Investigating Gentamicin Mechanism of Action with Fluorescence Microscopy: Relationships between Colony Growth, Antibiotic Accumulation, and Membrane Permeability Public
Rankin, Jillian (Fall 2020)
Abstract
Aminoglycosides constitute a powerful, broad-spectrum class of antibiotics. While aminoglycosides are known to exert their antimicrobial activity through ribosome targeting, the precise mechanism of action through which they produce cell death remains undefined. In particular, there is no clear scientific consensus regarding the role of membrane damage in initiating growth arrest. In this study, we probed aminoglycoside mechanism of action by using fluorescence microscopy to evaluate the interrelationships between membrane damage, growth arrest, and antibiotic accumulation in Escherichia coli microcolonies. A fluorophore-conjugated aminoglycoside, gentamicin-Texas Red (GTTR), was used to assess antibiotic accumulation, while SYTOX Green, a nucleic acid stain impermeant to living cells, was used to assess membrane permeability. To further clarify the role of membrane integrity in influencing growth inhibition, treatment groups with variable degrees of induced membrane permeability were compared. Specifically, given that magnesium availability is known to influence outer membrane stability for Gram-negative organisms, colonies cultured in magnesium-supplemented conditions were compared against those cultured in relatively low magnesium conditions. Furthermore, outcomes for colonies treated with colistin, an antibiotic known to induce membrane damage, were compared against those of magnesium-supplemented colonies. We found that, in the context of gentamicin treatment, both low magnesium growth conditions and treatment with colistin induced greater degrees of membrane permeability throughout the experimental timeline, as well as increased rates and extents of gentamicin accumulation, relative to the magnesium-supplemented group. However, we found that these outcomes were not reliable predictors of growth rate or the onset of growth inhibition. Furthermore, our analysis of the temporal relationships between changes in membrane permeability, accumulation, and growth inhibition suggests that the onset of significant losses in membrane integrity are not required to initiate growth arrest. Additional study is required to further clarify the role of membrane damage in gentamicin-induced growth arrest and to ultimately delineate the precise mechanism of action of aminoglycoside antibiotics.
Table of Contents
CHAPTER I. BACKGROUND 1
Introduction to Aminoglycosides 1
Ribosome-Targeting by Aminoglycosides 1
Proposed Mechanisms of Aminoglycoside Bactericidal Activity 2
Implications 3
CHAPTER II. EXPERIMENTAL APPROACH 5
Motivation 5
Use of Gentamicin 5
Use of Fluorescence Microscopy 7
Membrane Permeability Treatments 8
CHAPTER III. METHODS 11
Experimental Procedure 11
Image Analysis 16
Statistical Analysis 27
Accounting for Colony Heterogeneity 28
CHAPTER IV. RESULTS 30
General Findings 30
Measures of Membrane Permeability 31
Measures of Gentamicin Accumulation 36
Measures of Growth and Growth Inhibition 42
Relationships between Permeability, Accumulation, and Early Growth 45
Relationships between Permeability, Accumulation, and Growth Inhibition 49
CHAPTER V. DISCUSSION 57
Summary and Implications 57
Future Directions 60
APPENDIX A. COLONY DATA IN THE CONTEXT OF CELL HETEROGENEITY 63
APPENDIX B. TABLES 66
REFERENCES 71
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