Graduate Thesis Or Dissertation
 

A study of the lipids of Pseudomonas aeruginosa sensitive and resistant to chloramphenicol and quaternary ammonium compound

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  • From a population of cells of Pseudomonas aeruginosa sensitive 33 parts per million (ppm) and 0.1 mg per ml of quaternary ammonium (QAC) and chloramphenicol respectively, pure strains resistant to 750 ppm and 50 mg per ml of these antibiotics were isolated. Lipids from the sensitive and resistant cells grown under various environmental conditions were extracted and characterized. Increased lipid biosynthesis as a possible factor in the resistance of this bacterium to chloramphenicol and QAC also was investigated. Sensitive cells, QAC resistant cells grown in the absence of germicide, resistant cells grown in the presence of QAC, and chloramphenicol resistant cells grown in the absence of and presence of the antibiotic were grown in ten- to 30-liter volumes at room temperature, 32 C, and also in a New Brunswick Fermacell fermentor at 37 C. Cells were washed three times and lyophilized. Lipids present were extracted from the lyophilized cells, weighed, and characterized by thin layer and gas liquid chromatography. Total, free and bound lipids were quantitated; free fatty acids from these lipid classes also were quantitated and identified. Resistant cells grown in media containing 207 parts per million QAC at room temperature contained 25% more total lipid than sensitive cells. Resistant cells grown in the absence of QAC at room temperature retained their resistance but did not produce more lipid than sensitive cells. Cells grown at 32 C in the presence of QAC contained 8% more lipid than sensitive cells but less than cells grown at room temperature under the same conditions. There was no increased lipid production in QAC resistant cells grown at 37 C in a Fermacell fermentor. Cells grown in chloramphenicol-containing medium (2.5 mg per ml) at room temperature developed 28% more lipid than sensitive cells grown in antibiotic-free medium. Thin layer chromatography plates of the lipid classes extracted from various cell types, contrary to total lipid analyses, revealed no significant differences in amount and type of lipid present. Also, separation and analysis of the phospholipid fraction demonstrated no alteration in the kind or amount of phospholipid produced by the cells as a consequence of mutation to antibiotic resistance. Chromatograms of methyl esters of fatty acids of phospholipid, free fatty acid, triglyceride and hydrocarbon fractions of the various cell types indicated that the same fatty acids were present in the lipid extracts. There was a 10% increase of the C-18:1 fatty acid in both the phospholipid and free fatty acid fractions of the free lipids of cells grown in a Fermacell fermentor in the presence of chloramphenicol or QAC. However, there was no compensatory decrease in any single lipid but instead there was a general decrease of lipid. Sensitive Ps, aeruginosa cells contained 14% free lipid and 3 to 6% bound lipid. The major phospholipid was phosphatidylethanolamine which comprised 59% of the total phospholipid fraction of the sensitive cells. C-16 amounted to 34%, C-18:1 19% and 16:1 and C-18 each approximately 5% of the fatty acids present. These four fatty acids accounted for 63% of the lipid present in the sensitive cells. Cells resistant to QAC produced no pigments; those resistant to chloramphenicol produced two pigments, pyorubrin and fluorescin; sensitive cells produced only one pigment, pyocyanin.
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