Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0–6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.

Clostridioides difficile ( C. difficile ) is a gram-positive, anaerobic spore-forming bacterium and a major cause of antibiotic-associated diarrhea. Humans are naturally resistant to C. difficile infection (CDI) owing to the protection provided by healthy gut microbiota. When the gut microbiota is disturbed, C. difficile can colonize, produce toxins, and manifest clinical symptoms, ranging from asymptomatic diarrhea and colitis to death. Despite the steady-if not rising-prevalence of CDI, it will certainly become more problematic in a world of antibiotic overuse and the post-antibiotic era. C. difficile is naturally resistant to most of the currently used antibiotics as it uses multiple resistance mechanisms. Therefore, current CDI treatment regimens are extremely limited to only a few antibiotics, which include vancomycin, fidaxomicin, and metronidazole. Therefore, one of the main challenges experienced by the scientific community is the development of alternative approaches to control and treat CDI. In this Frontier article, we collectively summarize recent advances in alternative treatment approaches for CDI. Over the past few years, several studies have reported on natural product-derived compounds, drug repurposing, high-throughput library screening, phage therapy, and fecal microbiota transplantation. We also include an update on vaccine development, pre- and pro-biotics for CDI, and toxin antidote approaches. These measures tackle CDI at every stage of disease pathology via multiple mechanisms. We also discuss the gaps and concerns in these developments. The next epidemic of CDI is not a matter of if but a matter of when. Therefore, being well-equipped with a collection of alternative therapeutics is necessary and should be prioritized.

Receptor-binding proteins (RBPs) are located at the viral tail and mediate the initial recognition of phage to a specific bacterial host. Phage RBPs have co-evolved with numerous types of host receptors resulting in the formation of a diverse assortment of cognate pairs of RBP-receptors that function during the phage attachment step. Although several Clostridioides difficile bacteriophages have been discovered, their RBPs are poorly described. Using homology analysis, putative prophage-tail structure (pts) genes were identified from the prophage genome of the C. difficile HN10 strain. Competition and enzyme-linked immunosorbent assays, using recombinant PtsHN10M, demonstrated the interaction of this Pts to C. difficile cells, suggesting a role as a phage RBP. Gel filtration and cross-linking assay revealed the native form of this protein as a homotrimer. Moreover, truncated variants indicated that the C-terminal domain of PtsHN10M was important for binding to C. difficile cells. Interaction of PtsHN10M was also observed to the low-molecular weight subunit of surface-layer protein A (SlpA), located at the outermost surface of C. difficile cells. Altogether, our study highlights the function of PtsHN10M as an RBP and potentially paves the way toward phage engineering and phage therapy against C. difficile infection.

Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment.