Characterization of a Novel Endolysin Sb1-LysAEJ as a Biocide and Food-contact Surface Disinfectant for the Control...

Staphylococcus aureus (S. aureus) is a globally prevalent pathogen that causes foodborne diseases, posing a serious threat to the food industry and human health. The rapid bactericidal action, high specificity, and low resistance potential of bacteriophage-derived endolysins make them promising alternatives to conventional antimicrobials. In this study, we identified a 1503 bp gene encoding the phage endolysin LysAEJ from the genome of S. aureus phage Sb1 and cloned it into the pET28a expression vector. The encoded endolysin LysAEJ, consisting of 494 amino acid residues (55.5 kDa), was expressed in E. coli BL21 (Rosetta). LysAEJ demonstrated strong bactericidal activity against S. aureus under conditions of pH 9.0-12.0, 4.0-45.0 °C, and in the presence of Ca²⁺. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 25 µg/mL and 50 µg/mL, respectively. Time‑kill assays using CFU enumeration showed that 100 µg/mL LysAEJ reduced viable S. aureus counts by > 4 log within 30 min. LysAEJ effectively reduced the bacterial load of S. aureus in artificially contaminated milk by approximately 3.9 log CFU/mL and on ham surfaces to below the detection limit (10¹ CFU/cm²) within 6 h at room temperature. It also exhibited high efficiency in removing S. aureus biofilms from food‑contact surfaces (polystyrene, glass, stainless steel), reducing biofilm mass by 83%. Confocal laser scanning microscopy (CLSM) with acridine orange/ethidium bromide staining showed effective killing of cells within the biofilm matrix. Scanning electron microscopy (SEM) revealed significant perforations and morphological damage to cells. Collectively, LysAEJ demonstrates excellent specificity and antimicrobial activity, making it a promising biocontrol agent against S. aureus and its biofilms in diverse food processing environments.