Purpose: To develop a procedure by which surrogates can be identified for use in validating leafy greens washing processes.
Methods: A multi-step screening procedure including growth in nutrient media, chlorine susceptibility, and re-growth after chlorine exposure was developed for identifying potential surrogates for E. coli O157:H7. Potential strains were obtained from enrichment cultures of chlorine-washed lettuce (5-10 ppm for 30 sec), as well as enrichments of commercial probiotics and lactic acid bacterial (LAB) species. E. coli O157:H7 associated with leafy greens outbreaks were used as reference strains. Initial selection was based on growth at 25 and 37°C, with elimination of strains showing low OD600 turbidity. Lag phase time and max OD600were compared using Bioscreen-C. The remaining candidates were exposed to chlorine (0-10 ppm) for 30 sec in a minimum inhibitory concentration (MIC) format. After neutralization and incubation MICs were compared to reference strains. Growth and re-growth of exposed cultures were also monitored on agar and by Bioscreen-C. Strains with higher chlorine susceptibility by three dilution levels or having atypical re-growth compared to references were eliminated. The pool was further narrowed after 16S-RNA typing and elimination of potentially unsafe strains.
Results: A pool of 80 isolates, including 60 from the chlorine-exposed lettuce, 8 probiotic enrichment isolates and 12 LAB were tested. Atypical growth compared to references resulted in elimination of ~ 15 candidates. Approximately 50 isolates were eliminated after chlorine exposure and re-growth. Several of the remaining 15 candidates have been 16S-RNA typed and will be considered for evaluation in bench-scale washing processes.
Significance: A practical screening method for identifying surrogates was developed.