A Novel Bacteriophage Targeting mcr-9 Enterobacter kobei with Potential Application in Fresh Leafy Greens

The presence of opportunistic pathogens, such as the Enterobacter cloacae complex (ECC), in fresh vegetables poses a significant health risk, particularly amid the ongoing antibiotic resistance crisis. Traditional chemical decontamination methods are often ineffective and these are associated with issues such as cross-resistance between antibiotics and biocides, highlighting the need for alternative approaches. This study describes the isolation of a novel phage, FENT2, with anti-ECC activity, obtained from cattle farm sewage. Belonging to the Seunavirus genus, FENT2 did not carry genes associated with lysogenic cycle, antimicrobial resistance, or virulence factors. The phage demonstrated lytic activity against the host strain E. kobei AG07E, which harbored the mcr-9 gene, exhibiting a narrow host range that also included E. ludwigii strains. In vitro assays using BioTrac (SY-LAB) impedance technology confirmed the sustained lytic activity of FENT2 under food-related stress conditions, including pH levels from 5 to 7 and NaCl concentrations up to 2%. Furthermore, FENT2 demonstrated bactericidal potential on lettuce leaves, achieving 1 log reduction in bacterial counts of the host strain after 30 min immersion treatment. These findings highlight FENT2 as a promising candidate for biocontrol applications, offering a sustainable alternative to conventional decontamination methods for reducing antimicrobial-resistant ECC contamination in fresh produce.