Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Candida tropicalis à uma levedura diplÃide causadora de infecÃÃes superficiais e/ou sistÃmicas, as quais podem ser adquiridas de maneira endÃgena ou exÃgena e podem acometer diversos ÃrgÃos. No Brasil, dentre as espÃcies de Candida spp., a C.tropicalis à a segunda espÃcie mais comumente isolada e no Cearà à pouco estudada. Nos dias atuais, temos vivenciado um aumento significativo das infecÃÃes fÃngicas invasivas. PorÃm as drogas antifÃngicas disponÃveis no mercado sÃo restritas a um pequeno nÃmero quando comparadas as antibacterianas. Logo, este fato unido ao aumento da frequÃncia de resistÃncia cruzada faz necessÃria a busca por novas estratÃgias terapÃuticas. A amiodarona (AMD) à usada classicamente para tratar pacientes com arritmia. Trabalhos recentes tÃm demonstrado uma ampla atividade antifÃngica desta droga quando associado ao fluconazol (FLC). No presente estudo induzimos resistÃncia em sete cepas de Candida tropicalis e avaliamos um eventual sinergismo entre FLC e AMD. A avaliaÃÃo da interaÃÃo das drogas foi determinada atravÃs do cÃlculo da Fractionary Inhibitory Concentration (FICI) e por meio da tÃcnica de citometria de fluxo, onde tambÃm avaliamos o provÃvel mecanismo de aÃÃo desse sinergismo. Os isolados utilizados no estudo pertencem ao LaboratÃrio de BioprospecÃÃo Experimental em Leveduras (LABEL) da UFC. Para o cumprimento da metodologia, as cepas foram recuperadas do estoque e identificadas por biologia molecular. A induÃÃo foi realizada adaptando-se o protocolo de Pinto e Silva (2009). Os testes de sensibilidade foram realizados utilizando-se o teste de microdiluiÃÃo em caldo padronizado pelo CLSI, segundo o documento M27-A3. Para avaliar o sinergismo utilizou-se a tÃcnica do checkboard. Por fim, para verificar o possÃvel mecanismo de aÃÃo do sinergismo, determinamos a integridade de membrana, potencial transmembrana mitocondrial, formaÃÃo de espÃcies reativas de oxigÃnio (ROS), teste do cometa, anÃlise da oxidaÃÃo de bases purinas e caspases. As cepas de C.tropicalis apÃs ~100 dias atingiram um CIM > 64Âg/mL. Os testes de sensibilidade apresentaram CIM > 64Âg/mL tanto para o FLC como para AMD. Das cepas testadas, seis apresentaram sinergismo. O tratamento FLC+AMD alterou a integridade da membrana plasmÃtica e mitocondrial, aumentou os nÃveis de ROS intracelularmente, causou danos ao DNA e, consequentemente, conduziu morte celular por apoptose. / Candida tropicalis is a diploid yeast causing superficial infections and / or systemic, which can be acquired endogenously or exogenously and can affect several organs. In Brazil, among the species of Candida spp. The C.tropicalis is the second most common species isolated in Cearà and is rarely studied. Nowadays, we have experienced a significant increase in invasive fungal infections. But antifungal drugs on the market are restricted to a small number when compared to the antibacterial. Therefore, this fact coupled with the increased frequency of cross-resistance is necessary to search for new therapeutic strategies. Amiodarone (AMD) is traditionally used to treat patients with arrhythmia. Recent studies have shown a broad antifungal activity of this drug when combined with fluconazole (FLC). In the present study we induced resistance in seven strains of Candida tropicalis and evaluate a possible synergism between FLC and AMD. The evaluation of the interaction of the drugs was determined by calculating the Fractionary Inhibitory Concentration (FICI) and by flow cytometry, where we also evaluated the likely mechanism of action of this synergism. The isolates used in this study belong to the Laboratory of Experimental Bioprospecting in yeast (LABEL) the UFC. For the fulfillment of the methodology, the strains were recovered from stock and identified by molecular biology. The induction was carried out adapting the protocol of Pinto and Silva (2009). Sensitivity tests were performed using the broth microdilution test standardized by the CLSI, the document M27-A3. To evaluate the synergism used the technique of checkboard. Finally, to determine the possible mechanism of action of synergism, we determine the membrane integrity, mitochondrial transmembrane potential, formation of reactive oxygen species (ROS), comet assay, analysis of the oxidation of purine bases and caspases. Strains C.tropicalis after ~ 100 days reached an MIC> 64μg/mL. The sensitivity tests were MIC> 64μg/mL both the FLC as AMD. Of the strains tested, six showed synergism. The treatment changed the FLC + AMD plasma membrane integrity and mitochondrial increased levels of intracellular ROS, cause DNA damage and therefore led cell death by apoptosis.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-03-04T15:24:08Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE KLEDOALDO - vers digital2.pdf: 4135407 bytes, checksum: f3e6e51484b19ae1c63ca97b8a12fe1e (MD5) / Made available in DSpace on 2016-03-04T15:24:08Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE KLEDOALDO - vers digital2.pdf: 4135407 bytes, checksum: f3e6e51484b19ae1c63ca97b8a12fe1e (MD5) Previous issue date: 2015-03-30 / CAPES / Uma grande heterogeneidade da epidemia pelo HIV-1 é observada no Brasil, onde prevalecem os subtipos B, F1 e C e os recombinantes BF e BC. O objetivo principal deste estudo foi caracterizar as cepas do HIV-1 circulantes no estado de Pernambuco, Nordeste do Brasil, através de análises filogenéticas, avaliando-se as características sociodemográficas, laboratoriais e evolutivas entre os subtipos B e não-B. As sequências da região pol do HIV-1, que totalizaram 169 amostras, foram obtidas de dois estudos anteriores. Todos os pacientes eram maiores de 18 anos e virgens de terapia antirretroviral. O Alinhamento e a edição manual das sequências foram realizados pelo CLUSTAL X e BioEdit software, respectivamente. Para as inferências filogenéticas e de recombinação gênica foram utilizados os softwares MEGA 5 e SIMPLOT, respectivamente. Pesquisas sorológicas para determinação das co-infecções foram realizadas para os seguintes agentes infecciosos: HBV, HCV, HTLV e sífilis, pelo método de quimiluminescência. Na região analisada (pol), os resultados mostraram uma grande frequência do subtipo F do HIV-1 (31.4%) e a circulação de uma cepa H e AG. As frequências dos subtipos B e C foram 60.9% e 1.2%, respectivamente. Foram identificados um recombinante BC e 8 recombinantes BF (4.7%), com estruturas genômicas diferenciadas. Co-infecção HIV-HBV foi mais frequente entre homens que fazem sexo com homens (HSH) portadores do subtipo B do HIV-1, enquanto que co-infecção HIV-sífilis foi associado a HSH com subtipos não-B. O subtipo B foi associado ao sexo masculino, a uma maior carga vira, maior escolaridade e menor contagem de células T CD4+. Houve uma baixa frequência de mutações de resistência transmitidas (2.96%). Códons sob pressão seletiva positiva são mais frequentes em contagem de células T CD4+ ≥ 200 e em mulheres heterossexuais. Nossos resultados demonstram que a epidemia do HIV-1 em Pernambuco se caracteriza por uma alta proporção de subtipos não-B circulantes, o que revela a importância no monitoramento e melhor conhecimento do papel destas variantes na epidemia, no tratamento antirretroviral, formulação de vacinas, progressão à doença e transmissibilidade. / A great heterogeneity of HIV-1 epidemic is observed in Brazil, where subtypes B, F1 and C and recombinant forms BF prevails and BC. The aim of study was to characterize HIV-1 strains circulating in the state of Pernambuco, northeastern Brazil, through phylogenetic analysis, evaluating also the socio-demographic, laboratory and evolutionary characteristics between subtypes B and non-B. The sequences with pol region of HIV-1, totaling 169 samples were obtained from two previous studies. All patients were over 18 year old and antiretroviral naïve therapy. The alignment and manual editing of the sequences were performed by CLUSTAL X and BioEdit software, respectively. For the phylogenetic and genetic recombination inferences were used MEGA 5 and SIMPLOT software, respectively. Serological assaus of co-infections were performed for the following infectious agents: HBV, HCV, HTLV, and syphilis by chemiluminescence. In the analyzed region (pol), the results showed a high frequency of the HIV-1 subtype F (31.4%) and a strain H and AG. The frequency of the subtypes B and C were 60.9% and 1.2%, respectively. They identified a recombinant BC and eight BF recombinant (4.7%) with different genomic structures. Co-infection HIV-HBV was more frequent among men who have sex with men (MSM) with HIV-1 subtype B, while co-infection HIV-syphilis was associated with MSM with subtypes non-B. The subtype B was associated with male gender, higher viral load, higher education and lower T cells count. There was a low frequency of transmitted resistance mutations (2.96%). Codons under positive selection pressure are more common in T cells count ≥ 200 and heterosexual women. Our results demonstrate that the HIV-1 epidemic in Pernambuco is characterized by a high proportion of circulating non-B subtypes, which shows the importance of monitoring and better understanding of the role of these variants in epidemic in antiretroviral therapy, vaccine formulation, disease progression and transmissibility.

Orientadores: Marcelo de Carvalho Ramos, Maria Cecilia Barisson / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T01:15:13Z (GMT). No. of bitstreams: 1 Milan_Arlete_M.pdf: 2380396 bytes, checksum: 8b3168431347ab625bac1135be8ebb1c (MD5) Previous issue date: 2007 / Resumo: Introdução: A Pseudomonas aeruginosa é um importante agente de infecção nosocomial e motivo de grande preocupação por apresentar, com freqüência, resistência a diversos antimicrobianos. Os principais mecanismos de resistência encontrados são: inativação por enzimas, alterações na permeabilidade da membrana celular e promoção de efluxo. O conhecimento dos mecanismos de resistência que operam nesses microrganismos, bem como o estudo epidemiológico-molecular desses isolados, pode contribuir para a melhor compreensão e conseqüente controle dessas infecções no ambiente hospitalar. Objetivos: Determinar a Concentração Inibitória Mínima do Imipenem de Pseudomonas aeruginosa resistentes a esse antimicrobiano, isoladas de pacientes internados em hospital; Determinar a frequência de produção de metalo-beta-lactamases e da expressão da porina Opr D2 de Pseudomonas aeruginosa resistentes ao Imipenem, isoladas de pacientes internados em hospital; Genotipar e estudar a relação genética de Pseudomonas aeruginosa resistentes ao Imipenem, isoladas de pacientes internados em hospital. Materiais e Métodos: Foram estudadas 138 cepas isoladas de diversas amostras clínicas, identificadas bioquimicamente por técnicas de rotina e por automação. A triagem para a detecção de amostras produtoras de MBL's foi realizada pela fita de Etest combinada com EDTA. A extração e eletroforese de proteínas da parede celular foi executada para avaliar a expressão de porina Opr D2. Para a genotipagem foi utilizada a técnica de PFGE. Resultados e Discussões: Foram estudados 138 isolados, encaminhados como resistentes ao Imipenem. Em 128 desses isolados a resistência foi confirmada através do teste de difusão com disco e com a determinação do MIC (n = 98), através do Etest. A pesquisa da porina Opr D2 foi realizada em todos os isolados resistentes, ou seja, 128 casos. Em 68 deles não havia a expressão dessa proteína. Quando combinados os resultados da pesquisa de metalo-beta-lactamases e da deleção da porina Opr D2, encontramos 24 isolados que exibiam ambos os mecanismos de resistência. Da análise genotípica de 117 isolados, mostrou um acentuado polimorfismo, mesmo em isolados obtidos em um mesmo paciente. Não foram caracterizados surtos. Conclusões: A maioria das cepas de Pseudomonas aeruginosa resistentes ao Imipenem, isoladas em ambiente hospitalar, possui elevada resistência; A produção de meta-lobeta-lactamases constitui um mecanismo de resistência importante nesses isolados; A deleção da porina Opr D2 de membrana celular foi observada na maioria dos isolados. Em cerca de um terço deles havia também produção de metalo-beta-lactamase; A conglomeração desses isolados foi baixa, não caracterizando surtos em todos os casos; A variabilidade genotípica dos isolados foi intensa, mesmo em um determinado paciente, indicando que as ações relativas ao controle da disseminação desse patógeno deve ser de natureza rotineira, com as precauções de contato de universais e com o controle do uso de antimicrobianos / Abstract: Objective: Metallo-ß-lactamase production and Opr D2 channels expression was investigated in Imipenem resistant Pseudomonas aeruginosa recovered from hospital acquired infections. Design: Descriptive study in a convenient sample of organisms. Setting: Two 400-bed general teaching hospital, both tertiary care teaching hospitals, in Campinas, Brazil. All Imipenem resistant P. aeruginosa, recovered from March, 2000 through December 2004 from hospitalized patients were collected. Methods: Disk diffusion tests were used to confirm Imipenem resistance. Etest MBL was done to check for MBL production, and. Imipenem MIC's. Opr D2 expression was checked using cellular membrane proteins electrophoresis PFGE-genotyping was done in all isolates. Results: A sample of 128 Imipenem resistant Pseudomonas aeruginosa isolates was collected during the study period. Most isolates exhibited Imipenem MIC's = 256 µg / mL. Macrorestriction analysis (SpeI) using pulsed field gel electrophoresis (PFGE) showed a substantial polymorphism. Only 15 strains could be allocated to seven clusters, six with two isolates and one with three isolates. Ninety-nine Imipenem resistant isolates were screened for MBL production, and 87 were screened for both MBL, and porin Opr D2 expression. Sixty four isolates showed MBL production. Conclusion: Dissemination of MBL producing-genotypically heterogenous Pseudomonas aeruginosa strains was documented in the hospitals studied. Lack of Opr D2 combined with MBL production contributed to the high imipenem-resistance rates observed / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

No description available.

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus cotrimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to cotrimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Cotrimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium. IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.

No description available.

Antibiotic resistance is increasing rapidly world wide. Resistance determinants have evolved long before antibiotics were used. Though horizontal gene transfer and mutation play a major role in the dissemination of antibiotic resistance genes their evolutionary origins remain obscure. A model system was used to investigate how they might arise in the first case. Plasmid borne erythromycin resistant clones were selected through marker rescue from genomic libraries of DNA from Gram positive organisms, maintained in E. coli. EryR determinants were recovered from nine libraries of 23 screened. Clone pMP1 (DNA from Mycobacterium parafortuitum) was the most resistant, with an MIC of 400 ìg/ml for erythromycin and 12 ìg/ml for azithromycin. Antibiotic resistance was not expressed in Rhodococcus erythropolis. Restriction maps were constructed for clones pMP1 and pMCX (DNA from Mycobacterium avium). Clone pMP1 was sub-cloned and the fragment carrying the EryR determinant (~2.4 kb) was sequenced. Analysis revealed 2 open reading frames (ORF1 and ORF2). ORF1 showed highest similarity to an FixB/FixA proteins and ORF2 showed similarity to a methyl transferase. Key words: antibiotic resistance,erythromycin, Gram positive DNA / Prof. E.R. Dabbs

>Magister Scientiae - MSc / Drug-resistant infections are a global health crisis and drastically hinder the treatment options to effectively combat disease. Today, natural products remain an important source of novel drug candidates. Micro-organisms, in addition to being a source of bioactive natural products, represent a sustainable source of these compounds. As the marine environment is largely underexplored, the oceans represent a potential source of novel NPs. This study aimed at the discovery of novel NPs from bacteria associated with novel marine invertebrate species endemic to the South African coast, including a sponge Spongia (Spongia) sp. 001RSASPN and a tunicate, Pseudodistoma africanum Millar, 1954. The methodology comprised of culture-dependent and culture-independent strategies. The former involved the isolation of bacteria associated with the invertebrate species and subsequent screening for anti-microbial activity against a panel of indicator strains including a multi-drug resistant E. coli strain. Anti-bacterial activity was detected in 6.1% and 4% of bacterial isolates from the sponge and tunicate isolates respectively. The culture-independent strategy involved the use of PCR to select bioactive strains likely to contain novel NRPS or PKS secondary metabolite pathways. An NRPS A- domain exhibiting low sequence identity (65%) to reference sequences in the NCBI database was amplified from isolate PE8-15, a strain belonging to the genus Bacillus. This predicted a novel NRPS pathway within this strain. In addition, this isolate exhibited the most diverse anti-microbial profile including anti-bacterial and anti-fungal activity (A.fumigatus ATCC 46645). Therefore, as the most promising candidate, the genome of PE8-15 was sequenced following which 10 secondary metabolite pathways including bacteriocins (5), NRPS (3), siderophore (1) and a terpene pathway were identified. The A-domain amplified from PE8-15 originated from Cluster 4, and NRPS pathway predicted to encode a lipopeptide. Lipopeptides are an important class of compounds with a range of industrial applications in the pharmaceutical, cosmetic as well as food industry. The identification of potentially novel secondary metabolite pathways from even well- studied groups of organisms demonstrates the importance of sequence-based methods in natural product discovery. Furthermore, this study highlights the South African coast as a rich source of microbial natural products and should be exploited further for drug discovery.

Magister Public Health - MPH / The introduction of antiretroviral therapy (ART) brought new hope to HIV patients as it has transformed a fatal disease to a chronic manageable condition. In 2009 there were over 920,000 Malawians infected with HIV and 110,000 new infections. Malawi like other countries in the sub-Saharan Africa has made great strides in ensuring access to ART. The government of Malawi introduced free antiretroviral therapy (ART) in June 2004. By 2010, a total of 250,987 patients in the country were receiving ART. The success of ART requires, amongst others, a sustained adherence rate to medication of more than 95% to prevent viral replication and the development of drug resistant HIV strains. Identifying the factors that influence adherence is essential for the long-term success of public ART programmes. This study explored patient, socio-economic, cultural, and religious and health systems factors that influence adherence to ART in Zomba district in Malawi. An explorative qualitative study was conducted amongst ART patients and health workers in four health facilities in Zomba district of the Southern Region of Malawi. Data collection was through individual in-depth interviews with 25 ART patients and semi-structured key informant interviews with 13 health workers that were actively involved in the ART programme. Data was audio-recorded and transcribed verbatim. Thematic and content analysis of transcribed data was done. The study found high individual commitment, having social support from family and friends and continuous good counselling to be facilitators to adherence to ART. HIV-related stigma and discrimination, none disclosure of HIV status, lack of partner support, travelling to attend funerals and religious beliefs were noted barriers to adherence. Health system factors such as congestion in the clinic, negative staff attitudes and a lack of privacy at the pharmacy were also identified as barriers to clinic attendance and keeping appointments. Although pill burden was not mentioned, patients reported drug reactions as a barrier to adherence. Although there is good road network in the district, transport cost was still mentioned as a hindrance to treatment adherence. Treatment success was reported to be both a facilitator and a barrier to adherence. HIV-related stigma and discrimination among people need to be addressed to increase support to PLWHIV and encourage disclosure of HIV status. The improvement of the socio-economic status of ART patients needs to be addressed to reduce dependence on support from other people and provide money to make follow-up appointments. The health systems need to reduce clinic congestion and waiting times so that patients are not deterred from accessing ART.

No description available.