Design and evaluation of a phage cocktail to control agriculture-associated multidrug-resistant Escherichia coli contamination on lettuce.

Multidrug-resistant (MDR) Escherichia coli from agricultural environments is an emerging threat to food safety and public health. Bacteriophages have emerged as promising alternatives to antibiotics for controlling MDR bacteria. However, studies evaluating phage biocontrol against phenotypically characterized MDR E. coli isolates from agricultural environments remain limited. This study aimed to characterize E. coli isolates recovered from agricultural environments and to develop and evaluate a phage cocktail against a target MDR isolate. A total of 11 E. coli strains were isolated and assessed for antibiotic susceptibility, biofilm formation, and swimming motility. Among them, KAE09 was selected as the target MDR isolate based on its resistance to multiple antibiotics, including streptomycin, tetracycline, oxolinic acid, and ciprofloxacin, together with strong biofilm-forming ability and high motility. To control this isolate, we developed a phage cocktail consisting of two E. coli phages, ELP2 and ELT3, with previously reported distinct receptor specificities. Cross-resistance analysis showed no reciprocal cross-resistance between ELP2 and ELT3 in KAE09. The phage cocktail significantly inhibited bacterial growth, reduced preformed biofilms, and suppressed biofilm formation of KAE09. The cocktail also effectively reduced viable cell counts of the isolate on lettuce at room temperature and under refrigeration. These findings highlight the potential of the phage cocktail as an effective biocontrol agent for reducing MDR E. coli contamination on fresh produce.