Assessing the Feasibility of Employing a Combination of a Bacteriophage-Derived Endolysin and Spore Germinants to Treat Relapsing Clostridioides difficile Infection

Abstract

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. Whereas the vegetative form of the pathogen is susceptible to treatment with antibiotics, its ability to persist in the gut as antibiotic-resistant spores means that reinfection can occur in cases were the individual fails to re-establish a protective microflora. Bacteriophages and their lysins are currently being explored as treatment options due to their specificity, which minimizes the disruption to the other members of the gut microflora that are protective. The feasibility of employing recombinant endolysins to target the vegetative form of C. difficile has been demonstrated in animal models. In this study, we cloned and expressed the enzyme active domain of LysCD6356 and confirmed its ability to lyse the vegetative forms of a diverse range of clinical isolates of C. difficile, which included members of the hypervirulent 027 ribotype. Lytic activity was adversely affected by calcium, which is naturally found in the gut and is released from the spore upon germination. Our results suggests that a strategy in which the triggering of spore germination is separated in time from the application of the lysin could be developed as a strategy to reduce the risk of relapsing C. difficile infections.

The long and sinuous road to phage-based therapy of Clostridioides difficile infections

“…Hence, combination therapy that includes spore germinants could promote germination, therefore ensuring maximum killing of the infected strain. This strategy has, however, been shown to interfere with endolysin activity, as the massive release of calcium during germination is shown to inhibit the activity of LysCD6356, at least in vitro ( 38 ). The importance of this observation to the in vivo condition requires further investigation.…”

“…Of note, one common observation that has been reported with all C. difficile phage endolysins is that the catalytic domain of the enzyme alone (EAD) is sufficient for full activity. In fact, the removal of the CBD was reported to increase the lytic activity of C. difficile ( 30 – 32 , 36 , 38 ). It was also shown that the CBD from the CD16/50L endolysin was responsible for trapping the endolysin within cellular debris after lysis.…”

“…Full-length endolysins were shown to be very specific toward C. difficile , as little or no activity was noted on other commensal bacteria, including related Clostridia. However, most truncated endolysins comprising only the EAD displayed a slightly broader host range, killing all C. difficile isolates tested, in addition to a few other species, in particular Clostridium sordellii, Clostridium bifermentans, Bacillus subtilis , and Listeria monocytogenes ( 30 – 32 , 36 , 38 ).…”