Listeria monocytogenes Bacteriophages from Colombia Reveal Putative New Phage Species with Biocontrol Potential

In this study, we isolated and characterized novel bacteriophages targeting L. monocytogenes from food-processing environments in Colombia and evaluated their potential as biocontrol agents. Eighteen phages were isolated and initially characterized based on host range and efficiency of plating (EOP), from which, based on lytic and host range performance, six phages were selected and formulated into three phage cocktails (C1, C2, and C3). Infection curve assays at a multiplicity of infection (MOI) of 0.1 and 1 showed that cocktails C1 and C2 produced isolate-dependent and transient bacterial population reduction at 4-5 h postinfection, lowering OD₅₅₀ values to near zero (0.03-0.04). Cocktail C3 (composed of the six phages selected to formulate the cocktail) exhibited the strong and most sustained activity, reducing OD₅₅₀ to zero at 4-5 h postinfection and maintaining control for 6-7 h. One-step growth curves revealed latent periods ranging from 45 to 60 min and burst sizes of approximately 15-49 PFU per infected cell. Scanning transmission electron microscopy (STEM) confirmed the siphovirus morphotype for the six evaluated phages. Stability analysis at environmental and refrigeration temperatures showed minimal viability losses (≤1.2 log), under mildly acidic conditions (pH 4; <1.5-log reduction), and in the presence of curing salt (220 ppm; ≤0.5-log reduction), while strong inactivation was observed under alkaline conditions (pH 9; ∼6-8.5-log reduction). Comparative genomic analyses revealed that four sequenced phages (SP-LM003, SP-LM004, SP-LM007, and SP-LM010) are closely related to members of the genus Psavirus but remain genomically distinct from currently recognized ICTV reference species, supporting their classification as putative novel species. Although lysogeny-associated genes were identified, no closely related integrated prophages were detected among publicly available L. monocytogenes genomes, suggesting the presence of potentially incomplete or atypical lysogeny modules. Cocktail C3 efficacy in pork ham stored at 4 °C demonstrated a growth-inhibitory effect, maintaining bacterial counts over storage time at the tested MOIs (1,000 and 10,000). The results show that the formulated cocktail C3 is effective against L. monocytogenes in vitro, suggesting it could be used as a phage-based biocontrol strategy in ready-to-eat meat.