Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci

Abstract

The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont <i>Enterococcus faecalis</i> is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control <i>E. faecalis</i> populations are needed. We previously characterized an <i>E. faecalis</i> type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to <i>E. faecalis</i> for the selective removal of antibiotic resistance genes. Using <i>in vitro</i> competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant <i>E. faecalis</i> by several orders of magnitude. Finally, we show that <i>E. faecalis</i> donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants <i>in vivo</i> Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.