The role of interspecific competition on the evolution of phage resistance in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a pathogen of increasing medical concern due its inherent tolerance and ability to overcome antibiotics. Because of this, bacteriophages are increasingly being considered and applied as alternative therapeutics. P. aeruginosa however can evolve resistance to phages through a range of different means, including CRISPR-Cas adaptive immunity. Yet experimental studies on phage resistance evolution have almost exclusively been done using clonal bacterial populations. This, despite bacteria commonly thriving in complex microbial communities with potentially significant consequences for phage resistance and virulence evolution. Here, I first summarise the existing literature on P. aeruginosa ecology, virulence, antibiotic resistance, phage therapy and resistance, before I present experiments looking at how interspecific competition affects bacteria-phage co-evolution. I demonstrate how growth in the presence of other bacterial species (Staphylococcus aureus, Burkholderia cenocepacia, and Acinetobacter baumannii) causes P. aeruginosa to evolve higher levels of CRISPR-based immunity than when in monoculture. This again has important knock-on effects for P. aeruginosa virulence, which becomes attenuated if the bacterium evolves surface-based resistance. Next, to understand the causative mechanism(s) underlying the selection for CRISPR-based resistance in polyculture, I look at the evolution of phage resistance in conditioned media. I show that a greater proportion of P. aeruginosa clones evolve phage resistance through CRISPR-Cas when cultured in the conditioned media from A. baumannii. This suggest that the effect of this competitor species is caused by changes to the chemical environment, such as resource depletion and toxin secretion. Finally, I examine how phage and CRISPR-Cas immune systems shape microbial community structure. I find that A. baumannii takes over to become the dominant species in the presence of phage, regardless of the presence or absence of a CRISPR-Cas system in the P. aeruginosa genome. Additionally, phage has a diversity maintaining effect, with all four community members persisting for longer in the presence of phage. Collectively, this thesis sheds light on how interspecific competition shapes the evolution of phage resistance, and vice versa.