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Gentamicin accumulation by sensitive strains of Escherichia coli and Pseudomonas aeruginosa
Abstract:
Gentamicin accumulation with time shows multiphasic kinetics in strains of Escherichia coli and Pseudomonas aeruginosa. All but first phase accumulation may be prevented or reduced by inhibitors of electron transport, by a sulfhydryl poison, by agents which uncouple electron transport and oxidative phosphorylation and by an inhibitor of protein synthesis. The phases of accumulation which are sensitive to these inhibitors are required for loss of cell viability. Gentamicin can be extracted from cells in an unchanged form as judged by paper chromatography and is concentrated 4 to 250 times over extracellular concentrations within the bacterial cell. Gentamicin accumulation has been shown to occur before there is any evidence or release of acid-soluble 3H-adenine from cells. These data demonstrate that productive gentamicin accumulation capable of causing cell death is by active transport.
Insights
Gentamicin accumulation in bacteria occurs via active transport, a process essential for its cell-killing effects. Inhibitors of cellular processes can reduce gentamicin uptake, impacting bacterial viability.
Area of Science:
- Microbiology
- Bacterial Physiology
- Antimicrobial Resistance
Background:
- Gentamicin is a crucial antibiotic for treating Gram-negative bacterial infections.
- Understanding gentamicin's cellular uptake mechanisms is vital for optimizing its efficacy and combating resistance.
- Previous studies suggested complex interactions between gentamicin and bacterial cells.
Purpose of the Study:
- To elucidate the mechanism of gentamicin accumulation in Escherichia coli and Pseudomonas aeruginosa.
- To determine the relationship between gentamicin accumulation, bacterial metabolism, and cell viability.
- To identify the transport system responsible for intracellular gentamicin uptake.
Main Methods:
- Monitoring gentamicin accumulation over time in bacterial strains.
- Utilizing metabolic inhibitors (electron transport inhibitors, sulfhydryl poisons, uncouplers, protein synthesis inhibitors) to assess transport dependency.
- Analyzing gentamicin's chemical form within cells using paper chromatography.
- Measuring intracellular gentamicin concentrations relative to extracellular levels.
- Assessing cell viability and release of acid-soluble 3H-adenine.
Main Results:
- Gentamicin accumulation exhibits multiphasic kinetics in both Escherichia coli and Pseudomonas aeruginosa.
- Later phases of gentamicin accumulation are sensitive to metabolic inhibitors, indicating an energy-dependent process.
- Inhibitor-sensitive accumulation phases correlate with loss of bacterial cell viability.
- Gentamicin is concentrated intracellularly (4-250x) and remains chemically unchanged.
- Accumulation precedes significant release of acid-soluble 3H-adenine, suggesting it's not a general indicator of cell damage.
Conclusions:
- Gentamicin accumulation in bacteria is primarily mediated by an active transport system.
- This active transport is essential for the bactericidal activity of gentamicin.
- The findings provide a mechanistic basis for understanding gentamicin's efficacy and potential resistance pathways.