Date of Award


Document Type

Open Access Honors Thesis

Degree Name

Bachelor of Science (BS)




Michael Shiaris


Rajini Srikanth

Subject Categories



Enterococcus faecalis is a Gram-positive aerotolerant anaerobic bacterium primarily found as a member of the human and animal intestinal flora. Enterococcus faecalis along with 27 other species comprise the genus Enterococcus. Out of the whole genus E. faecalis and E. faecium are the second most common nosocomial cause for urinary tract and wound infections. The treatment of the infections is made even more complex due to E. faecalis resistance to last-resort drugs like vancomycin. An inverse relationship has been observed in the species between antibiotic resistance and presence of the CRISPR-cas locus. CRISPR-Cas (clustered, regularly interspaced short palindromic repeats and associated Cas proteins) systems function as an adaptive and specific immune response for many Bacteria, including Enterococcus faecalis. The CRISPR-Cas system incorporates a small segment of foreign DNA into the spacer regions in the bacterial chromosome, which allows for future targeting and defense against the phage. Three CRISPR systems have been identified in E. faecalis, two of them with corresponding Cas proteins (CRISPR1-Cas and CRISPR3-Cas) and one without (CRISPR2). CRISPR2 is assumed to be an orphan locus and is thought to be inactive due to the lack of associated Cas proteins. As it is presumed to lack a beneficial function, a reason for the high level of conservation of CRISPR2 loci in E. faecalis remains unknown. One hypothesis is that CRISPR2 can interact with Cas proteins from other CRISPR types (CRISPR1 or CRISPR3), which can insert spacer DNA from lytic phage into its genome. To test this hypothesis, E. faecalis lytic bacteriophage was isolated from an activated sewage sample obtained at Deer Island Water Treatment Plant and used to challenge E. faecalis strains containing different arrangements of CRISPR-Cas systems (CRISPR1-Cas/CRISPR2 and CRISPR2 only). Bacteriophage-resistant strains were generated by mixing a high concentration of phage with E. faecalis strains containing different CRISPR system combinations. Strains surviving the phage challenge were further tested to ensure that they were resistant to the phage. CRISPR arrays in both the original and the resistant bacterial strains were PCR-amplified. The wild type and the mutant array PCR products were sequenced and examined for new spacers, indicating CRISPR adaptation for phage resistance of the mutant strains. No spacer acquisition was observed in the mutant strains compared to the original, indicating that CRISPR2 was not involved in phage-resistance.

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