During the investigation of the effect of age upon endogenous metabolism, advanced stationary phase cultures of Pseudomonas aeruginosa were found to be susceptible to cold-shock. The phenomenon was apparent through an increased oxygen uptake and an initial absence of extracellular ammonia during subsequent respiration at 30 C, which were shown to be due to the presence of an oxidizable substrate in the suspending fluid. Intracellular enzymes were released following the exposure of these cells to the cold, and a partial protection against damage was afforded by the addition of magnesium ions to the washing and suspending buffer. The storage of a reserve material for utilization during endogenous metabolism could not be demonstrated in cells grown for various periods of time in a chemically-defined medium which contained glucose in excess of that required for growth. Further, when not previously exposed to the cold, an immediate evolution of ammonia was observed when this organism was respired at 30 C irrespective of the medium in which it was cultured or the age at which it was harvested. The ribosome complement was seen to diminish during the prolonged incubation of cultures grown in the chemically-defined media, and was found to disappear almost completely when 48 hr cells, harvested from defined or complete media, were respired at 30 C for a further 48 hr. Chemical fractionation during the respiration period revealed an increase in the concentration of deoxyribonucleic acid and a decrease in the concentrations of ribonucleic acid and protein. Glucosamine was not found to be a major metabolite in the endogenous respiration of this organism. Progressive starvation resulted in a reduction in the constituitive enzymes and/or cofactors involved in the oxidation of glucose, and an ability to adapt to, and oxidize α-ketoglutarate was evident after a period of starvation which had reduced the ribosome complement to negligible proportions. Endogenously produced ammonia and acid-soluble UV-absorbing material were reincorporated upon the addition of an exogenous substrate following respiration for 48 hr, and a concurrent increase was recorded in the concentration of 50S ribosomes. / Land and Food Systems, Faculty of / Graduate

Pseudomonas aeruginosa was shown to constitutively degrade arginine via the arginine dihydrolase pathway to ornithine, which was converted both to glutamate and to putrescine. The conversion of ornithine to glutamate appeared to be the major route of arginine degradation in this organism, and was induced to higher activity after growth of the cells with arginine as the sole source of carbon and nitrogen. P. aeruginosa did not further degrade putrescine constitutively. However, growth of the cells in arginine resulted in a partial induction of succinic semialdehyde dehydrogenase, an enzyme functioning in putrescine degradation. The anabolic ornithine transcarbamylase of P. aeruginosa was repressed after growth of the organism in the presence of arginine. Pseudomonas putida and Pseudomonas fluorescens also possessed the ability to constitutively convert arginine to putrescine via the intermediates, citrulline and ornithine. However, these organisms did not oxidize arginine to the same extent as did P. aeruginosa. P. aeruginosa grew in a mixture of glucose and arginine in the presence of ammonium ions without exhibiting a diauxie effect. Glucose and arginine were oxidized concomitantly when supplied as a mixed substrate, by both growing cells and resting cell suspensions. However, assimilation studies showed that the two substrates were used to serve somewhat different biosynthetic needs. Growth of P. aeruginosa in arginine caused an increase in the rates of transport of arginine, lysine, ornithine and citrulline. Kinetic studies of arginine uptake demonstrated the presence of two uptake systems with different affinities for arginine. Inhibition studies indicated that arginine was transported by two uptake systems: a permease specific for arginine, and, with a lower affinity, for ornithine; and a general permease, which transported all the basic amino acids. Polyamines appeared to be transported by an uptake system which was induced to higher levels after growth of the cells with either arginine or putrescine as the sole source of carbon and nitrogen. P. aeruginosa was found to maintain a stable pool of putrescine when supplied with exogenous ¹⁴C-arginine or ¹⁴C-putrescine, even when the organism had previously been induced to degrade these substrates. A physical fractionation of the cells indicated that the major portion of this pool was located in the soluble cytoplasm. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Thesis (M.S. )--Marshall University, 2009. / Title from document title page. Includes abstract. Document formatted into pages: contains 155 p. Includes bibliographical references p. 151-152.

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains xv, 205 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 163-190).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains ix, 132 p.; also includes graphics (some col.). Includes bibliographical references (p. 112-132). Available online via OhioLINK's ETD Center

Orientador: Marcelo de Carvalho Ramos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T01:23:41Z (GMT). No. of bitstreams: 1 Cacci_LucianaCamila_M.pdf: 1733904 bytes, checksum: 2616f843895d251e8e236cdd873fe436 (MD5) Previous issue date: 2007 / Resumo: Introdução: As metalo beta-lactamases, presentes em Pseudomonas aeruginosa assim como em diversos microrganismos Gram negativos, são grande ameaça ao tratamento dos pacientes portadores de infecções causadas por este microrganismo. Objetivos: A produção de MBLs e a relação genética foram investigadas em isolados de P. aeruginosa resistentes ao Imipenem, recuperados de infecções hospitalares. Descrição do estudo: Estudo restropectivo em uma amostra de microrganismos. O estudo foi conduzido em dois hospitais universitários, em Campinas. Todos os isolados de P. aeruginosa resistentes ao Imipenem foram coletados de pacientes hospitalizados no período de Março de 2000 a Dezembro de 2004. Métodos: O método da disco-difusão foi utilizado para confirmar a resistência ao Imipenem. O E-test MBL@ foi feito para verificar a produção de MBLs e a Concentração Inibitória Mínima (CIM) do Imipenem. Os ftagmentos das seqüências dos genes blaIMP-I, blaVIM-I, blaVIM-2 e blaSPM-I foram amplificados. Resultados: Cento e vinte e oito isolados resistentes ao Imipenem foram coletados durante o período do estudo. A maioria dos isolados exibiu CIM do Imipenem maior ou igual a 256 ug/mL. A análise por macrorestrição com a enzima SpeI através do Pulsed Field Gel Electrophoresis (PFGE) mostrou um polimorfismo significativo. Apenas 15 cepas puderam ser distribuídas em sete "clusters", seis com dois isolados e um com três isolados. Noventa e oito isolados resistentes ao Imipenem foram triados para a pesquisa da produção de MBL. Setenta isolados apresentaram produção de MBL e o fragmento do gene blaSPM-l pôde ser amplificado em 12 isolados. Nas cepas restantes nenhum outro tipo de MBL referente aos genes blaIMP-l, blaVIM-I, blaVIM-2 foi encontrado. Conclusão: A disseminação de cepas de P. aeruginosa produtoras de MEL genotipicamente heterogêneas foi documentada nos hospitais estudados. Apenas a metalo beta-lactamase SPM-1 foi encontrada entre essas cepas / Abstract: Objective: Genetic relatedness and Metallo-lactamase production 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 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. E-test MBL@ was done to check for MBL production, ando Imipenem MIC's blasPM-l, .blaIMP-l, blaVIM-l and blaVIM-2 sequences were amplified. Results: A sample of 128 Imipenem resistant P. aeruginosa isolates was collected during the study period. Most isolates exhibited Imipenem MIC's > 256 ug/mL. Macrorestriction analysis (Spel) 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-eight Imipenem resistant isolates were screened for MBL production. Seventy isolates showed MBL production, and blasPM-l sequence could be amplified from 12 isolates. In the remaining strains no other MBL-type from blaIMP-l, blaVIM-l and blaVIM-2 investigated in the study was demonstrated. Conclusion: Dissemination of MBL producing-genotypically heterogenous Pseudomonas aeruginosa strains was documented in the hospitals studied. Only SPM-l metallo-_-lactamase was found among these strains / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

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published_or_final_version / Pathology / Doctoral / Doctor of Philosophy