CRISPR-Cas Systems and Bacteriophages: Alternative Therapies to Combat Antibiotic-resistant Enterococcus Faecalis

The increasing demand for antibiotics has created selective pressure for the emergence of multidrug-resistant (MDR) pathogens. Enterococcus faecalis is normally found as a commensal of the gastrointestinal (GI) tract of healthy humans. However, E. faecalis is also an opportunistic pathogen and a leading cause of hospital-acquired infections (HAI). E. faecalis intrinsic and acquired antibiotic resistance can make these infections very difficult to treat. Genomic analyses have revealed that E. faecalis strains may become more virulent through horizontal gene transfer (HGT) of mobile genetic elements (MGEs), such as the highly transmissible E. faecalis pheromone-responsive plasmids (PRPs). MGEs can also decrease cellular fitness. Therefore, bacteria encode many defense systems to limit their transfer. A well-studied defense mechanism is the adaptive defense system CRISPR-Cas. In addition to the rise of MDR pathogens, the development of new antimicrobial drugs has been limited, pushing modern medicine toward a post-antibiotic era. The void created by the limited number of antibiotics that are still effective against infections has led to exploring alternative therapies. Among them are CRISPR-Cas-based antimicrobials. Multiple studies, including one in the Palmer lab, have established that CRISPR- Cas can effectively remove antibiotic resistance from a bacterial population in a sequence-specific manner. Most of these studies have used model strains; therefore, there is limited understanding of how effective CRISPR-Cas antimicrobials are against non-model strains. Another proposed alternative is bacteriophage therapy. Studies on the molecular mechanism involved in enterococcal phage infection and phage resistance are limited. My research aimed to establish the efficacy of CRISPR-Cas antimicrobials against non-model strains, which are the intended target for CRISPR- based therapy, and to further elucidate the mechanisms involved in E. faecalis phage infection and the host genomic alterations to become phage-resistant. Conjugation assays were used to compare the efficacy of our previously engineered plasmid-based CRISPR-Cas antimicrobials against a recent collection of E. faecalis fecal isolates, referred to here as “wild” isolates. It was discovered that the wild isolates could i) antagonize the CRISPR-Cas antimicrobial donor strain via competitive factors and ii) prevent CRISPR-Cas antimicrobial transfer, effectively avoiding CRISPR-Cas targeting. Additionally, a 14-day coevolution study was performed using the E. faecalis strain SF28073 and two genetically distinct E. faecalis phages, followed by whole-genome sequencing to elucidate novel mutations resulting from the pressure imposed by phage infection. The results revealed mutations in genes encoding macromolecules that may be associated with phage infection, many of them previously unreported. Results from the first study emphasize the need to continue studying CRISPR-Cas antimicrobials in the context of wild isolates to determine potential limitations. The second study serves as a basis for the continued research of enterococci- phage coevolution. Both studies are critical to developing viable CRISPR-Cas and phage therapies.