Efficacy of plasmid-encoded CRISPR-Cas antimicrobial is affected by competitive factors found in wildEnterococcus faecalisisolates

Abstract

Enterococcus faecalis is a leading cause of hospital-acquired infections. These infections are becoming more difficult to treat due to the increasing emergence of E. faecalis strains resistant to last resort antibiotics. Over the past decade, multiple groups have engineered the naturally occurring bacterial defense system CRISPR-Cas as a sequence-specific antimicrobial to combat antibiotic-resistant bacteria. We have previously established that the type II CRISPR-Cas system of E. faecalis can be reprogrammed as a CRISPR-Cas antimicrobial and delivered to antibiotic-resistant recipients on a conjugative pheromone-responsive plasmid. Using a co-culture system, we showed sequence-specific depletion of antibiotic resistance from E. faecalis model strains, both in vitro and in vivo. Although this and other studies have demonstrated the potential use for CRISPR-Cas as an antimicrobial, most have deployed the system against model bacterial strains. Thus, there is limited knowledge on how effective these potential therapies are against recently isolated and uncharacterized strains with limited laboratory passage, which we refer to here as wild strains. Here, we compare the efficacy of our previously established CRISPR-Cas antimicrobials against both E. faecalis model strains and wild E. faecalis fecal isolates. We demonstrate that these wild isolates can antagonize the CRISPR-Cas antimicrobial donor strain via competitive factors like cytolysin. Furthermore, we show that the wild isolates can effectively prevent delivery of the CRISPR-Cas antimicrobial plasmids, consequently avoiding CRISPR-Cas targeting. Our results emphasize the requisite to study CRISPR-Cas antimicrobials against wild strains to understand limitations and develop delivery systems that can endure competitive interspecies interactions in the gut microenvironment and effectively deliver CRISPR-Cas antimicrobials to their intended targets.

A diverse repertoire of anti-defense systems is encoded in the leading region of plasmids

“…Notably, plasmids have been tested as potential conjugative delivery systems for various biotechnological applications, such as targeting antibiotic-resistance bacteria using CRISPR nucleases. However, these attempts resulted in low conjugation efficiency, especially in complex microbial communities like the human gut [87][88][89] . These studies emphasize that improving conjugation efficiency is vital for future applications.…”

Functional and genetic adaptations contributing toEnterococcus faecalispersistence in the female urinary tract

“…Each isolation group is comprised of publicly available genome assemblies obtained from NCBI that met inclusion criteria described in Materials & Methods (Figure 1C). The majority of urinary strains (74%) were sequenced as part of this study (Figure 1B), while 31% of gut isolates were obtained from Dallas, Texas, fecal surveillance (33,34) and 41% of blood isolates were obtained from a Wisconsin hospital outbreak in the 1980s (24). Proportions of complete assemblies available in the gut (31%) and blood (9%) isolation groups are substantially lower than in the urine group (70%) due to the genome availability at the time of curation (Figure 1C).…”

“…In all three cases, urine isolates were predicted least frequently to be resistant whereas blood isolates were predicted to be most frequently resistant. This in part may be due to bias in the selection criteria of sequenced E. faecalis blood strains -most from hospital outbreaks, surveillance, et cetera (24,33) -that target Vancomycin-resistant and MDR strains. Aminoglycoside resistance was substantially more common in blood isolates (72%) compared to gut (35%) and urine (20%) strains (Figure 5B).…”