The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

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No. of bitstreams: 1 Tese - Milena Rodrigues Soares Mota.pdf: 10830184 bytes, checksum: b6f2977c2b749cd675523693c33de0ff (MD5) Previous issue date: 2011-03-25 / Não Informada / This study describes the therapeutic potential of extracts and standardized fractions of Arrabidaea chica leaves. A. chica is a Bignoniaceae popularly known as “crajiru”. The genus Arrabidaea occurs in Tropical America, from the Southern Mexico to the Southern Brazil. The red color of its dried leaves is attributed to two flavonoidal pigments: carajurina (main pigment) and carajurona. Chemical studies described the isolation of saponins and flavonoids from the plant leaves; purified 3- desoxyanthocyanidins were reported as anti-inflammatory. The infusion or decoction of the plant leaves is used in the folk medicine to treat anemia, inflammation and in skin wound-healing. In this work, the antimicrobial activity of the standardized extracts and fractions of A. chica cultivated at Embrapa Amazônia Ocidental, Manaus, were evaluated against fungal and bacterial microorganisms grown either from local domestic dogs and cats, or from human samples supplied by the Microorganism Collection of FIOCRUZ, in Manaus, Brazil. The plant dried leaves were extracted with increasing polarity solvents and progressively purified in preparative thin layer chromatography (TLC) and silica-gel column; the semi-purified extracts were standardized in TLC. The agar diffusion method; bioautography; minimum inhibitory concentrations tested against Staphylococcus epidermidis (CBAM 293), Staphylococcus aureus (CBAM 324), Pseudomonas aeruginosa (CBAM 232), Escherichia coli (CBAM 002), Trichophyton mentagrophytes (CFAM 1288), Microsporum canis (CFAM 1289), Malassezia pachydermatis (CFAM 1290) e Candida albicans (CFAM 1285). The standardized fractions were effective against all these microorganisms, but more intensively against Microsporum canis and Staphylococcus epidermidis. The results might favour the use of the standardized sub-fractions of A. chica as topic phytotherapic agent to treat canine external otitis. So far oleanolic and ursolic acids were identified as the main compounds in the active semi-purified fraction but other compounds of the leaves extract were not discarded. Later studies will consider the veterinarian use of the standardized extract, of the active pure entities and the convenience of the natural active antibiotic mix. / Este estudo analisou o potencial terapêutico de extratos e frações purificadas da planta amazônica Arrabidaea chica visando seu uso tópico como medicamento e eficácia comprovada em doenças cutâneas. A. chica Verl., é uma Bignoniaceae conhecida popularmente como crajiru. O gênero Arrabidaea ocorre na América tropical, do sul do México ao sul do Brasil. A cor avermelhada da folha seca e sua propriedade tintorial são devidas a dois pigmentos flavonoídicos: a carajurina, que é o pigmento principal e a carajurona. Dela foram isolados saponinas e flavonóides; As 3-desoxiantocianidinas, descritas na planta parecem possuir atividade antiinflamatória. A medicina popular utiliza o decocto ou a infusão das folhas para tratar anemia, inflamações e na cicatrização da pele. Neste trabalho, a atividade antimicrobiana de extratos e frações padronizadas da A. chica cultivada na Embrapa Amazônia Ocidental, em Manaus/AM, foi avaliada contra fungos e bactérias de amostras clínicas coletadas de animais domésticos e contra amostras humanas depositadas na coleção de Microrganimos da FIOCRUZ, Manaus/AM. Para isso, as folhas secas da planta foram extraídas com solventes de polaridade crescente, as frações foram progressivamente purificadas em cromatografia de placa ou coluna de sílica-gel, os extratos semi-purificados foram padronizados em cromatografia líquida de alta eficiência acoplada à espectrometria de massas. Os testes de difusão em ágar, bioautografia e concentração inibitória mínima foram usados para avaliar a atividade antimicrobiana das subfrações padronizadas frente aos microrganismos Staphylococcus epidermidis (CBAM 293), Staphylococcus aureus (CBAM 324), Pseudomonas aeruginosa (CBAM 232), Escherichia coli (CBAM 002), Trichophyton mentagrophytes (CFAM 1288), Microsporum canis (CFAM 1289), Malassezia pachydermatis (CFAM 1290) e Candida albicans (CFAM 1285). As frações padronizadas foram ativas contra todos esses microrganismos, com melhores resultados contra M. pachydermatis e S. epidermidis. Os resultados foram favoráveis à utilização das subfrações padronizadas na formulação de um produto fitoterápico para uso tópico em otite canina. Nas frações ativas foram identificados os ácidos oleanólico e ursólico. Estudos posteriores deverão avaliar a possibilidade de uso humano das frações purificadas ou dos compostos identificados.