Biocontrol of Listeria Monocytogenes in Milk: Isolation and Characterization of a Novel Bacteriophage Cocktail

Abstract

Listeria monocytogenes is a significant foodborne pathogen known for its persistence in food processing environments and resistance to various stress conditions. This study aimed to isolate and characterize bacteriophages specific to L. monocytogenes and evaluate their potential as biocontrol agents in milk. A total of 43 phages were isolated from diverse wastewater sources across Türkiye. Based on their host range, lytic activity, and growth kinetics, two phages (LM1116TR and LM1418TR) were selected for further comprehensive phenotypic and genomic characterization. Host range assays revealed high specificity toward L. monocytogenes, with lytic activity observed against multiple serotypes (1/2a, 1/2b, 1/2c, and 4b). Transmission electron microscopy revealed that the phages exhibit a myovirus-like morphotype and belong to the class Caudoviricetes. Whole-genome sequencing confirmed that two phages had complete circular genomes lacking antimicrobial resistance genes. Thermal stability tests showed the phages remained detectable after 1 h at 40 degrees C, 50 degrees C, and 60 degrees C but were sensitive to acidic pH, especially to pH 3. Significant reductions in L. monocytogenes counts were observed following phage cocktail treatment, reaching up to 4.22 log CFU/mL in vitro (37 degrees C) and 1.66 log CFU/mL in UHT milk (4 degrees C) (p < 0.001). Additionally, one-year storage tests at +4 degrees C, -20 degrees C, and -80 degrees C in tryptic soy broth (TSB), SM buffer, physiological saline and Tris-Buffered Saline (TBS, 1 & times; ) identified TSB with 20% glycerol as the most suitable for preserving phage stability at subzero temperatures. These results show the potential of new phages as effective and stable biocontrol agents against L. monocytogenes in food safety applications, supporting their potential as biocontrol agents, particularly within the dairy industry.