Development & Exploration of Quantitative Metrics and Selection-Based Strategies for the Facilitation of Phage Therapy and Agricultural Biocontrol Targeting Infections by members of the Burkholderia cepacia complex & Burkholderia gladioli
Creators: Lauman, Philip
Publisher: name: University of Alberta Library
Year: 2024

<pre><code>    The increasing global spread of multidrug resistant bacterial pathogens is an imminent menace to public health and threatens virtually all aspects of modern medicine. Antibiotic resistance is a major cause of mortality worldwide and is predicted to cause roughly 10 million fatalities per year by 2050. Particularly concerning are the members of the Burkholderia cepacia complex (Bcc) and the related pathogen Burkholderia gladioli (Bg), a group of opportunistic, hospital-transmitted species which cause morbidity in patients who are immunocompromised or afflicted with chronic illnesses such as cystic fibrosis, while Bg is additionally a phytopathogen infecting several crop species important to global food security. These species are extraordinarily resistant to a wide range of antimicrobial compounds and treatment options are often limited, meaning novel approaches to targeting these problematic pathogens are urgently required. 
One promising alternative is the medicinal use of phages, viruses that destroy pathogenic bacteria while leaving commensals and host cells unharmed, in what is called phage therapy. In nature, phages have co-evolved with bacteria for billions of years, are environmentally abundant, and destroy roughly half of the global bacterial population every 48h, meaning phage therapy is simply the application of a natural antibacterial agent to a human problem. Selection pressures imposed upon bacterial populations through phage predation differ substantially from those exerted by antibiotics or  immune systems, meaning that phages represent a distinct mode of treatment and can additionally be combined with other antimicrobial agents to increase antibacterial efficacy using selection-based treatment strategies. The multipartite interactions between phages, bacterial hosts, and environmental factors are complex, however, and the development of selection-based strategies and even straightforward therapeutic approaches can therefore be challenging and requires the initial characterization of various phage, bacterium, and host properties. Here we developed several quantitative variables measuring parameters related to phages, their bacterial hosts, and the interactions between the two, and subsequently applied these metrics to investigate both straightforward and selection-based approaches to phage therapy targeting Bcc species and Bg. 
In Part I, we developed and mathematically formalized several parameters including Efficiency of Phage Activity (EPA), Growth Reduction Coefficient (GRC), Stable Lysogenization Frequency (f(s.lys)), Mortality Coefficient (Cm), Reduction of Mortality Coefficient (R Cm), and Phage Stability Index (IPS), and employed these metrics to study ten previously-characterized Burkholderia phages in the context of in vitro and in vivo interactions with Bcc species. Notably, we observed that lysogenization-capable (LC) phages exhibit enormous variability in terms of the frequency with which they form stable lysogens (f(s.lys)), and that this property is inversely correlated with GRC, a measure of the ability of a phage to control bacterial growth in vitro. We subsequently developed a series of equations defining in vitro antibacterial synergy in terms of GRC and found  that high-f(s.lys) LC phages form powerful synergistic interactions with other phages as well as with the antibiotic ciprofloxacin. These findings challenge the longstanding paradigm of excluding LC phages from medicinal use, and reveal novel therapeutic roles for these ubiquitous phages.
In Part II, we employed these parameters to explore the antibacterial effects of Burkholderia phages against Bg in various contexts, and additionally developed a novel ex planta maceration index (xPMI) to gauge the virulence of Bg in agriculturally relevant crops. We identified that Bcc phages can cross-infect Bg strains, control their growth in vitro, and reduce Bg infection-associated mortality in vivo and tissue morbidity ex planta, thereby revealing the previously-unexplored potential of phage therapy against this organism. Crucially, we determined that the highly efficacious Burkholderia phage KS12 utilizes the O-antigen as a receptor and therefore selects for LPS-truncated, outer membrane-permeabilized survivors that are sensitized to the effects of antimicrobial compounds and have attenuated virulence in vivo and ex planta. Moreover, KS12 synergizes with colistin to destroy Bg populations in vitro, suggesting that this combination could be utilized as part of a selection-based therapeutic strategy. Importantly, KS12 does not elicit a pro-inflammatory response directly but might do so indirectly through lysis-driven release of bacterial LPS, thereby possibly contributing to immune activation against invading pathogens. 
Taken together, this work introduces new approaches to investigating phage-bacterium-host dynamics in several contexts, challenges a major paradigm within the field, and highlights the potential of Burkholderia phages for therapeutic applications and agricultural biocontrol targeting Bcc species and Bg.
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