CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure

The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system CRISPR-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its anti-plasmid efficacy in an agricultural niche – manure. We show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. CRISPR-Cas was found to be an effective barrier against resistance plasmid transfer in manure, with improved effect as time progressed. CRISPR-based antimicrobials to cure resistant E. faecalis of erythromycin resistance was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting, and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a OneHealth approach.