Identification and characterisation of barriers to antimicrobial resistance gene transfer

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Antimicrobial resistance (AMR) is a mounting global public health issue, causing hundred of thousands of deaths annually, which is ever increasing. AMR genes are often carried on transferable sections of DNA called mobile genetic elements (MGE) which can be shared between bacteria through horizontal gene transfer (HGT). The widespread use of antibiotics in hospitals has therefore provided a selective pressure that has aided the rapid spread of AMR genes, leading to the establishment of resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) However, HGT in S. aureus is not completely understood, particularly for hospital and community associated MRSA (HA-MRSA and CA-MRSA respectively).In S. aureus, the primary method of HGT is through a process called transduction. Bacteria-infecting viruses (bacteriophage) mis-package non-phage DNA into viral particles, which then transfer the DNA to a new host. Bacteria have evolved a range of systems to block MGE and bacteriophage invasion and - important examples are restriction modification (R-M) and CRISPR-Cas systems, which target and degrade foreign DNA. These systems therefore also act as barriers to HGT. Our knowledge of the molecular process of transduction in S. aureus is incomplete, with little new research emerging.The aim of this project is therefore to improve our understanding of the mechanism and barriers of HGT in MRSA. In order to investigate the barriers of transduction HGT in S. aureus, the J. Lindsay team at St. George's, University of London have developed an assay to allow the identification of potential HGT barrier genes. The assay uses a mutant library of 1952 non-essential genes in a strain of CA-MRSA. It then allows for the quantification of the rate of HGT of AMR genes. A significant increase in HGT implies that the knocked-out gene was acting to block transduction, identifying it as a gene candidate, of which several have already been identified. This project will therefore continue this research and optimise the assay.Another project goal is the characterisation of these candidate genes using bioinformatic and molecular techniques. Computational analysis of the gene sequence, as well as the gene operon, would allow for the identification of conserved domains and orthologues with similar DNA sequences, giving us an idea of the gene function. This can then be further explored through molecular experimentation, or by focussing on modular components of the assay. The distribution of identified genes within sequenced S. aureus populations will also provide insight into their role in evolution.Bacteriophage insert their genome into the host chromosome upon infection. The genome can remain inactive in the chromosome as a prophage until a change conditions causes it to excise, replicate, create viral particles, lyse the host cell and spread. Most S. aureus have 1-4 prophage in their genome. However, clinical and HA-MRSA prophage are poorly understood. The R-M system blocks HGT of MGEs from strains different from the host. A goal of this project is therefore to knock this system out in our mutant library. This will allow us to repeat the HGT rate assay with strains other than our CA-MRSA, including MRSA isolated directly from patients, allowing us to compare HGT rates between isolates. This give us the opportunity to sequence and characterise clinical MRSA prophage.This project aims to improve our understanding of the molecular process of HGT in S. aureus through bacteriophage, allowing us to better understand MRSA population evolution dynamics globally and in clinical settings. As the tools R-M and CRISPR-Cas demonstrate, greater research into MRSA HGT barriers could also lead to the discovery of new biotechnology.This project involves the use of quantitative skills, interdisciplinary skills and whole organism physiology.