Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

David C. Rowley

Abstract

Antimicrobial resistance is a growing threat to human health both worldwide and in the United States. Most concerning is the emergence of multi-drug resistant (MDR) bacterial pathogens, especially the ‘ESKAPE’ pathogens for which treatment options are dwindling. To complicate the problem, approvals of antibiotic drugs are extremely low and many research and development efforts in the pharmaceutical industry have ceased, leaving little certainty that critical new antibiotics are nearing the clinic. New antibiotics are needed to continue treating these evolving infections. In addition to antibiotics, approaches that aim to inhibit or prevent antimicrobial resistance could be useful. Also, studies that improve our understanding of bacterial pathophysiology could lead to new therapies for infectious disease. Natural products, especially those from the microbial world, have been invaluable as resources for new antibacterial compounds and as insights into bacterial physiology. The goal of this dissertation is to find new ways to treat resistant bacterial infections and learn more about the pathophysiology of these bacteria. Investigations of natural products to find molecules able to be used as new antibiotics or to modulate resistance and other parts of bacterial physiology are crucial aspects of the included studies.

The first included study, which is reported in chapter two, details a chemical investigation of a marine Pseudoalteromonas sp. Purification efforts of the microbial metabolites were guided by testing against a resistance nodulation of cell division model of efflux pumps expressed in E. coli. These pumps play an important role in the resistance of MDR Gram negative pathogens such as Pseudomonas aeruginosa and Enterobacteriaceae. Through this process, 3,4-dibromopyrrole-2,5-dione was identified as a potent inhibitor of the RND efflux pumps and showed synergistic effects against the E. coli strain with common antibiotics including fluoroquinolones, beta-lactams, tetracyclines, aminoglycosides, and chloramphenicol. The efflux pump inhibitory mechanism was further proved through an accumulation assay with the Hoechst dye 33342.

In chapter three, we report the discovery of a 1,2-benzisoxazole with new antibacterial activity against MDR A. baumannii, a pathogen with a critical need of new treatments. This compound was produced by bacterial fermentation and synthetic preparation and shows minimum inhibitory concentrations as low as 6.25 μg/mL against a panel of four clinically relevant A. baumannii strains. Key structure activity relationships were demonstrated using synthetic analogs of the lead 1,2-benzisoxazole. We advocate for further studies to advance the development of this compound.

The third study, describes an in vitro quiescent state of uropathogenic E. coli (UPEC) and bacteria-produced signals that can prevent this state. Quiescence was seen in the classic UPEC strain CFT073 only when grown on glucose M9 minimal medium agar plates seeded with ≤106 CFU. Interestingly, this quiescent state is seen in ~80% of E. coli phylogenetic group B2 multilocus sequence type 73 strains, as well as 22.5% of randomly selected UPEC strains isolated from community acquired urinary tract infections in Denmark. Furthermore, it was determined that CFT073 forms a high persister cell fraction under these growth conditions. Both the persistent and quiescent states were inhibited significantly by a cocktail of lysine, tyrosine, and methionine at concentrations relevant to those in human urine. The use of CFT073 mini-Tn5 metabolic mutants (gnd, gdhA, pykF, sdhA, and zwf) showed that both quiescence and persistence require a complete TCA cycle, but that the dormant states differ in that persistence requires a non-functional rpoS gene and quiescence does not. These results suggest that interference with these central metabolic pathways may be able to mitigate UPEC infections.

In the fifth chapter, cranberry oligosaccharides and related compounds were determined to be able to reduce the quiescent and persistent phenotypes of UPEC CFT073. This is the first report describing components of cranberry juice with the ability to modulate these important physiological aspects of UPEC and further suggests that cranberry oligosaccharides may be vital to the effectiveness of cranberry juice products in urinary tract infections.

Available for download on Friday, July 27, 2018

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