Healthcare associated infections are a major problem for patients and a global health challenge. Clostridium difficile infection (CDI) is particularly problematic, for example the UK has ~20,000 infectious diarrhoeal cases every year, and progressive complications can lead to death in ~3% of infected patients. This observed high morbidity and mortality, is attributed to the emergence of severe and antibiotic-resistant strains, and inadequate treatment options. Conventional treatment of CDI is limited to three antibiotics, and there are problems with efficacy, cost and with the observation that they can trigger dysbiosis (microbial imbalance) that leads to recurrent infection.
To effectively control CDI, novel specific antimicrobials are urgently needed, preferably those that will target C. difficile and not disrupt the ‘commensal’ microbiota. Previously, we have isolated a set of bacteriophages (phages) that target C. difficile and these phages have significant therapeutic promise (1-3). It is clear however that C. difficile has multiple ways of evading phages. These defence methods need to be understood within the context of using phages as therapeutics. In this PhD the student will determine how C. difficile most commonly develops resistance to phages. They will subject key C. difficile strains to phages and determine rates of receptor-based resistance, CRISPR based resistance and of lysogeny.
The overall hypothesis to be tested in this project is that C. difficile has multiple ways of becoming resistant to phages, and that all methods contribute to phage evasion in this bacterial species.
Methodology and approaches
Objective 1. To identify all genes involved in phage resistance student will use transposon mutagenesis to create a library of relevant strains of C. difficile, with at least one fatal mutation per gene. They will subject the library to phages identify all the genes involved with phage resistance. They will use cutting edge bioinformatic analysis approaches to determine key genes of interest.
Objective 2. From the set of mutants generated above the receptor-based mutants will be confirmed by adsorption assays.
Objective 3. Rates of lysogeny will be determined subjecting C. difficile to high dosages of phages as described in 1, and then using PCR to identify how often phages have integrated. Rates for our key 7 phages will be tested in different strain backgrounds.
Objective 4. To determine the importance of the C. difficile CRISPR system the student will take strains of C. difficile that have active CRISPR regions and use PCR to determine how the CRISPRs respond to phage infection.
This PhD will give for the first time an assessment of how frequently different phage defence systems are deployed. Data from this basic science will inform future healthcare translation.