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ARS Home » Plains Area » Lincoln, Nebraska » Agroecosystem Management Research » Research » Publications at this Location » Publication #409014
Research Project: Managing Manure as a Soil Resource for Improved Biosecurity, Nutrient Availability, and Soil Sustainability

Location: Agroecosystem Management Research

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

Author
item UPRETI, CHAHAT - The University Of Texas At Dallas
item KUMAR, PRANAV - The University Of Texas At Dallas
item Durso, Lisa
item PALMER, KELLI - The University Of Texas At Dallas

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 10/2/2023
Publication Date: 10/2/2023
Citation: Upreti, C., Kumar, P., Durso, L.M., Palmer, K.L. 2023. CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure. bioRxiv. https://doi.org/10.1101/2023.09.26.559507.
DOI: https://doi.org/10.1101/2023.09.26.559507

Interpretive Summary: The development and use of antibiotics has revolutionized human health and livestock production. However, due to the use and misuse of antibiotics, we are now facing a global issue of antibiotic resistance. More than a million people each year die from antibiotic-resistant infections. One of the ways in which bacterial pathogens become resistant to antibiotics is via the transfer of resistance genes from one bacterium to another, on DNA fragments called plasmids. In order to protect themselves from foreign genetic elements such as plasmids, bacteria have evolved several defense systems, one of which is called CRISPR-Cas. It has been shown previously that bacteria that possess a functioning CRISPR-Cas system are significantly less likely to be multi-drug resistant. This has several potential applications since we can leverage CRISPR presence or CRISPR-based technologies to tackle antibiotic resistance. However, much of this work has been done on strains from clinical origins. Bacteria from agricultural niches are also exposed to a wide variety of antibiotics, and multi-drug resistance is a concern in these niches, but how prevalent CRISPR-Cas is in them is not well known. In this work, we try to answer this question, by focusing on the bacterium Enterococcus faecalis. E. faecalis is both clinically relevant, as one of the major causes of hospital acquired infections in US, and also agriculturally relevant, since it colonizes most animals and has been implicated in transfer of resistance from food animals to humans. We found a clear similarity in CRISPR type diversity in E. faecalis strains from humans and animals. To test whether CRISPR-Cas is functional in this environment, we tested its ability to block plasmid transfer in manure and found that its efficacy is lower than in lab conditions initially, but surpasses lab conditions after 48 hours. Finally, we test CRISPR-Cas-based antimicrobials in the manure niche, and find that they effectively immunize bacteria against the acquisition of resistance-carrying plasmids. Our work is among the first to explore prevalence and efficacy of CRISPR-Cas in agricultural environments, and lays the foundation for further CRISPR-based interventions to tackle antibiotic resistance.

Technical Abstract: 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 shows 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. Importance: Antibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas - a bacterial defense system - to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium – Enterococcus faecalis. We show that CRISPR-Cas is prevalent, functional in manure, and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas based strategies for control of antibiotic resistance in agricultural settings.