Current antibiotic treatments for Clostridioides difficile infection (CDI), which causes the chronic, degenerative disease colitis, eliminate beneficial bacteria in the gut along with the harmful pathogen. Researchers at North Carolina State University (NCSU) have taken the first step toward developing a therapy that attacks C. difficile while sparing other microbes.
Notoriously difficult to resolve, CDIs are responsible for an estimated 500,000 illnesses annually in the U.S., according to the CDC. Antibiotic use — the primary risk factor for CDI — alters the composition of the gut microbiome, making conditions for infection favorable. Antibiotics are also the standard treatment for CDI. That may contribute to recurrent infections, which occur in about 30% of cases, NCSU researchers report.
“Patients with recurrent CDI typically require a fecal microbiota transplant [FMT] to clear it, but we do not know the long-term consequences of giving a FMT to patients,” says Casey Theriot, PhD, Associate Professor of Infectious Disease at NCSU College of Veterinary Medicine. “This is evident from recent deaths due to transfer of antibiotic-resistant bacteria in donor stool. C. difficile continues to be on the CDC’s most urgent threat list today. Therefore, it will be important to develop novel, non-antibiotic treatments that target this pathogen while sparing the rest of the gut microbiota for clearance.”
Theriot and colleagues recently demonstrated proof of concept for just such a therapy.
“We are trying to develop and test new, innovative therapeutics for Clostridioides difficile infection. These new therapies are specific to the pathogen, largely sparing the rest of the gut microbiota.”
— Casey Theriot, PhD, Associate Professor of Infectious Disease at NCSU College of Veterinary Medicine
Tapping the Potential of Genome Editing
Working with startup company Locus Biosciences, Theriot and colleagues in the NCSU Veterinary Medicine and Agriculture and Life Sciences colleges used genome-editing technology CRISPR-Cas to target C. difficile using a specially engineered virus called a bacteriophage. The team used the bacteriophage to deliver a CRISPR cargo designed to destroy C. difficile using Cas3 proteins.
“The very nature of CRISPR-Cas systems is to provide DNA-encoded, RNA-mediated, nucleic acid targeting as part of the adaptive immune system in bacteria,” the authors write. “Conveniently, this immune system can be reprogrammed to target the host chromosome rather than an invasive bacteriophage or plasmid, leading to lethal cleavage of the genome by the CRISPR-Cas machinery. Our approach here was to deliver a CRISPR array containing a self-targeting sequence, which redirects the endogenous Cas machinery toward the genome, using CRISPR phage as a delivery vector.”
Precise targeting of C. difficile is crucial to preserving colonization resistance, which is the gut microbiota’s natural defense mechanism against harmful pathogens, according to Theriot. Antibiotic use subjects beneficial bacteria in the gut to continuous disturbance, making CDI recurrence more likely.
A Successful First Step
In vitro tests and in vivo tests in C. difficile-infected mice demonstrated the effectiveness of the team’s novel therapy — the CRISPR-Cas systems destroyed the target bacteria, although the treatment did not eliminate all of the C. difficile bacteria in the mice. Rather, the CRISPR-Cas systems temporarily reduced the mice’s bacterial load. Two days after receiving treatment, C. difficile levels in the mice rebounded. Results of the study were published in mBio, a journal of the American Society for Microbiology.
Demonstrating the ability to reduce C. difficile levels in mice, albeit temporarily, is an important step on the road to developing a viable treatment. Now, the researchers’ goal is to permanently prevent infection recurrence, which they hope to accomplish by adjusting the bacteriophage.
“Novel therapeutics that are tailored to one’s gut microbiota are on the horizon for CDI and other infectious diseases,” Theriot says. “Right now, I think the most important thing that physicians can continue to do is practice prudent use of antibiotics, as our next big challenge will be combatting antibiotic-resistant pathogens in the hospital setting. We need to be innovative when developing future therapeutics.”