<i>Clostridium perfringens</i>: A Potential Pathobiont in Inflammatory Bowel Disease

The role of gut microbiota in the pathogenesis of inflammatory bowel diseases [IBD] has garnered much attention. While many studies have demonstrated a reduced diversity and altered gut microbiome in IBD patients compared to healthy individuals,1 no specific bacterium has been conclusively identified as causative. Studies have frequently observed reductions in Firmicutes, Bacteroidetes, Lactobacillus, and Eubacterium, while Proteobacteria [especially Enterobacteriaceae], including adherent-invasive Escherichia coli [AIEC], are often increased in IBD patients.2 Several specific strains have been suggested to be involved in IBD; for instance, certain E. coli strains have been associated with the development of IBD, while others, such as Faecalibacterium prausnitzii, are probably protective.3–6 However, the exact nature of their involvement and whether they have a causative role is incompletely understood. Therefore, major gaps remain, including identifying specific bacterial species associated with IBD, enhancing our mechanistic understanding of the involvement of gut microbiota, and translating such findings into developing effective interventions. In a notable paper published in this issue of JCC, Kuo et al.7 aimed to identify potential pathobionts [bacteria behaving as pathogens under specific conditions] using both culture-independent [utilizing 16S rRNA gene sequencing] and culture-dependent [with strict anaerobic collection, to allow for further in vitro experiments] methods on stool, colonic [endoscopic] washes, and/or biopsy samples collected from nine children [seven with IBD]. Clostridium perfringens was identified in eight of nine cases and was more abundant in biopsies than in stool, suggesting its importance in interacting with the host mucosal immune system. C. perfringens is a Gram-positive, spore-forming, toxigenic anaerobic bacterium. It has been identified in up to 15% of antibiotic-associated diarrhoea cases and is well established in causing various diseases, from food poisoning/infectious diarrhoea to necrotizing/gangrenous colitis in preterms and adults.8,9 However, studies assessing its association with IBD have been limited.8 To mitigate potential biases arising from the small cohort of patients, Kuo et al. evaluated the presence of this bacterium in stool and biospies in larger published datasets comprising healthy adults and IBD patients. They observed a higher prevalence of C. perfringens in IBD adult patients [19–27%] compared to healthy individuals [5%], suggesting a potential association with IBD. The efforts that went into characterizing how the identified putative pathobionts might contribute to tissue damage and symptoms in the IBD setting are certainly impressive. Amongst the wealth of experiments performed, we will highlight a few. Since bleeding is common in inflamed bowel, the authors assessed the microbial haemolytic activity [which could provide pathobionts a survival advantage in IBD]; supernatants from five of the nine patients exhibited haemolytic activity. Interestingly, C. perfringens was present within microbial communities with haemolytic activity from the five patients and following incubation with red blood cells [RBCs] these strains indeed lysed RBCs. Kuo et al. then further explored virulence factors and exotoxin production of the isolated strains, employing whole genome sequencing and Western blot techniques. Their findings revealed that the strains encode numerous virulence factors, including perfringolysin O A [pfoA], a toxin associated with haemolytic activity. Notably, strains exhibiting the highest haemolytic activity all carried this gene. They assessed the PFO protein in supernatants and observed its production. To further substantiate the role of this gene in the haemolytic potential of the strains, the authors generated genetic insertion mutants of C. perfringens. They discovered that disrupting pfoA decreased the haemolytic potential, indicating that PFO is the primary contributor to the haemolytic activity observed in these strains. Furthermore, Kuo et al. conducted in vitro experiments to elucidate additional toxicity traits of C. perfringens. They assessed the impact of the bacterial supernatant on cell viability of a variety of intestinal cell types, including colonic epithelial cell lines [T84, HT29, and Caco2], adult colonic organoid-derived monolayer epithelial cells, and some cells beneath the gut epithelium, including endothelial cells, neuroblasts, and neutrophils. While all the epithelial cell types demonstrated relative resilience to C. perfringens supernatant and purified toxins [PLC, PFO, PFO2], except for Caco2 cells, other cell types did not. Considering the results from their toxicity experiments, the authors concluded that PFO toxins are the primary contributors to the cell toxicity and haemolytic activity in the supernatant of C. perfringens. Moreover, they compared the toxicity of C. perfringens to that of 75 different bacterial strain supernatants commonly found in the gastrointestinal tract. The findings underlined that only C. perfringens could diminish cell viability, highlighting the cytotoxic nature of this strain. Kuo et al. also questioned whether the supernatant of these strains can activate neuronal cells. This exploration was prompted by the common occurrence of pain in IBD and published evidence that these toxins activate neuronal cells and induce pain during infection.10 Interestingly, they observed sensitivity of primary mouse dorsal root ganglion neurons to C. perfringens supernatants encoding pfoA, showing an ability to activate sensory neurons, as well as morphological and functional impairment of these neurons. While very preliminary, these findings suggest that C. perfringens toxins could potentially contribute to abdominal pain in IBD patients. What is unique about this paper is the amount and quality of work, with complementary approaches, supporting the novel findings. The current study sheds light on specific strains within the microbiota of IBD patients, with multiple virulence traits, including producing toxins with haemolytic activity that could also impact neurons. It provides compelling evidence supporting C. perfringens as a previously under-recognized pathobiont, relevant to IBD. To further validate the toxicity of this bacterium and define pathogenic mechanisms, in vivo experiments, particularly germ-free animal studies, could serve as a valuable complement to the current findings. As the authors rightfully suggest, evaluating the abundance of the bacterium and its toxins within the tissue of IBD patients could offer additional insights. This exploration could be expanded further by conducting a more extensive cohort study involving IBD patients and diverse populations. Subsequently, considering C. perfringens [or its toxin] as a potential therapeutic target in IBD may become plausible, and developing strategies to eliminate or control its presence could emerge as a promising avenue for future research. This is probably just one example of microbe-directed therapies that should be incorporated into the IBD treatment armamentarium. The Wine lab is funded by operating grants including from the Canadian Institutes of Health Research [CIHR] and Weston Family Foundation. The authors declare there are no conflicts of interest. N.A.F. and E.W. wrote the editorial.

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