The Role of CRISPR-Mediated Phage Resistance in the Development of Phage-Based Biocontrol for Erwinia amylovora

Abstract

In the post-antibiotic era, resistance in pathogenic bacteria is projected to significantly hinder crop production and become one of the leading causes of death. This has necessitated the development of therapies to address antibiotic resistant microbes and prolong the period for which antibiotics remain a viable treatment option. A prominent alternative technology that has recently re-emerged is the use of bacterial viruses known as phages. Phages selectively lyse their bacterial hosts during the replication process but must avoid phage resistance mechanisms to eliminate a bacterial population. In this dissertation, the impact of phage resistance on biocontrol efficacy is examined using the phytopathogen Erwinia amylovora. The primary source of acquired phage immunity in bacteria is the CRISPR-Cas system. However, the absence of methodologies to study Erwinia phages, and a lack of genomic data for E. amylovora, has previously hindered this avenue of research. Quantitative real time PCR assays were developed to simultaneously monitor both the E. amylovora and phage populations. The individual steps of the phage lytic cycle during infection were characterized by further modification of this methodology. Through this, phage candidates ΦEa46-1-A1 and ΦEa21-4, that previously demonstrated high biocontrol potential, were shown to produce a large number of progenies over a short period of time. A comparative genomic analysis using 127 sequenced isolates of E. amylovora was then completed. This study proposed three primary clades of E. amylovora which infect apples in North America. A novel bioinformatic pipeline was subsequently developed to analyse the CRISPR regions of E. amylovora and the activity of the CRISPR-Cas system was then confirmed. While each clade of E. amylovora exhibited a unique CRISPR arrays, none of the identified CRISPR spacers provided inherent protection against any biocontrol candidate. CRISPR-mediated phage resistance was confirmed in E. amylovora against biocontrol candidate ΦEa21-4 but only in isolates with primed CRISPR-Cas systems. Still, phage resistance to ΦEa21-4 was observed through an unknown resistance mechanism in wild-type isolates. Overall, this work demonstrates new techniques to improve trial outcome prediction and lays the foundation for further investigation into the phage resistance mechanisms of E. amylovora.