Quantitative Comparison of Pathogen Enrichment Strategies: Toward the Harmonization of Methods for the Recovery of Shigella from Produce

Introduction: In the absence of a growth medium selective for Shigella only, the ability of these pathogens to grow in the absence of oxygen has been the enrichment strategy used in most methods for their recovery from foods. Success is dependent on their ability to grow and successfully compete with the resident microflora.

Purpose: To compare the influence of aerobic and anaerobic environments, and the impact of various chemical oxygen scavengers, on the growth efficiency of Shigella in competition with the natural flora from various produce types

Methods: Resazurin was added to the growth medium to demonstrate anaerobic conditions. Ten freeze-stressed Gfp-expressing Shigella cells were inoculated into the normal flora of spinach, field-grown tomato, parsley, cilantro and lettuce, using five replicates plus one unspiked control sample per experiment. Numbers of fluorescent bacteria and total bacteria were obtained pre and post enrichment, from serial dilutions onto non-selective Tryptic Soy Agar, to calculate growth factors and enrichment factors for each enrichment condition tested.

Results: The current anaerobic ISO method for Shigella was the most efficient method to recover the pathogen, with several logs difference in both the growth and the enrichment factors, in competition with the spinach natural flora, compared to the less efficient aerobic US Food-Emergency-Response-Network Shigella method. From the various modifications performed, i.e., nitrogen flush, nitrogen bubbling, adding oxyrase or various oxygen chemical scavengers, only the addition of nitrate had a more consistent positive effect on the recovery of Shigella from produce, in anaerobic condition.

Significance: Here we present a way to quantify the growth of a foodborne pathogen of interest, in competition with background microflora, leading to optimization of enrichment conditions for successful detection and/or recovery. We will further explore nitrate respiration as a selective step to anaerobic enrichment of Shigella, using additional food types.