em forma de comprimidos, com doses fixas combinadas (DFC) 4 em 1, para a fase de tratamento intensivo, e, 2 em 1, para fase de manutenção. Devido à grande incidência da tuberculose no Brasil e no mundo, à emergência de cepas resistentes e à implantação de novas terapias pelo Ministério da Saúde, este trabalho objetivou avaliar a suscetibilidade aos tuberculostáticos associados ou individualmente dos isolados clínicos de Mycobacterium tuberculosis do Hospital Universitário de Santa Maria. A suscetibilidade aos antimicrobianos isolados ou associados (rifampicina, isoniazida, pirazinamida e etambutol) foi avaliada através do método de microdiluição em caldo (MMC) e comparados ao método das proporções (MP), método padrão ouro para suscetibilidade de micobactérias. O MMC mostrou-se ser um método rápido, de fácil realização e boa correlação com o MP. Foram encontrados vários isolados clínicos de M. tuberculosis resistentes a um, dois ou três fármacos quando testados frente aos quatro fármacos isoladamente. Porem, quando estes foram, testados contra os quatro fármacos da DFC associados nenhuma cepa foi considerada resistente. Este fato vem de encontro ao conceito da DFC a qual pretende unir os quatro medicamentos anti-TB para combater a resistência dos bacilos.