The Black Soldier Fly Hermetia illucens Larva Presents an Antimicrobial Activity in Response to Clostridioides difficile Exposure.

Clostridioides difficile C. difficile infection (CDI) is the primary cause of antibiotic-associated diarrhea and is highly resistant to antibiotics. The black soldier fly Hermetia illucens (BSF) has a large antimicrobial peptide (AMP) repertoire effective against various infectious diseases. This study evaluated the activity of BSF hemolymph-extracted AMPs, with particular focus on defensin-like peptide 4 (DLP4), against C. difficile isolates. In the current study, BSF larvae (BSFL) were injected with or fed pathogenic C. difficile strain 630. The effects of post-infection hemolymph and of synthetic AMPs on 18 clinical antibiotic-resistant C. difficile isolates were assessed using the disk diffusion test. Real-time RT PCR was conducted to measure expression of five hemolymph AMP genes in infected larvae. Bacterial counts were measured after DLP4 exposure and cytotoxicity of DLP4 against Caco-2 epithelial cells was tested using the XTT method. Bacterial outer layer damage was evaluated by scanning electron microscopy. Hemolymph extracted from C. difficile-infected BSFL had a significant inhibitory effect on C. difficile growth. All five AMPs were upregulated following exposure to pathogenic C. difficile. DLP4 was the most potent AMP, with MICs in the range of 64-250 µg/ml for the different clinical isolates. DLP4 exhibited rapid-onset bactericidal activity (1 hour), by inducing holes in the bacterial outer layer, while having no cytotoxic effect on human epithelial cells. In conclusion, BSF hemolymph-derived DLP4 bears antimicrobial properties against antibiotic-resistant C. difficile isolates. DLP4 may represent a promising and novel approach for the treatment of C. difficile infections.