Mining and multifaceted applications of phage lysin for combatting Vibrio parahaemolyticus

Vibrio parahaemolyticus, a prevalent foodborne pathogen found in both water and seafood, poses substantial risks to public health. The conventional countermeasure, antibiotics, has exacerbated the issue of antibiotic resistance, increasing the difficulty of controlling this bacterium. Phage lysins, as naturally occurring active proteins, offer a safe and reliable strategy to mitigate the impact of V. parahaemolyticus on public health. However, there is currently a research gap concerning bacteriophage lysins specific to Vibrio species. To address this, our study innovatively and systematically evaluates 37 phage lysins sourced from the NCBI database, revealing a diverse array of conserved domains and notable variations in similarity among Vibrio phage lysins. Three lysins, including Lyz_V_pgrp, Lyz_V_prgp60, and Lyz_V_zlis, were successfully expressed and purified. Optimal enzymatic activity was observed at 45℃, 800 mM NaCl, and pH 8-10, with significant enhancements noted in the presence of 1 mM membrane permeabilizers such as EDTA or organic acids. These lysins demonstrated effective inhibition against 63 V. parahaemolyticus isolates from clinical, food, and environmental sources, including the reversal of partial resistance, synergistic interactions with antibiotics, and disruption of biofilms. Flow cytometry analyses revealed that the combination of Lyz_V_pgp60 and gentamicin markedly increased bacterial killing rates. Notably, Lyz_V_pgrp, Lyz_V_pgp60, and Lyz_V_zlis exhibited highly efficient biofilm hydrolysis, clearing over 90 % of preformed V. parahaemolyticus biofilms within 48 h. Moreover, these lysins significantly reduced bacterial loads in various food samples and environmental sources, with reductions averaging between 1.06 and 1.29 Log CFU/cm² on surfaces such as stainless-steel and bamboo cutting boards and approximately 0.87 CFU/mL in lake water and sediment samples. These findings underscore the exceptional efficacy and versatile application potential of phage lysins, offering a promising avenue for controlling V. parahaemolyticus contamination in both food and environmental contexts.