Abstract
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.
Importance Enterococcus faecalis is a leading cause of hospital-acquired infections and known disseminator of drug resistance among Gram-positive bacteria. One of the main means of antibiotic resistance dissemination among E. faecalis populations is mediated by plasmids. We have previously shown that strains with an active CRISPR-Cas system can reduce plasmid acquisition and thus limit the transmission of antibiotic resistance determinants. In this study, we observed subpopulations with transient co-existence of active CRISPR-Cas and a plasmid target. Through serial passage and targeted sequencing analysis on these populations, we demonstrate that antibiotic treatment plays a key role in shaping the E. faecalis genome, resulting in compromised genome defense that facilitates acquisition of other resistance plasmids. These results are significant because they show how antibiotic selection for a plasmid can alter the evolutionary trajectory of E. faecalis populations rendering them vulnerable to the acceptance of multiple resistance plasmids.