Deep sequencing analysis of CRISPR-escaping plasmid transconjugants in Enterococcus faecalis

Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Some E. faecalis strains possesses CRISPR-Cas systems, which reduce the conjugation frequency of pheromone-responsive plasmids. However, many transconjugants still arise, and we have demonstrated in previous studies that E. faecalis can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze CRISPR array dynamics over time in transconjugants which possess both a functional CRISPR-Cas system and a CRISPR-Cas target. In the presence of antibiotic selection for the plasmid, we found that plasmids ultimately escape CRISPR defense via the emergence of compromised CRISPR-Cas defense in host populations. As a consequence, these populations have enhanced abilities to acquire a second antibiotic resistance plasmid. In the absence of antibiotic selection, plasmids are lost from wild-type but not Δcas9 host populations over time. We conclude that the adaptive immune system of E. faecalis becomes compromised under antibiotic selection for MGEs, generating populations with enhanced abilities to undergo horizontal gene transfer.