The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed April 27, 2010). Thesis advisor: Dr. Kent S. Gates. Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Corrigenda pasted onto back end-papers. Bibliography: leaves 163-182. Analyses the proteins involved in Shigella flexneri O-antigen biosynthesis at the molecular level in order to gain a more concise understanding of the biosynthesis machinery and how it functions.

Orientador: Angelo Berchieri Junior / Coorientador: Oliveiro Caetano de Freitas Neto / Banca: Wanderley Dias da Silveira / Banca: Gerson Nakazato / Banca: Manoel Victor Franco Lemos / banca: Marcos tulio de Oliveira / Resumo: O tifo aviário, causado por Salmonella Gallinarum biotipo Gallinarum, é uma infecção caracterizada pela alta mortalidade nos lotes de aves suscetíveis acometidos, enquanto S. Gallinarum biotipo Pullorum, o agente da pulorose, infecta as aves de produção industrial com as quais desenvolve relação mais branda. Ainda é escasso o conhecimento sobre os mecanismos moleculares que sustentam essas diferentes interações patógeno-hospedeiro. Nesse estudo, objetivou-se investigar o efeito de deleção parcial das sequências codificantes dos genes idnT (transportador de L-idonato ou D-gluconato), idnO (5-cetogluconato redutase) e ccmH (heme liase necessária na montagem de citocromos do tipo C) sobre a patogenicidade de S. Gallinarum 287/91 (SG287/91), uma vez que seus ortólogos são pseudogenes conservados em S. Pullorum. Os clones mutantes SG∆idnTO, SG∆ccmH e SG∆ccmHidnTO foram obtidos por meio da técnica de mutação sítio-dirigida, denominada de recombinação Lambda-Red e testados em dois experimentos independentes com aves comerciais semipesadas de postura suscetíveis ao tifo aviário. No 1º experimento não se observou alteração da patogenicidade dos clones mutantes após inoculação oral, pois todos os animais infectados desenvolveram sinais clínicos típicos do tifo aviário e vieram a óbito ao longo de 12 dias pós-infecção (dpi). Apesar dos 100% de mortalidade, as infecções desenvolvidas pelos clones SG∆idnTO e SG∆ccmHidnTO levaram os animais a óbito dentro de 48 horas desde o aparecimento d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fowl typhoid, caused by Salmonella Gallinarum biovar Gallinarum, is an infectious disease which elicits high mortality into a flock of susceptible birds whereas S. Gallinarum biovar Pullorum, the aetiological agent of pullorum disease, infects poultry of commercial importance with which such a bacterium sets off a more permissive host-pathogen interaction. Little is known about the molecular mechanisms driving these distinct interplays with the host. Herein, we aimed at investigating the effect of partial deletions in the idnT (L-idonate / D-gluconate transporter), idnO (5-ketogluconase reductase) and ccmH (heme liase involved in the c-type cytochrome maturation) coding sequences on S. Gallinarum 287/91 (SG287/91) pathogenicity since they are conserved pseudogenes in S. Pullorum genomes. SG∆idnTO, SG∆ccmH and SG∆ccmHidnTO mutant strains were constructed through a one-step inactivation technique, known as Lambda-Red-mediated recombination, and tested on two independent experiments by using a commercial brown egg-producing layer line susceptible to fowl typhoid. On the experiment 1, no changing was observed in the pathogenicity of the mutant strains upon oral inoculation as the infected animals developed typical fowl typhoid clinical signs and died along 12 days post-infection (dpi). In spite of causing 100% mortality, SG∆idnTO and SG∆ccmHidnTO killed all the animals within 48 hours since the clinical signs appearance while SG287/91 did so in 6 days, indicating an increased virulence by these mutant strains. On the experiment 2 every mutant strain were able to invade the host system from the intestine albeit SG∆idnTO and SG∆ccmHidnTO were recovered from livers and SG∆idnTO alone from spleens at higher numbers than was SG287/91, supporting the hypothesis of increased virulence for those clones ha... (Complete abstract click electronic access below) / Doutor

Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all cases these virulence factors were significantly decreased in the mutant. Even supplementing with uracil did not attain wild type levels. Starvation of the pyrimidine mutant for uracil caused increased specific activity of the pyrimidine enzymes, suggesting that regulation of the pyrimidine pathway occurred at the level of transcription. This effect has also been reported for P. oleovorans. The present research consolidates the idea that pyrimidine auxotrophs cause decreased pathogenicity in P. aeruginosa. Such a finding may open the search for chemotherapy targets in cystic fibrosis and burn victims where P. aeruginosa is an infecting agent.

Pseudomonas aeruginosa are medically important bacteria that are notorious for causing nosocomial infections. To gain more knowledge into understanding how this organism operates, it was decided to explore the pyrimidine biosynthetic pathway. Pyrimidine synthesis, being one half of the DNA structure, makes it a very important pathway to the organism’s survivability. With previous studies being done on various genes in the pathway, pyrE has not been studied to the fullest extent. To study the function of pyrE, a site directed mutagenesis was done to completely knock out pyrE, which encodes the protein orotate phosphoribosyl transferase that is responsible for converting orotate into orotate monophosphate (OMP). A mutation in this step leads to accumulation and secretion of orotate into the medium. Analyzing virulence factors produced by the mutant and comparing to the wild type, some intriguing features of the mutant were discovered. One of the findings was the over expression of virulence factors pyoverdin and pyocyanin. Pyocyanin over expression, based on the results of this study, is due to the accumulation of orotate while over production of pyoverdin is due to the accumulation of dihydroorotate. The other virulence factors studied were motility assays, exoproducts, and growth analysis. All virulence factor production was reduced significantly in the mutant compared to the wild type. The casein protease assay showed absolutely no production of proteases in the mutant. The conclusion is that orotate accumulation leads to a significant reduction in virulence factor production in Pseudomonas aeruginosa. In addition to that, it was found that excess orotate in the wild type led to a decrease in quorum sensing regulated products.

Orientador: Mara Corrêa Lelles Nogueira / Banca: Dirce Yorika Kabuki / Banca: Mariza Landgraf / Banca: Fátima Pereira de Souza / Banca: Aripuanã Sakura Aranha Watanabe / Resumo: A resistência bacteriana aos antimicrobianos é um importante problema de saúde pública. Nos últimos anos tem sido frequente o isolamento de bactérias resistentes a partir de animais de produção e alimentos de origem animal. O objetivo desse estudo é identificar em Enterococcus spp. isolados de carnes (suína, bovina e frango) e de queijo Minas Frescal, o perfil de suscetibilidade aos antimicrobianos e a diversidade de genes de resistência aos antimicrobianos e de genes de virulência. Os resultados são comparados ao perfil observado em Enterococcus spp. isolados de pacientes internados em um hospital terciário no estado de São Paulo. Foram avaliados 102 Enterococcus spp. isolados de alimentos e 46 isolados clínicos. Entre os isolados de alimentos, 39% apresentaram resistência à tetraciclina, 32% à eritromicina, 17,5% à estreptomicina, 13% à norfloxacina, 10% ao cloranfenicol, 8% à ciprofloxacina e à fosfomicina, 6% à levofloxacina, 5,5% à quinupristina-dalfopristina, 4% à linezolida e à penicilina, 3,5% à moxifloxacina, 3% à ampicilina e 2% à gentamicina. Todos os Enterococcus spp. isolados de alimentos foram sensíveis à teicoplanina e à tigeciclina. Entre os isolados clínicos, 67,5% apresentaram resistência à ciprofloxacina, 56,5% à penicilina, 43,5% à ampicilina, 37% à estreptomicina, 24% à vancomicina e à teicoplanina, e 11% à gentamicina. Cepas multirrestentes foram detectadas entre os isolados de alimentos e de amostras clínicas. Nos Enterococcus isolados de alimentos resistentes à tetraciclina foram detectados os genes tet M, tet L, tet K e tet C. Os genes ant6-Ia e aac(6')-Ie/aph(2")-Ia foram detectados em cepas de alimentos e de origem clínica. Com relação aos genes de virulência, entre os Enterococcus isolados de alimentos, 48% apresentaram o gene efaA, 42% o gelE, 37,5% o ace, 15% o as, 13% o esp e 11% o... / Abstract: Bacterial resistance to antibiotics is a major public health. In recent years there has often been the isolation of resistant bacteria from production and animal food animals. The aim of this study is to identify in Enterococcus spp. isolated from meat (pork, beef and chicken) and Frescal Minas cheese, susceptibility profile to antimicrobials and the diversity of antimicrobial resistance genes and virulence genes. The results are compared to the profile observed in Enterococcus spp. isolated from patients admitted to a tertiary hospital in São Paulo. One hundread two Enterococcus spp. were isolated from food and 46 were from clinical isolates. Resistances to different classes of antimicrobials has been observed, and among the isolates of food were resistant to tetracycline (39%), erythromycin (32%), Streptomycin (17.5%), norfloxacin (13%), chloramphenicol (10%), ciprofloxacin and fosfomycin (8%), levofloxacin (6%), quinupristin-dalfopristin (5.5%), penicillin and linezolid (4%), moxifloxacin (3.5%), ampicillin (3%) and gentamicin (2%). All Enterococcus spp. isolates from foods were sensitive to teicoplanin and tigecycline. Among clinical isolates, were resistant to ciprofloxacin (67.5%), penicillin (56.5%), ampicillin (43.5%), streptomycin (37%), vancomycin and teicoplanin (24%) and gentamicin (11%). Multidrug-resistant strains were detected among isolates from food and clinical samples. In Enterococcus spp. tetracycline resistant, the genes tet M, tet L, tet K and tet C were detected. The genes ant6-la and aac(6')-Ie/aph(2")-Ia were detected in strains of food and clinical origin. Regarding the virulence genes among Enterococcus isolates from food, 48% had the gene efaA, 42% the gelE, 37.5% the ace, 15% the as, 13% the esp and 11% the cyl. Among the clinical isolates, 61% had the ace, 56.5% the efaA, 43.5% the gelE, 11% the cyl and esp, and 6,5% the as. The hyl gene was ... / Doutor

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Abstract not available