P1-147 A Multi-step Screening Procedure for Selecting Surrogate Organisms for Use in Validating Fresh Produce Washing Processes

Monday, August 4, 2014
Exhibit Hall D (Indiana Convention Center)
Arlette Shazer, U.S. Food and Drug Administration, Bedford Park, IL
Diana Stewart, U.S. Food and Drug Administration, Bedford Park, IL
Kaiping Deng, U.S. Food and Drug Administration, Bedford Park, IL
Mary Lou Tortorello, U.S. Food and Drug Administration, Bedford Park, IL
Introduction: Chlorine-containing washes are used in post-harvest processing of fresh produce.  Validation of this preventative control will be required under the Food Safety Modernization Act, but safe-to-use surrogates which mimic the pathogen of concern are unavailable.

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.