Evolutionary outcomes of plasmid-CRISPR conflicts in an opportunistic pathogen

The persistence of antibiotic resistance plasmids in pathogens is a global health concern. Plasmid persistence results from host-plasmid co-evolution that enhances plasmid stability, where the role of CRISPR-Cas is not well understood. Enterococcus faecalis is an opportunistic pathogen that disseminates antibiotic resistance via conjugative plasmids. Some E. faecalis possess CRISPR-Cas that limit acquisition of resistance plasmids; however, transconjugants arise despite CRISPR-Cas activity. We utilized in vitro evolution to investigate how the conflict between CRISPR-Cas and plasmid targets is resolved. We observed a cost to maintain both the plasmid and functional CRISPR-Cas. Under antibiotic selection, heterogeneous populations with compromised CRISPR-Cas emerged, which benefited acquisition of other plasmids. Using targeted sequencing, we demonstrate RecA-independent allelic heterogeneity provides an evolutionary basis for the emergence of compromised CRISPR-Cas. Overall, antibiotic selection for plasmids targeted by CRISPR-Cas results in host mutations that stabilize plasmid maintenance and reduce the barrier to future horizontal gene transfer events.