The Impact of Genome Defense Systems on Antibiotic Resistance Dissemination in the Opportunistic Pathogen Enterococcus faecalis

Since the advent of the modern antibiotic era there has been an increasing number of bacteria that are resistant to antibiotics. The growing antibiotic resistance and the lack in development of new antibiotics poses a serious public health threat worldwide. One of the first steps to combat this problem is to understand the molecular details associated with antibiotic resistance. Enterococcus faecalis is one of the most common causes of healthcare associated infections (HAI). E. faecalis is an opportunistic pathogen that normally resides in the gastrointestinal (GI) tracts of humans and other animals. This bacterium possesses intrinsic antibiotic resistance and can also acquire resistance to other antibiotics through horizontal gene transfer (HGT). Some multi-drug resistant (MDR) E. faecalis strain have acquired resistance vancomycin, an antibiotic of last-resort, leaving very few treatment options. It has been shown that MDR E. faecalis have expanded genomes enriched with mobile genetic elements (MGEs) that were acquired through horizontal gene transfer (HGT). Plasmid conjugation is one of the most common forms of HGT in this species. The pheromone responsive plasmids (PRPs) are a group of narrow-host range plasmids that are rapid disseminators of antibiotic resistance in clinical isolates of E. faecalis. PRPs have high conjugation frequencies and often encode virulence factors that have been associated with enhanced pathogenicity. The acquisition of foreign DNA is usually associated with a fitness cost to the host cell, therefore prokaryotes encode defense systems that limit HGT. The two forms of genome defense studied here are restriction modification (R-M) and CRISPR-Cas. Interestingly, MDR E. faecalis lack active CRSIPR-Cas systems, leading to the hypothesis that MDR E. faecalis emerge due to the lack of barriers to HGT. The primary focus of the research presented here was to establish a role for R-M and CRISPR-Cas in providing genome defense in E. faecalis and to elucidate the interactions between CRISPR-Cas and MGEs. Conjugation assays were used to determine that R-M and CRISPR-Cas work cooperatively to significantly limit PRP acquisition in a natively drug-susceptible E. faecalis isolate, T11. Through in vitro evolution and deep sequencing analysis, it was concluded that under antibiotic selection for a PRP, the CRISPR-Cas system of T11 becomes compromised, providing a potential mechanism for the emergence of MDR E. faecalis. Finally, a mouse model of E. faecalis colonization was used to show that CRISPR-Cas provides defense against PRP acquisition in vivo. Overall, the data presented here have shown that R-M and CRISPR-Cas are capable of limiting MGE acquisition in E. faecalis justifying the claim that MDR E. faecalis emerge due to the absence of genome defense. This coupled with the in vivo activity of CRISPR-Cas prove that the CRISPR-Cas systems associated with E. faecalis strains could be harnessed for use as alternative therapies to treat E. faecalis infections.