Biocontrol of Escherichia coli O157: H7 on fresh-cut leafy greens

Abstract

The effect of a bacteriophage cocktail (EcoShield™) that is specific against Escherichia coli O157:H7 was evaluated against a nalidixic acid-resistant enterohemorrhagic E. coli O157:H7 RM4407 (EHEC) strain on leafy greens stored under either (1) ambient air or (2) modified atmosphere (MA; 5% O₂/35% CO₂/60% N₂). Pieces (~2 × 2 cm²) of leafy greens (lettuce and spinach) inoculated with 4.5 log CFU/cm² EHEC were sprayed with EcoShield™ (6.5 log PFU/cm²). Samples were stored at 4 or 10°C for up to 15 d. On spinach, the level of EHEC declined by 2.38 and 2.49 log CFU/cm² at 4 and 10°C, respectively, 30 min after phage application (p ≤ 0.05). EcoShield™ was also effective in reducing EHEC on the surface of green leaf lettuce stored at 4°C by 2.49 and 3.28 log units in 30 min and 2 h, respectively (p ≤ 0.05). At 4°C under atmospheric air, the phage cocktail significantly (p ≤ 0.05) lowered the EHEC counts in one day by 1.19, 3.21 and 3.25 log CFU/cm² on spinach, green leaf and romaine lettuce, respectively compared with control (no bacteriophage) treatments. When stored under MA at 4°C, phages reduced (p ≤ 0.05) EHEC populations by 2.18, 3.50 and 3.13 log CFU/cm², on spinach, green leaf and romaine lettuce. At 10°C, EHEC reductions under atmospheric air storage were 1.99, 3.90 and 3.99 log CFU/cm² (p ≤ 0.05), while population reductions under MA were 3.08, 3.89 and 4.34 logs on spinach, green leaf and romaine lettuce, respectively, compared with controls (p ≤ 0.05). The results of this study showed that bacteriophages were effective in reducing the levels of E. coli O157:H7 on fresh leafy produce, and that the reduction was further improved when produce was stored under the MA conditions.

Keywords: E. coli O157:H7; MAP; bacteriophage; leafy green; modified atmosphere packaging.

Figure 1. Effect of EcoShield™ on EHEC on spinach surface. (A) 4°C; (B) 10°C. Data shown as mean ± SD. NP: no phage; P: Phage treatment.

Figure 2. Effect of EcoShield™ on EHEC on green leaf lettuce at 4°C. Experiment was repeated twice in duplicates. NP: No phage; P: Phage treatment.

Figure 3. Bacteriophage stability on fresh produce stored at 4°C. Results are the mean of 3 replicates ± SD.

Figure 4. Recovered EHEC counts on green leafy produce contaminated with EHEC, treated with bacteriophage cocktail, and stored at 4°C under; (A) MAP with atmospheric air; (B) MAP with 5% O₂, 35% CO₂, 60% N₂ gas. Experiment was repeated twice in duplicates for each produce. Spi: Spinach; GLL: Green leaf lettuce; RL: Romaine lettuce; A: Air package; G: Gas package; NP: No phage; P: Phage treatment.

Figure 5. Recovered EHEC counts on green leafy produce contaminated with EHEC, treated with bacteriophage cocktail, and stored at 10°C under; (A) MAP with atmospheric air; (B) MAP with 5% O₂, 35% CO₂, 60% N₂ gas. Experiment was repeated twice in duplicates. Spi: Spinach; GLL: Green leaf lettuce; RL: Romaine lettuce; A: Air package; G: Gas package; NP: No phage; P: Phage treatment.

Figure 6. Partial percentages of O₂ and CO₂ during MAP experiment. (A) MAP with atmospheric air; (B) MAP with 5% O₂, 35% CO₂, 60% N₂ gas.