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<p>The current rise in multi-drug resistant Gram-negative bacterial infections is of particularconcern. Gram-negative pathogens are difficult to treat due to their intrinsic resistome.The outer membrane (OM) of Gram-negative bacteria serves as a permeability barrier tomany antibiotics, due in large part to the lipopolysaccharide (LPS) component that isunique to these organisms, and in addition to, the OM is lined with a number of multidrugresistant efflux pumps. As the clinical effectiveness of first line therapies declines inthe face of this resistance, novel strategies to discover new antibiotics are required. Theidentification of new antibiotic targets is one method currently being applied to meet thischallenge. This work examines the permeability barrier of Escherichia coli as a possibletarget for antibiotic adjuvants. A structure-function analysis of GmhA and GmhB, whichcatalyze the first and third conserved steps in LPS ADP-heptose biosynthesis, wasperformed. The active site residues of each of these enzymes were identified viacrystallographic, mutagenic, and kinetic analyses. Potential mechanisms have beenproposed, offering insight into the function of these potential adjuvant targets. In addition,a whole screen of E. coli was performed to identify compounds that potentiatenovobiocin, an antibiotic with limited activity against Gram-negative pathogens due toOM permeability. Four small molecules were found that were able to synergize withnovobiocin. One of these, A22, is known to alter bacterial cell shape, suggesting a newpathway for antibiotic adjuvants to combat Gram-negative infection. Together, thesestudies highlight the varied targets available for novel therapeutic strategies.</p> / Doctor of Philosophy (PhD)

Widespread antimicrobial resistance, particularly in Gram-negative pathogens, is a serious threat facing the global community. Aminoglycosides are inactivated by enzymes such as aminoglycoside N-acetyltransferase-3 (AAC(3)) and O-nucleotidyltransferase-2” (ANT(2”)), while the New Delhi metallo-b- lactamase-1 (NDM-1) degrades carbapenems. Inhibition of these enzymes should result in bacteria becoming once again susceptible to aminoglycosides and carbapenems. This thesis describes the development of inhibitors to these enzymes, in an effort to rescue the utility of aminoglycoside and carbapenem drug classes through adjuvant therapy. High-throughput screening of protein kinase libraries identified two AAC(3)-Ia inhibitors with a common 3-benzylidene-2-indolinone core. New methods for purification of AAC(3)-Ia and monitoring its activity were developed. A chemical library was built around this scaffold and assessed for SAR. It was found that the initial hit (Z)-methyl 3-(3,5-dibromo-4-hydroxybenzylidene)-2- oxoindoline-5-carboxylate was the most active against AAC(3)-Ia, and alterations to either the 3,5-dibromo-4-hydroxybenzyl warhead or methyl ester substituent resulted in a decrease in activity. Previous whole-cell screening had identified two protein kinase inhibitors with a biphenyl isonicotinamide scaffold as inhibitors of ANT(2”)-Ia. A convergent parallel synthesis was developed, involving Suzuki and amide couplings and protecting group strategies. This methodology was used to assemble a focused chemical library for SAR analysis. Stepwise removal of extraneous complexity from the initial hits yielded a selective ANT(2”)-Ia inhibitor which demonstrated in vivo synergy with gentamicin. Aspergillomarasmine A (AMA) is a natural product with activity against NDM-1. Several derivatives of AMA have been synthesized to assess SAR, but the specific contributions of individual carboxylic acids have yet to determined due to difficulties accessing position 6. A synthetic approach was developed via reductive amination using Garner’s aldehyde as a serine equivalent. This strategy was used to synthesize an AMA analog with a hydroxyl group in place of the carboxylic acid in position 6. Additionally, an imine-promoted isomeric resolution was discovered. / Thesis / Doctor of Philosophy (PhD) / Antibiotics, such as aminoglycosides and carbapenems, are losing their effectiveness against bacteria responsible for deadly diseases. This is often due to resistance enzymes such as aminoglycoside N-acetyltransferase-3 (AAC(3)) and O- nucleotidyltransferase-2” (ANT(2”)), which inactivate aminoglycosides, and the New Delhi metallo-b-lactamase-1 (NDM-1), which destroys carbapenems. If these enzymes are blocked, the antibiotics should work against bacteria again. In order to develop compounds that will inhibit these enzymes, sets of similar compounds are made and tested. Patterns of what chemical groups improve or worsen inhibitory activity are noted and used to make another set of compounds in an iterative process. This thesis describes the development of inhibitors of AAC(3)-Ia and ANT(2”)-Ia by this process. Additionally, a specific compound was made to test if a particular chemical group has a role in inhibiting NDM-1.

Graduation date: 2004

Cystic fibrosis (CF) is an autosomal recessive disorder affecting 70,000 individuals worldwide. This disease is characterized by the buildup of mucus in the airways leading to chronic lung infections resulting in pulmonary failure and death in 95% of CF patients. Routine surveillance of CF pathogens using traditional microbiology culture guides management and treatment of CF patients. Molecular profiling studies have revealed emerging pathogens that may play a role in CF lung disease by either directly causing infection or upregulating the virulence factors of classic CF pathogens, such as P. aeruginosa; however, routine CF culture protocols have not been modified to detect these organisms. The goal of this study was to expand the data relevant to the use of microbiology cultures for the management and treatment of CF patients at Virginia Commonwealth University Medical Center (VCUMC) by directly selecting for emerging CF pathogens in culture. This was accomplished by developing,optimizing, and implementing an agar to select for colistin-resistant non-fermenting Gram- negative rods (NF GNRS). In addition, McKay agar and anaerobic media were utilized to recover members of the Streptococcus anginosus group (SAG) and anaerobes in CF respiratory samples. The prevalences of SAG, anaerobes, and colistin-resistant NF GNRs recovered on study media from 75 adult and pediatric CF patients at VCUMC were 17.33%, 41.33%, and 4% respectively. Approximately 62% of patients culture-positive for SAG were also infected with P. aeruginosa and 53.8% of SAG recovered in culture were from CF patients experiencing PE. These findings further support the claim that interspecies interactions among emerging and classic CF pathogens may result in periods of clinical instability or PE. Twenty-eight of the 75 patients were culture-positive for Veillonella species, with the majority of samples collected during a period of surveillance. Four colistin-resistant NF GNRs were isolated on the study media alone. The selective nature of the study media prevented the mixed respiratory flora and classic CF pathogens from overgrowing and obscuring the growth of these colistin-resistant NF GNRs. The presence and role of emerging pathogens in the CF patient population at VCUMC warrants further investigation; therefore, the routine culture protocol needs to be revised to recover and select for those organisms thought to play a role in PE and lung function decline.

A linhagem queratinolítica Chryseobacterium sp. Kr6 mostrou-se com possibilidade de aplicação em processos envolvendo queratinólise, principalmente na hidrólise de penas de frango e depilação de couro bovino. No presente trabalho avaliou-se o efeito da composição do meio sobre o crescimento e atividade proteolítica deste isolado e uma protease queratinolítica (queratinase) foi purificada e caracterizada. O microrganismo mostrou-se adaptado à utilização de queratina como substrato durante o crescimento, produziu diferentes proteases dependendo do meio utilizado e a maior atividade proteolítica foi atingida quando utilizado meio de cultivo com penas como única fonte de carbono e nitrogênio. A adição de fonte extra de nutrientes resultou em uma parcial repressão catabólica. Uma protease extracelular (Q1) foi purificada cerca de 14 vezes utilizando cromatografia de interação hidrofóbica em Phenyl-Sepharose CL 4B e gel filtração em Superose H12R. Q1 mostrou ser uma proteína monomérica com peso molecular de 64 KDa determinado por SDS-PAGE e pH e temperatura ótimos de 8,5 e 50°C respectivamente. O perfil de inibição indica tratar-se uma metaloprotease e as seqüências internas dos peptídeos resultantes de digestão tríptica mostraram homologia ao sítio ativo e de ligação ao Zn da família M14 (Carboxipeptidase). A atividade proteolítica foi estimulada pela presença de íons Ca2+ e Mg2+ e inibida por Cu2+, Zn2+, Al2+, Hg 2+ e agentes redutores. Q1 apresentou atividade queratinolítica sobre o substrato keratin azure, mas não foi capaz de hidrolisar penas de frango sugerindo a necessidade de outras enzimas durante o processo de degradação de penas. Utilizando os iniciadores degenerados desenhados com base na seqüência dos peptídeos, foi amplificado um fragmento de 470 pb correspondente a uma região do possível gene desta metaloproteína utilizando DNA e cDNA como molde. A seqüência do fragmento pode estar sendo expressa, mas não apresentou similaridade e homologia a proteínas conhecidas e portando, indicativa de uma nova metaloprotease. / The strain Chryseobacterium sp. kr6 shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers and de-hairing of bovine pelts. The effect of media composition on the protease production and growth by this strain was studied and a keratinolytic protease (keratinase) was purified and characterized. The strain was adapted to use keratin as substrate to growth, produced different proteases in different media composition and the higher proteolytic activity was reached when used feather as only source of carbon and nitrogen. The addition of sources of nutrients has resulted in partially repressed catabolism. An extracellular protease Q1) was purified 14-fold by chromatography using the hydrophobic interaction Phenyl-Sepharose CL 4B column and gel filtração in Superose 12HR. SDS-PAGE indicated that the Q1 is a monomeric protein with molecular mass of 64 KDa. and optima pH and temperature were 8,5 e 50°C, respectively. The inhibition profile indicates to be a Zn-metalloprotease and analysis of tryptic peptides sequence revealed sequence homology to the conserved active site and Zn binding site, which may characterize keratinase Q1 as a member of M14 metalloprotease family (Carboxipeptidase). The activity was stimulated by of Ca2+ and Mg2+ and inhibited by Cu2+, Zn2+, Al2+, Hg 2+ and reducing agents. Q1 presented keratinolytic activity under substrate keratin azure, but was unable to hydrolyze poultry feather, suggesting the requirement by other enzymes in the feather hydrolysis mechanism. Degenerate primers amplified a 470 bp, corresponding to a probable gene region of this metalloprotein, with DNA and cDNA. The sequence is being expressed but do not showed similarity and homology to known proteins, thus indicating a new metalloprotease.

O estresse oxidativo, causado por níveis aumentados de espécies reativas de oxigênio (ROS), pode causar danos celulares. Várias enzimas, como as subunidades da alquil-hidroperóxido redutase (AhpC e AhpF) e as superóxido dismutases (SOD), são responsáveis por remover as ROS. Os mecanismos de regulação da expressão gênica de C. crescentus para os genes ahpC, sodA, sodB e sodC, foram analisados com fusões de transcrição ao gene repórter lacZ, permitindo a quantificação da expressão por ensaios da atividade de b-galactosidase, e RT-PCR quantitativo. As culturas foram cultivadas em meio PYE ou M2 e a expressão de cada gene foi avaliada na presença de peróxido de hidrogênio (H2O2), tert-butil hidroperóxido (tBOOH), paraquat, menadiona, pirogalol, FeSO4 ou DDPi. Em C. crescentus ahpC é induzido por peróxidos e regulado por OxyR. As SODs são induzidas principalmente por superóxidos, sodB e sodC possuem indução de fase na fase estacionária e possivelmente estão sob o controle do sigma J, enquanto o gene sodA é regulado por sigma F e sigma J. / Oxidative stress, caused by increased levels of reactive oxygen species, can lead to damage in all cellular components. Several enzymes, as subunit of alkyl hydroperoxide reductase (AhpC and AhpF) and superoxide dismutases (SOD), are responsible for removing ROS. Mechanisms of gene expression of C. crescentus for genes ahpC, sodA, sodB and sodC, were evaluated with transcription fusions with the lacZ reporter gene were constructed, allowing the quantification of expression by b-galactosidase activity assays, furthermore we analyze of the gene expression by qRT-PCR assay. The cultures were grown in PYE and M2 media, and gene expression was evaluated in the presence of hydrogen peroxide (H2O2), tert-butyl hydroperoxide (tBOOH), paraquat, menadione, pyrogallol, FeSO4 or DPPi. In C. crescentus ahpC is induced by peroxides and is under the control of OxyR. The SODs are mainly induced by superoxide, sodB and sodC are induction in stationary phase and are possibly under the control of sigma J, while the sodA gene is regulated by sigma F and sigma J.

O presente trabalho teve como objetivo realizar um estudo de vigilância epidemiológica de bactérias MR em isolados obtidos de amostras de búfalo de bubalinocultura e em animais de estimação apresentando sinais e sintomas clínicos de infecção urinária. O estudo relata resultados inéditos referentes à disseminação de bactérias MR, com alto índice de resistência a antimicrobianos de uso clínico e do agronegócio, constituindo o primeiro reporte mundial da emergência de cepas de Escherichia coli produtoras de <font face=\"Symbol\">b-lactamases de amplo espectro (ESBL) do tipo CTX-M-8 e AmpC plasmidial (pAmpC) CMY-2 na bubalinocultura e a presença de cepas de E. coli produtoras de ESBL do tipo CTX-M-15, CTX-M-8 e CTX-M-2, e pAmpC CMY-1, CMY-2 e DHA-1 em animais de companhia é relatada pela primeira vez no Brasil. Nos isolados de E. coli ESBL positivos, não foi constatada relação clonal. As cepas isoladas de búfalos pertencem aos grupos A e B1 e em animais de companhia foram identificados predominantemente os grupos filogenéticos de alta virulência B2 e D. / This study aimed to conduct an epidemiological surveillance on MDR among Gram-negative bacilli recovered from samples from buffalo and in pets exhibiting signs and symptoms related to urinary tract infection. The study reports the spread of MDR bacteria exhibiting a high resistance profile to veterinary- and human-use <font face=\"Symbol\">b-lactams and quinolones, in livestock of buffalos and in pets, constituting the first worldwide report of CTX-M-8-type extended-spectrum <font face=\"Symbol\">b-lactamase (ESBL)- and CMY-2-type plasmid AmpC (pAmpC)-producing E. coli strains in buffalo. Moreover, to the best of knowledge, this is the first report of CTX-M-15-, CTXM-8-, CTX-M-2, CMY-1, CMY-2- and DHA-1-producing E. coli strains in pets in Brazil. With respect to the origin of resistance, we found no clonal relatedness among MDR. E. coli isolates from buffalos belonging to groups A and B1 and in companion animals, the phylogenetic analysis of virulence in E. coli denoted the predominance of the highly virulent phylogenetic groups B2 and D.

A cana-de-açúcar (Saccharum sp.) é uma planta que ocupa posição de destaque entre as culturas de importância econômica no cenário internacional, principalmente no Brasil, que é o maior produtor mundial. O estudo da diversidade microbiana associada a plantas, principalmente aquelas com interesse comercial, apresenta-se como importante alternativa para melhorar as características e a sustentabilidade destas culturas. Neste sentido, bactérias endofíticas e rizobactérias tem sido alvo de muitos estudos, pois apresentam efeitos benéficos para as plantas, como promoção de crescimento e inibição de patógenos. Um dos grupos de bactérias que colonizam a cana-de-açúcar é composto por espécies do gênero Burkholderia. Este gênero é composto de bactérias que podem ser encontradas em diferentes nichos ecológicos, como o solo, a água, ou em associação com plantas, fungos, e outros animais, além de humanos. Na interação bactéria-planta, as espécies de Burkholderia podem colonizar a rizosfera e o interior das raízes hospedeiro, onde podem estimular o crescimento do vegetal, contribuir para sua nutrição, como também protegê-lo da ação de fitopatógenos. Sendo assim, isolados de Burkholderia spp. do interior das raízes (endofíticos) e da rizosfera de cana-de-açúcar foram avaliados quanto à capacidade de fixar nitrogênio, produzir AIA, sideróforos, enzimas de interesse biotecnológico, solubilizar fosfato inorgânico e inibir patógenos desta cultura. Estes isolados também foram caracterizados geneticamente por análise de restrição e seqüenciamento dos genes 16S rDNA e gyrB, além da caracterização genotípica por BOX-PCR. Os resultados indicam que os isolados possuem potencial para promoção de crescimento vegetal, inibição de patógenos e produção de lipases. Filogeneticamente, a maioria dos isolados pertencem ao complexo Burkholderia cepacia com similaridade à B. cepacia e B. cenocepacia. Considerando a ocorrência dos isolados como endófitos ou rizosféricos, as metodologias fenotípicas e genotípicas não foram capazes de distinguir os membros componentes destas comunidades. Este trabalho evidencia a ampla associação deste grupo com cana-de-açúcar, e destaca as possíveis aplicações que tais bactérias podem ter no cultivo e sustentabilidade desta cultura. / Sugarcane (Saccharum spp.) occupies a position of prominence among the economically important crops in the international scene, mainly in Brazil, which is the world\'s largest producer. The study of microbial diversity associated with plants, especially those with commercial interest, presents itself an important alternative to improve the performance and sustainability of these crops. In this sense, endophytic bacteria and rhizobacteria has been target of many studies, since they have beneficial effects for plants, such as plant growth promotion and inhibition of pathogens. One of the bacterial groups that colonize sugarcane is composed of species of the genus Burkholderia. This genus is composed of a bacterium that can be found in different ecological niches, such as soil, water, or in association with plants, fungi and other animals, as well as in humans. In the association bacterium-plant, the species of Burkholderia can colonize the rhizosphere and the inside of the host roots, which can stimulate the growth of the plant, contributing to its nutrition, but also protects it from the action of phytopathogens. Thus strains of Burkholderia spp. isolated from the inside of the roots (endophytic) and from the rhizosphere of sugarcane were evaluated for the ability to fix nitrogen, produce IAA, siderophores, enzymes of biotechnological interests, solubilize inorganic phosphate and inhibits pathogens of the same crop. These strains were also genetically characterized by the analysis of enzymatic restriction and sequencing of 16S rDNA and gyrB genes, in addition to the characterization by genotypic technique BOX-PCR. The results indicate that the strains have potential for plant growth promotion, inhibition of pathogens and production of lipases. Phylogenetically, the isolates were affiliated to Burkholderia cepacia complex, with mainly similarity to B. cepacia and B. cenocepacia. Considering the occurrence of isolated as endophytes or rhizosphere, the genotypic and phenotypic methods were not able to distinguish the members of these communities. This research work demonstrates the broad association that this group has with sugarcane, and highlights the possible applications that these bacteria may have in cultivation and sustainability of this crop.