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 Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis 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 E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro 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 E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo. 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.

CRISPR-Cas provides a barrier to horizontal gene transfer in prokaryotes. It was previously observed that functional CRISPR-Cas systems are absent from multidrug-resistant (MDR) Enterococcus faecalis, which only possess an orphan CRISPR locus, termed CRISPR2, lacking cas genes. Here, we investigate how the interplay between CRISPR-Cas genome defense and antibiotic selection for mobile genetic elements shapes in vitro E. faecalis populations. We demonstrate that CRISPR2 can be reactivated for genome defense in MDR strains. Interestingly, we observe that E. faecalis transiently maintains CRISPR targets despite active CRISPR-Cas systems. Subsequently, if selection for the CRISPR target is present, toxic CRISPR spacers are lost over time, while in the absence of selection, CRISPR targets are lost over time. We find that forced maintenance of CRISPR targets induces a fitness cost that can be exploited to alter heterogeneous E. faecalis populations.DOI: http://dx.doi.org/10.7554/eLife.26664.001

Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.

Antibiotic-resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this “CRISPR tolerance.” We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low cas9 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.

27The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced 28 by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired 29 infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont 30Enterococcus faecalis contributes to a high incidence of HAIs with limited antibiotic treatment 31 options. Therefore, novel therapies for the mitigation of E. faecalis HAIs is critically needed. We 32 previously characterized an E. faecalis Type II CRISPR-Cas system and demonstrated its utility 33 in the sequence-specific removal of antibiotic resistance determinants. Here we present work 34 describing the adaption of this CRISPR-Cas system into a constitutively expressed module 35 encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis 36 for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we 37show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can 38 reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific 39 manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the 40 murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of 41 magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials 42 are immune to uptake of antibiotic resistance determinants in vivo. Our results demonstrate that 43 conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from 44 probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of 45 polymicrobial communities in the mammalian intestine. 46 47 48 49 50 51 52 Introduction 53 Disruption of the intestinal microbiota can predispose individuals to infection by opportunistic 54 pathogens [1, 2]. Antibiotics facilitate such disturbances leading to the development of hospital-55 acquired infections (HAIs) [3, 4]. Enterococcus faecalis, a normal constituent of the healthy human 56 intestinal microbiota and historically used in probiotics and during food fermentation, is now a 57 leading cause of HAIs [5-7]. E. faecalis is an adept opportunist that can proliferate in the dysbiotic 58 intestine following antibiotic perturbation [8]. Antibiotic-resistant clinical isolates of E. faecalis 59 typically possess expanded genomes compared to susceptible isolates due to the acquisition of 60 mobile genetic elements [9, 10], and patients colonized with these multidrug-resistant (MDR) E. 61faecalis are at increased risk of acquiring bloodstream infections [11]. Thus, there is a need for 62 novel strategies to decolonize high-risk individuals of MDR E. faecalis [12]. 63 64 CRISPR-Cas systems are protective barriers in prokaryotes that function in the adaptive 65 immunity to mobile genetic elements [13][14][15]. The well-studied Type II CRISPR-Cas system 66 consists of a DNA endonuclease (Cas...