| Summary: | <i>Cronobacter sakazakii</i> (<i>C. sakazakii</i>) is a foodborne pathogen with the ability to survive and proliferate under cold-chain conditions, thereby posing serious risks to infants and public health. However, most reported phages targeting this pathogen demonstrate limited bacteriolytic activity at low temperatures. In this study, the lytic phage LPCS39 was isolated, characterized, and tested in food matrices including milk, reconstituted powdered infant formula (RPIF), and lettuce. LPCS39 exhibited a broad hiost range against multiple <i>Cronobacter</i> species, and <i>Enterobacter cloacae</i>, along with high stability across pH 3–11, thermal tolerance up to 60°C, a high burst size of 545 PFU/cell with a short latent period of 10 min, and a genome free of virulence or antibiotic resistance genes. In food matrices artificially contaminated with <i>C. sakazakii</i> (3 log<sub>10 </sub>CFU/mL), it effectively reduced <i>C. sakazakii</i> levels in lettuce, liquid milk, and RPIF at various temperatures, including ambient (23°C), physiological conditions (37°C), and retained antibacterial activity under refrigeration (4°C). Notably, at 4°C, LPCS39 reduced <i>C. sakazakii</i> to undetectable levels (<10 CFU/mL) within 2 h in lettuce, 6 h in liquid milk, and 9 h in RPIF at 4°C. To investigate its cold-adaptive features, <i>in silico</i> analyses were conducted. LPCS39 encoded 25 cold-adaptive proteins (8.8 % of total ORFs) with elevated Ala/Leu, Asp/Glu, and Arg/Lys ratios, compared to only 4 (1.5 % of total ORFs) in LPCS28. These findings provide novel insights into the molecular basis of phage cold adaptation, highlighting LPCS39 as a promising biocontrol agent for managing <i>C. sakazakii</i> contamination in cold-chain food systems.
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