Using Fluorescence Microscopy to Study Antibiotics and Factors that Influence Bacterial Susceptibility

This dissertation looks at the overall theme of antibiotics and factors that influence bacterial susceptibility through the application of the fluorescence microscopy tool Bacterial Cytological Profiling (BCP). The first chapter is a brief history of antibiotics and the rise of bacterial resistance, the use of bacteriophages as an alternative therapy, and an overview of current fluorescence microscopy techniques used in the field to study antibiotic mechanism of action (MOA) and susceptibility. Chapter 2 describes the integration of microfluidics and fluorescence microscopy to investigate antibiotic transport kinetics in Escherichia coli. Uptake of fluorescently-tagged azithromycin and roxithromycin were observed in different genetic backgrounds to characterize the extent to which the outer and inner membranes, and efflux, function as barriers to antimicrobial activity. Different growth media was used to further characterize antibiotic uptake by cells under different microenvironments, specifically the impact of bicarbonate on transport kinetics. Chapter 3 focuses on the application of BCP to probe the molecular interactions between antibiotic treatment and the jumbo phage φKZ during infection of Pseudomonas aeruginosa. A diverse set of antibiotic classes were assayed, including those commonly used to treat P. aeruginosa infections. Further investigation into cell-wall active antibiotics were completed to explore mechanistic explanations for observed increases in relative phage infection and phage nucleus localization. Chapter 4 is an overview of several collaborations with Victor Nizet’s lab at UCSD to examine antimicrobial synergies active against multi-drug resistant bacteria using BCP. This work included studies examining binding of the antimicrobial peptide LL-37 to Klebsiella pneumoniae during treatment with non-β-lactam β-lactamase inhibitors and the synergy between two protein synthesis inhibitors against Acinetobacter baumannii