Genome-mined endolysin LysMD30 in combination with colistin: synergistic antimicrobial and antibiofilm activities against Vibrio parahaemolyticus

ABSTRACT Vibrio parahaemolyticus is a foodborne pathogen that threatens seafood safety globally. Endolysins are promising antibacterial candidates with rapid action and low resistance induction. Through genome mining of 163 Vibrio phages, we identified 168 potential endolysin genes and expressed the first Vibrio -phage-derived L-alanyl-D-glutamate peptidase, LysMD30, in Pichia pastoris . Although LysMD30 exhibited peptidoglycan hydrolytic activity, it lacked an intrinsic bactericidal activity. However, it displayed broad-spectrum synergy with colistin against pathogenic Vibrio species ( V. parahaemolyticus , V. harveyi , and V. alginolyticus ), yielding fractional inhibitory concentration index values lower than 0.5. This synergism was marked by a fourfold reduction in the minimum inhibitory concentration of colistin, decreasing from 16–32 μg/mL to 4–8 μg/mL across all tested strains. The bactericidal kinetics indicated that the combination triggered rapid lysis within 5 min, and the majority of turbidity reduction occurred within 20 min. Moreover, the combination effectively eradicated biofilms on both polystyrene and stainless steel surfaces within 1 h, reducing the viable bacterial counts by >3 log units. In the Galleria mellonella infection model, the combination provided 100% protection against lethal Vibrio challenge while confirming its excellent in vivo safety. Furthermore, LysMD30 exhibited robust biochemical stability—maintaining 36.96% activity at 85°C, over 90% activity at pH 5.0–8.0, and exceeding 60% activity at 800 mM NaCl—while effectively delaying the emergence of colistin resistance in V. parahaemolyticus during serial passage. In summary, LysMD30 is a newly characterized and stable enzyme that synergizes powerfully with colistin and represents a promising biocontrol agent for Vibrio control in food and aquaculture. IMPORTANCE Vibrio pathogens pose a severe threat to seafood safety and aquaculture. Conventional intervention strategies often face significant limitations: physical treatments can adversely affect the organoleptic and nutritional quality of seafood, whereas chemical agents carry the risk of toxic residues and environmental pollution. Furthermore, the escalating antimicrobial resistance of Vibrio and its capacity to form biofilms on food-contact surfaces exacerbate these challenges. This study identified LysMD30 as the first Vibrio -phage-derived L-alanyl-D-glutamate peptidase that exerts a potent synergistic bactericidal effect with colistin. This combination significantly reduced the required antibiotic dosage, rapidly eliminated both pathogenic Vibrio planktonic cells and biofilms, and demonstrated excellent in vivo safety. Our findings expand the repertoire of Vibrio -targeting biocontrol agents and provide an eco-friendly and efficient strategy for Vibrio control in food processing and aquaculture, offering robust technical support for safeguarding seafood safety and public health.