Control of Escherichia coli in Fresh-Cut Mixed Vegetables Using a Combination of Bacteriophage and Carvacrol.

Abstract

The continual emergence of antibiotic-resistant bacteria and the slow development of new antibiotics has driven the resurgent interest in the potential application of bacteriophages as antimicrobial agents in different medical and industrial sectors. In the present study, the potential of combining phage biocontrol and a natural plant compound (carvacrol) in controlling Escherichia coli on fresh-cut mixed vegetable was evaluated. Four coliphages, designated Escherichia phage SUT_E420, Escherichia phage SUT_E520, Escherichia phage SUT_E1520 and Escherichia phage SUT_E1620, were isolated from raw sewage. Biological characterization revealed that all four phages had a latent period of 20-30 min and a burst size ranging from 116 plaque-forming units (PFU)/colony forming units (CFU) to 441 PFU/CFU. The phages effectively inhibited the growth of respective host bacteria in vitro, especially when used at a high multiplicity of infection (MOI). Based on transmission electron microscopy analysis, all phages were classified as tailed phages in the class of Caudoviricetes. Additionally, next generation sequencing indicated that none of the selected coliphages contained genes encoding virulence or antimicrobial resistance factors, highlighting the suitability of isolated phages as biocontrol agents. When a phage cocktail (~109 PFU/mL) was applied alone onto fresh-cut mixed vegetables artificially contaminated with E. coli, no bacteria were recovered from treated samples on Day 0, followed by a gradual increase in the E. coli population after 24 h of incubation at 8 °C. On the other hand, no significant differences (p < 0.05) were observed between treated and non-treated samples in terms of E. coli viable counts when carvacrol at the minimum inhibitory concentration (MIC) of 6.25 μL/mL was applied alone. When a phage cocktail at an MOI of ~1000 and MIC carvacrol were applied in combination, no E. coli were recovered from treated samples on Day 0 and 1, followed by a slight increase in the E. coli population to approximately 1.2-1.3 log CFU/mL after 48 h of incubation at 8 °C. However, total elimination of E. coli was observed in samples treated with a phage cocktail at a higher MOI of ~2000 and carvacrol at MIC, with a reduction of approximately 4 log CFU/mL observed at the end of Day 3. The results obtained in this study highlight the potential of combined treatment involving phage biocontrol and carvacrol as a new alternative method to reduce E. coli contamination in minimally processed ready-to-eat foods.