The characteristics and expression of laboratory derived chloramphenicol (CM) resistance in P. aeruginosa were examined. Resistant strains exhibiting single cell resistance of 1.5 to 2 mg/mL were readily isolated following one passage in CM at 150 to 1000 (mu)g/mL. Isogenic strains, selected on CM at 150 and 500 (mu)g/mL were chosen for detailed study. Resistance was not a consequence of drug detoxification or altered sensitivity of the target site. The resistant strains exhibited unusual phenotypic properties including pronounced variations in growth rate, CM susceptibility and cell morphology as a function of the composition of the growth medium. Growth in CM also resulted in significant alterations in amino acid transport and respiratory capacity, the extent of which varied with the strain, the growth medium and the concentration of CM. These drug and medium-dependent alterations were determined to reside in an increased and highly specific requirement for Ca('2+), Mg('2+), Mn('2+) or Sr('2+). Manipulation of the divalent cation concentration of a variety of growth media resulted in dramatic alterations in growth rate, resistance and amino acid transport. Ca('2+) was significantly more effective than the latter three ions. The expression of native and plasmid-mediated CM resistance was also modified by the external concentration of divalent cations. In view of the nature and specificity of the cation requirement, it was concluded that (1) divalent cation-mediated alterations of outer membrane permeability are fundamental to the expression of native and acquired CM resistance in P. aeruginosa; (2) laboratory-derived CM resistance involves envelope changes, such that interaction with divalent cations promotes more effective exclusion of CM. The latter conclusion is supported by other divalent cation-dependent alterations in envelope function in the resistant strains.

The characteristics of the effects of chloramphenicol on Pseudomonas aeruginosa were examined. Resistant strains were easily isolated following a single passage in chloramphenicol at 150 $ mu$g/ml to 500 $ mu$g/ml. Drug detoxification or altered sensitivity of the target site could not be the mechanism of resistance. This resistance to chloramphenicol was correlated with the addition of an outer membrane protein with a molecular weight of 49 kDa and the loss of two outer membrane proteins, one with the molecular weight of 19 kDa and the other of about 10 kDa. The highly specific requirement of the resistant strains for Ca$ sp{2+}$, Mg$ sp{2+}$, Mn$ sp{2+}$ or Sr$ sp{2+}$ described by Irvin and Ingram (1982) was confirmed by the observation that the outer membrane of the resistant cells contained twice as much Mg$ sp{2+}$ cation as the sensitive cells. Many other experiments designed to observe the effects of chloramphenicol on the outer membrane of P. aeruginosa failed. It was concluded that the observations made in this study strongly suggested a "re-structuring" of the outer membrane of P. aeruginosa, rendering the resistant cells more impermeable to chloramphenicol.

This diploma thesis addresses the presence and determination of chloramphenicol residues in biological materials. The theoretical part presents the literature retrieval containing information about veterinary medicaments with the banned use in food producing animals and also the sum of the legislative requirements concerning the presence of these substances in foodproducts and raw food materials of animal origin. The comparison was carried out between the existing analytical methods used for the determination of chloramphenicol residues in different biological materials, which are altogether based on the solid phase extraction for the extract cleaning and the new procedures for sample preparations using columns where the sorbent performs on the molecularly imprinted polymers principle.

A 27-year-old Peruvian woman living with human immunodeficiency virus (HIV) in clinical stage B3 and not on antiretroviral therapy presented with a ten-day history of fever, chills, night sweats and a two-day history of skin lesions. On physical examination, several erythematous-purplish lesions were found on the face and legs. Meningococcal infection was suspected and ceftriaxone was started. Blood culture grew nontyphoidal Salmonella enterica. A biopsy of the skin lesions showed leukocytoclastic vasculitis (LCV); therefore, corticosteroids were added. After two weeks of antibiotic and corticosteroid treatment, the lesions had resolved, but they recurred two days after treatment with prednisone was stopped. Corticosteroids and combination antiretroviral therapy were started simultaneously and the lesions resolved without recurrence. HIV infection has been associated with higher rates of skin lesions in salmonellosis. LCV has been described both in the setting of HIV infection and salmonellosis. However, our review of the literature found no previous cases of LCV in concurrent HIV and salmonellosis. / Revisión por pares / Revisión por pares

Thioesters are highly reactive centers for acyl-CoAs which allows them to be utilized in a variety of differing enzyme chemistries. As a result of this reactivity, structure-function studies of enzymes using acyl-CoA substrates is difficult. When acyl-CoAs are used in structure-function studies, they often result in a hydrolyzed CoA substrate fragment bound in the active site or require only one of multiple substrates in order to be bound. This results in a lack of information regarding enzyme interactions with the key thioester and acyl chain. To overcome this challenging problem, I have synthesized acetyl- and malonyl-CoA analogs where the thioester has been replaced by an ester (oxygen), amide (nitrogen), or carbonyl (carbon) in a way that is easier, cheaper, and more efficient than performed previously. In addition, we used our synthetic analogs to study a enzymes which span different acyltransferase mechanisms in a combination of kinetics and structure. With this work, it was determined that the amide analogs were stable in all enzymes it was utilized for, while the ester analogs were mostly stable, except the acetyl analog in KasIII, where it acted as a pseudo substrate. As such, these synthetic analogs may have future potential in either type of enzyme for structure-function studies, albeit limited for the acetyl ester analog.

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published_or_final_version / Pathology / Master / Master of Philosophy