Combination Antimicrobial Therapy: Synergistic Effect of a Cationic Zn-Containing Porphyrin with Lytic Bacteriophage PEV2 for Inhibition of Pseudomonas aeruginosa

Date of Award

2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Institution Granting Degree

University of Dayton

Cedarville University School or Department

Science and Mathematics

First Advisor

Jayne Robinson

Keywords

Pseudomonas aeruginosa, Biofilms, Bacteriophage, Phage-Antibiotic Synergy, Porphyrins

Abstract

Antibiotic resistance has been declared a global concern by the World Health Organization and is increasing the rate of mortality of once-treatable, common infections. Antibiotic resistance is conferred by multiple mechanisms both intrinsic (horizontal gene transfer) and extrinsic (production of biofilms). The eradication of biofilms produced by bacterial colonization remains a serious threat to human infections. Bacterial biofilms produce an extracellular matrix composed of proteins, polysaccharides, and extracellular DNA (eDNA). This matrix acts as a scaffold for growth and imparts a form of protection against predators, harsh conditions, and chemicals (e.g., bacteriophage, pH, and antibiotics). The biofilm-associated cells of Pseudomonas aeruginosa (PsA) are up to 1000-fold more resistant to antibiotics than planktonic cells. Additionally, PsA has been linked to many infections that can be mortally dangerous for individuals with compromised immune systems such as Cystic Fibrosis (CF). PsA colonization in individuals with CF causes a decreased quality of life. Thus, there is a search for alternative strategies for antimicrobial management. Our lab has produced a patented zinc-containing porphyrin, Zn(II)meso-5,10,15-triyl-tris(1-methylpyridin-1-ium)-20-(pentafluorophenyl) porphine tritosylate (ZnPor), which exhibits broad antibacterial activity against planktonic and biofilm-associated cells of PsA. ZnPor presents itself as a unique possible surrogate for traditional antibiotics by its interaction with eDNA of biofilms. ZnPor intercalates between base pairs and binds to the outside of the helix, resulting in a more porous biofilm that dissembles and detaches from substrata. Furthermore, ZnPor has potent photoactivity that increases both its bactericidal and viricidal properties when exposed to light. The ability to disrupt the inherent matrix structure makes biofilm-associated cells more accessible to other treatments such as antibiotics and bacteriophage infection. As highly specific against their selected targets, bacteriophage have many advantages against traditional antibiotics. Bacteriophage PEV2 is obligately lytic against PsA strain PAO1. Furthermore, it has been shown that PEV2 protects lung cells from infection and is not toxic in vitro. PEV2’s activity against PsA makes it a viable therapeutic option, thus this study is dedicated to the investigation of the synergistic effect between ZnPor and PEV2 against PsA. Chapter 1 of this dissertation details the issue of antibiotic resistance, the mechanisms of resistance, and how becoming resistant affects organisms like PsA. This is followed by an exploration of current strategies for biofilm mitigation such as bacteriophage therapy and photodynamic therapy where the properties of ZnPor are discussed. Chapter 2 investigated the efficacy of both ZnPor and phage PEV2 against both planktonic and biofilm-associated cells of PsA strain PAO1 and PA14. Biofilms were grown on multiple types of clinically relevant substrata such as polyethylene, titanium, and hydroxyapatite. The combinatorial treatment of both ZnPor and PEV2 had a greater effect against biofilms of PsA than either treatment alone. Furthermore, the combined treatment showed a significant decrease of biofilm-associated cells of strain PA14 which is remarkable since this strain is not susceptible to PEV2 phage. This has led to the hypothesis that ZnPor is able to permeabilize the cell membrane which permits PEV2 cell entry apart from the normal receptor. Additionally, we report for the first time, a porphyrin that retains dark toxicity against bacteriophage virus. Chapter 3 expanded upon the combination therapy of ZnPor and PEV2 and its potential on human lung cells. It was demonstrated that ZnPor decreased the PsA load in a murine model of infection without showing any signs of toxicity to mouse lung cells. H441 tissue cells, which originate from a papillary lung adenocarcinoma of alveolar tissue, were chosen because they secrete alveolar fluid when challenged with bacteria that mimics the natural lung environment. A combinatorial treatment of ZnPor and PEV2 was able to rescue lung cells from PsA infection and was comparable to an untreated and uninfected control. Chapter 4 considered the property of ZnPor to make cells permeable and determine if chemically forcing competence allows for bacterial cells to uptake phage PEV2 genome. It was demonstrated that PsA biofilm cells pre-treated with a minimum inhibitory concentration of ZnPor allowed for phage propagation to proceed and cell lysis to occur without the need for whole intact phage. Chapter 5 of this dissertation will summarize results and further explore future directions and goals for this project.

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