Heterosexual man: Development of a phage cocktail to control the top 7 shiga -toxin E. coli serogroups on fresh produce

Sorenne is afraid of bugs.

I told her the other day how Surgeoner made his students (and kids) stick their arms into boxes filled with mosquitoes – mossies as they’re called here – to see what dose of DEET worked, while the kids got bitten.

She became more terrified.

That’s OK, I have lots of parenting failures.

As the consumption of fresh produce increases worldwide, Shiga-toxigenic Escherichia coli (STEC) outbreaks linked to contaminated produce are becoming more frequent. Biocontrol of STEC using phages can be a safe and effective way to reduce STEC on fruits and vegetables.

Purpose: 1) Evaluate the effectiveness of 7 bacteriophages to reduce viability of E. coli serogroups O26, O45, O103, O111, O121, O145, and O157 on lettuce and sprouts at 4, 10 or 25 °C and storage for 1, 24, 48, and 72 h; 2) to assess STEC phage effectiveness to reduce STEC compared to a conventional chlorinated water wash (150ppm for fresh produce, 1000 ppm for seeds); and 3) to determine if phage-insensitive mutants arise after phage treatments. Methods: 1) Phages either individually or as a cocktail (~108 log PFU/ml) were sprayed to control 105 log CFU/ml STEC spot-inoculated onto fresh lettuce and sprouts that were stored for 1, 24, 48, and 72 h at 2, 10, and 25°C. Samples were collected and bacteria enumerated on McConkey (STEC total numbers) and Rainbow agar (STEC individual serogroup numbers) after each storage period. STEC isolates were confirmed using latex agglutination tests specific for STEC, PCR, and Immunomagnetic Separation (IMS) to recover any surviving STEC serogroups. 2) A scale-up experiment with STEC phage cocktail and chlorinated water applied to lettuce, sprouts, and mung bean seeds (MB) was undertaken to further assess the effectiveness of STEC phage cocktail to reduce STEC on larger quantities of produce and to compare its effectiveness to chemical disinfection. Lettuce, sprouts, and MB were treated with: i) chlorinated water wash (150 ppm for lettuce/sprouts, 1000 ppm for MB); ii) STEC phage cocktail; and iii) a combination of chlorinated water and STEC phage cocktail. Phage cocktails were delivered by immersion. After treatment, lettuce and sprouts were stored for 1, 24, 48, and 72 h at 2, 10, and 25 °C. MB were stored in the dark for 24 h at 25 °C. Seeds were germinated in sterilized water and the survival of STEC isolates was assessed by plating on Rainbow agar. 3) To determine if phage resistant mutants developed during STEC phage treatment, isolated colonies surviving after exposure to phage were randomly picked and tested for phage sensitivity using a microplate virulence assay. OD was measured after microplate incubation for 5 h at 37 °C. Results: 1) In spot-inoculation experiments, the highest STEC reduction (3.7 log10 CFU/g) caused by the phage cocktail was observed at 2 °C after 72 h of storage on lettuce; whereas on sprouts the highest reduction (2.45 log10 CFU/g) occurred at 25 °C after samples were stored for 1 h. All STEC O157, O26, and O103 were killed by spraying phages on both lettuce and sprouts. Overall STEC phages reduced STEC by > 2 log10 CFU/g on fresh produce. 2) During scale-up experiments, the combination of STEC phage cocktail and chlorinated water achieved the highest STEC reductions on produce. On MB, the highest reduction in STEC (1.69 log10 CFU/g) was observed after treatment with the STEC phage cocktail alone. On MB germinated sprouts, the reduction in total STEC was < 1 log10 CFU/g in all treatments. However, none of the treatments eliminated all 7 STEC serogroups. 3) Regarding phage resistance experiments, no STEC mutants were recovered. Conclusion: Results showed that the phage cocktail was able to reduce STEC O26, O45, O103, O111, O121, O145, and O157 serogroups alone, or in combination with chlorinated water on lettuce, sprouts, and MB seeds without the development of phage resistant mutants. However, for MB, the STEC phage cocktail was not effective at controlling STEC populations during germination.