Exploring the antimicrobial potential of hybrid endolysin L4-GS-C111 for the control of Bacillus cereus in food systems

Bacillus cereus is a major foodborne pathogen with limited effective control strategies. To enhance antibacterial efficacy against B. cereus , hybrid endolysins were engineered by fusing the enzymatically active domain (EAD) of LysB4 with those of CD27L and PHICD111_20024, amidase-type endolysins derived from Clostridioides difficile phages. All recombinant proteins were successfully expressed in Escherichia coli and retained their bacteriolytic activity. Among the four hybrid constructs, L4-GS-C111 and C111-GS-L4 exhibited stronger bactericidal activity against Bacillus species than that of their parental forms. Structural modeling predicted that the active sites of each endolysin were accessible in both L4-GS-C111 and C111-GS-L4, suggesting enhanced activity. Both constructs maintained stable lytic activity across a wide pH range (4.5–9.5) and varying NaCl concentrations (0–150 mM), outperforming LysB4_EAD under various changes in pH and salinity. L4-GS-C111 successfully inactivated B. cereus in soymilk with pH 6.5 and 15 mM NaCl, achieving a 4 log decrease within 12 h at 40 μM. L4-GS-C111 also effectively disrupted preformed B. cereus biofilms, as confirmed by confocal microscopy, but barely displayed any cytotoxicity toward Caco-2 cells. These results demonstrate that L4-GS-C111 exerts bactericidal activity against B. cereus in diverse environments, supporting its potential as a biocontrol agent for improving food safety.