Abstract
Enterococci are core members of the human gut microbiome but can also act as opportunistic pathogens that cause life threatening infections. Enterococci have established themselves as important hospital acquired infections (HAI), due to their resilience and high multidrug-resistant (MDR). This project focused on Enterococcus faecalis and Enterococcus faecium as these two enterococcal species are most abundant species isolated from the human gastrointestinal (GI) tract and most clinically relevant making up 90% of human enterococcal infections. In the post-avoparcin era the MLST-type ST108 dominated New Zealand poultry and early clinical vancomycin resistant enterococci (VRE). Upon further analysis a putative phase-variable Type I restriction modification (RM) system was discovered to be prevalent in ST108. For decades bacterial RM systems have been solely thought of as a bacterial defence mechanism. However, with recent technological advances in bacterial epigenetics, new evidence has emerged showing they contribute to many critical functions. Methylation by methyltransferase creates epigenetic profiles that have been associated with virulence in important human pathogens such as Haemophilus influenzae and Streptococcus pneumoniae. Phase-variable Type I RM systems encode multiple target sequence specificity (hsdS) genes. Phase-variation is mediated through reversible site-specific recombination of two or more hsdS genes, producing multiple hsdS subunits. This means the cognate methyltransferase and restriction endonuclease can now bind multiple recognition sequences in a HsdS-specific manner. This generates a number of different epigenetic profiles within a single population, creating phenotypic flexibility. In this project we used a combination of bioinformatics and molecular techniques to explore the role of Type I RM mediated phase variability in enterococci. We generated an hsd locus knockout in E. faecalis and during the troubleshooting process we discovered a putative stress-induced regulatory mechanism for this locus. We found that while phase-variable Type I RM systems are not prevalent in E. faecium, a recently isolated healthcare-associated MLST-type ST1421 does encode a phase-variable Type I RM system and hypothesize that this may be contributing to its increasing dominance in the clinic.