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Mechanisms of resistance to silver in Pseudomonas aeruginosa

Hitch, Geeta; (1999) Mechanisms of resistance to silver in Pseudomonas aeruginosa. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

A silver-sensitive clinical isolate of Pseudomonas aeruginosa with multiple resistance to gentamicin, tobramycin and mezlocillin was subcultured in increasing concentrations of silver nitrate to induce silver resistance. A resistance to silver of 1000μg/ml was achieved over a period of several months. Resistance to silver increased resistance to arsenate, copper, mercury and zinc metal ions with a decrease in the resistance to cobalt and lead ions. The resistance to silver was lost on repeated subculturing of Pseudomonas aeruginosa in the absence of silver; however the multiple resistance to the antibiotics remained stable. The presence of an unstable, non-conjugative plasmid possibly conferring silver resistance was also detected. The growth of cells in the presence of varying concentrations of silver resulted in death of a percentage of the viable population. At the very high concentration of silver (1000μg/ml), the percentage of cell death was 40% within the first hour of exposure to silver with a lag phase of 23 hours which was reduced to 9 hours on repeated subculturing. Silver uptake was extremely rapid in the first hour of exposure. Evidence of possible active efflux of excess silver ions by the Ag-r cells was observed during studies with the uncoupling agent carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). Transmission electron microscopic studies of the silver resistant cells revealed the presence of electron dense particles consisting of silver on the cell surface, in the trilaminar structure of the cell walls and in the cell cytoplasm. The composition of the outer membrane proteins of Ag-r cells differed to that from the control cells. High concentrations of silver were found to be associated with all outer membrane proteins. Analysis of low molecular weight proteins, on Sephadex G50 columns showed these proteins to be associated with increasing amounts of silver which correlated with an increase in the external concentration of silver. A silver binding protein with a MW of 7400 daltons was extracted and analysed for silver binding sites using 1H NMR spectroscopy. The protein was not classified a typical metallothionein due to the presence of several aromatic amino acids. An increase in production of extracellular carbohydrate was observed in Ag-r cells exposed to increasing amounts of silver. Isolation and purification of these exopolysaccharides showed high amounts of bound silver. Scanning electron microscopy showed silver resistant cells surviving in cocoons of extracellular mucopolysaccharide. There appear to be several mechanisms by which silver resistance is conferred in Pseudomonas aeruginosa. One or more of these mechanisms may be expressed by a plasmid conferring silver resistance.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Mechanisms of resistance to silver in Pseudomonas aeruginosa
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Health and environmental sciences; Pseudomonas aeruginosa
URI: https://discovery.ucl.ac.uk/id/eprint/10105266
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