Effects of Postbiotic from Bacteriocin-Like Inhibitory Substance Producing Enterococcus faecalis on Toxigenic Clostridioides difficile

Abstract

Objective: To determine the activities of postbiotics, prepared from bacteriocin-like producing Enterococcus faecalis (E. faecalis) against Clostridioides difficile (C. difficile) and its spores, and to assess the safety of postbiotics in vivo. Material and Methods: Bacteriocin production of E. faecalis PK1201 was screened by proteolytic enzyme treatment, and bacteriocin-encoding genes were characterized by whole genome sequencing. A postbiotic of E. faecalis PK1201 was prepared using neutralized cell-free supernatant or bacterial cell lysate, which was then used to screen for antimicrobial activity via agar well diffusion. The lyophilized cell-free supernatant (LCFS) was further determined for its minimum inhibitory concentration (MIC) against C. difficile 630. The morphological changes of C. difficile were observed under scanning electron microscopes (SEM). Subsequently, the LCFS at sub-MIC and MIC were used to evaluate anti-spore germination activity. Finally, the safety of postbiotics was accessed using the Galleria mellonella model. Results: E. faecalis PK1201 carried enterolysin A encoding gene. For postbiotic preparation, only LCFS exhibited antimicrobial activity, and the activity was completely lost after proteinase K treatment; indicating the existence of bacteriocin. LCFS showed anti-C. difficile with MIC of 18.2±6.9 mg/mL. The SEM images demonstrated shorter destruction of C. difficile cells after being treated with LCFS. Interestingly, LCFS at the MIC and sub-MIC revealed anti-spore germination activity against toxigenic C. difficile compared to the control. LCFS showed no acute toxicity in G. mellonella at the tested concentration. Conclusion: The powerful activity and safety of LCFS shed light on the role of postbiotics in pharmaceutical products to control CDI.