A treatment for clostridioides difficile based on cell wall lysins isolated from C. difficile specific bacteriophages

Abstract

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. While the vegetative form of the pathogen is susceptible to antibiotic treatment, its ability to persist in the gut as antibiotic-resistant spores leads to reinfection in cases where protective microflora is not reestablished. Utilizing recombinant endolysins as treatment of CDI is promising because of their activity against antibiotic and bacteriophage resistant strains. In this study, the recombinantly expressed endolysin LysCD6356 and its enzymically active domain (EAD) were shown to lyse the vegetative forms of a panel of clinical isolates of C. difficile from the UK, including hypervirulent 027 ribotype strains. While these results were promising endolysins, like antibiotics and bacteriophages, endolysins have no effect against the spore form of the pathogen, which is responsible for recurrent infections following successful treatment. To address this issue, a combination of germinants and a bactericidal agent were employed to target the more sensitive form of the pathogen. In this study, exposing of C. difficile spores to germinants followed by endolysin several hours later proved to be the most effective approach suggesting a promising approach to treat relapsing CDI. Additionally, the binding of LysCD6356 and its EAD to the spore and vegetative forms of C. difficile would leave the endolysin ideally placed to target the emerging vegetative cells. This also raise the possibility of developing a diagnostic tool that could be used to detect the pathogen. Bioinformatic studies revealed a significant similarity between LysCD6356 and its EAD to CD27L and CD271-179, respectively, suggesting a possibility of sharing common motifs. However, there was no similarity of LysCD6356 and its EAD with previously published spore binding domains, suggesting the presence of a new spore binding region. An attempt to investigate the binding of endolysins to spore surface proteins, CdeC and CdeM, was unsuccessful possibly due to deficiencies in the experimental approach or the fact that the endolysins recognize other spore surface targets. The combination of our strategy of targeting spores with more traditional approach, such as fecal microbiota transplant, could provide more efficient treatment of relapsing CDI