Enterococcus faecalis CRISPR-Cas Is a Robust Barrier to Conjugative Antibiotic Resistance Dissemination in the Murine Intestine.

CRISPR-Cas systems are barriers to horizontal gene transfer (HGT) in bacteria. Little is known about CRISPR-Cas interactions with conjugative plasmids, and studies investigating CRISPR-Cas/plasmid interactions in models relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogens , and it is essential to identify strategies to reduce the spread of these elements. We use enterococci as models to understand the interactions of CRISPR-Cas with conjugative plasmids. is a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessed CRISPR-Cas anti-PRP activity in the mouse intestine and under different conditions. We observed striking differences in CRISPR-Cas efficiency versus With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, while , the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standard experiments do not adequately model the antiplasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparent antiplasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor-to-recipient ratios, production of a plasmid-encoded bacteriocin, and the time point at which matings are sampled. Our results are clinically significant because they demonstrate that barriers to HGT encoded by normal (healthy) human microbiota can have significant impacts on antibiotic resistance dissemination. CRISPR-Cas is a type of immune system in bacteria that is hypothesized to be a natural impediment to the spread of antibiotic resistance genes. In this study, we directly assessed the impact of CRISPR-Cas on antibiotic resistance dissemination in the mammalian intestine and under different conditions. We observed a robust effect of CRISPR-Cas on but not dissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizer We conclude that standard experiments currently do not appropriately model the conditions where antibiotic resistance dissemination occurs between strains in the intestine. Moreover, our results demonstrate that CRISPR-Cas present in native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.