Date of Award
Doctor of Philosophy (PhD)
Bacteriophages (phages) are prolific, ubiquitous viruses that infect bacterial cells. Phages have proven to be invaluable tools of the biological sciences, enabling the discovery and description of some of the foundations of molecular biology and genetics; excellent systems for the study of evolutionary dynamics; and their seemingly limitless diversity promises to produce interesting new biology for many years to come. As natural predators of bacteria, phages might additionally be employed for phage therapy, the therapeutic use of phages to treat bacterial infections. Lytic phages impose strong selective pressure on bacteria to evolve resistance to phage infection. Under certain environmental conditions this selection pressure can result in the evolution of phage resistance at reduced fitness; this is an example evolutionary trade-off, or the evolution of a certain trait at the detriment of the fitness. In particular, it is possible that phage-imposed selection on bacterial surface expressed molecules may result in reduced antibiotic resistance and/or attenuated virulence of opportunistic bacterial pathogens. In this thesis, we will examine the idea of phage-imposed trade-offs on opportunistic bacterial pathogens. A published review paper serves as the first chapter and an introduction to phage biology and phage therapy. We present the history of the discovery of phages as well as the early reports of phage therapy. We highlight recent animal studies, case reports and clinical trials that have investigated the therapeutic use of phage. Finally, we discuss new approaches to phage therapy as well as identify interesting question and potential hurdles that will likely underscore a modern approach to phage therapy. In chapter 2, transposon insertional sequencing (INSeq) is used as a new high-throughput method to identify phage receptors to identify new phage that use antibiotic resistance or virulence factors as receptors. Preliminary experiments using characterized phages T2, T4, T6 and T7 demonstrate that the top results of INSeq screens with phage are involved in phage binding. These screens were extended to enable receptor identification for six newly isolated phages, R3, U115, EC35, EC14, 8S and P2. Adsorption assays and efficiency of plaquing assays to demonstrate phage binding and infection are used to validate the results of the INSeq screens with uncharacterized phage. In summary, this chapter establishes the use of INSeq screens as a high-throughput method to identify phage receptors which should allow for identification of phage that target virulence factors or molecules contributing to antibiotic resistance. Evolutionary dynamics between Pseudomonas aeruginosa and a lytic phage, OMKO1 that selects against antibiotic resistance, are examined in a short-term coevolution experiment in chapter 3. Time shift assays showed that coevolution of the three experimental populations followed arms race dynamic. Interestingly, only one experimental population demonstrated the predicted trade-off between phage resistance and antibiotic resistance. Whole genome sequencing of bacterial population allowed for identification of mutations underlying bacterial evolution and the trade-off. Results from this study demonstrate that evolution may not be as repeatable or predictable as previous experimental evolution studies have suggested. In chapter 4, we identify a new Shigella flexneri phage that selects for phage-resistant bacteria that are attenuated for virulence. We isolate and characterize a new Myoviridae phage, A1-1 which uses OmpA of S. flexneri as a receptor. Assays to interrogate membrane permeability including, live-dead staining, minimum inhibitory concentrations to various antibiotics and measurements of total lipopolysaccharide (LPS) quantity demonstrate that phage A1-1 selects for two different phenotypes of resistant mutants: OmpA deficient and altered LPS. Whole genome sequencing reveals that the five phage-resistant mutants examined in this study have mutations in either ompA or in genes involved in LPS biosynthesis genes. Using bacterial plaque assays, we show that all five resistant mutants are attenuated for intercellular spread, indicating that in the case of phage A1-1, phage resistance trades off with bacterial virulence. While the previous two chapter highlight phage selection resulting in a trade-off, the final chapter investigates a system in which phage selection results in a trade-up with antibiotic resistance. Tsx is a nucleoside porin in Escherichia coli which also serves as a receptor for phage T6, phage U115 and albicidin, a DNA gyrase inhibitor. We show that selection for resistance to any of these three antibacterials results in cross-resistance to the other two. Competition assays show resistance to these antibacterials does not result in a fitness cost relative to wild-type. In all 29 of the resistant mutants observed in this study, mutations in tsx were found. The trade-up between phage resistance and antibiotic resistance in this study highlights the need for rigorous studies of phage-bacteria interactions prior to deployment of phage therapy.
Kortright, Kaitlyn, "Phage Selection Against Antibiotic Resistance or Virulence in Opportunistic Bacterial Pathogens" (2021). Yale Graduate School of Arts and Sciences Dissertations. 69.