Evaluation of Non-pathogenic Escherichia coli Isolates to be Used in Field Trials Examining the Persistence of Pathogens in Untreated Soil Amendments

Introduction: The proposed FDA “Produce Rule,” part of the Food Safety Modernization Act (FSMA), states that untreated soil amendments (UTSA) must be applied to agricultural fields 270 days before harvest of produce crops to minimize pathogen transfer.  A framework document which evaluated experimental designs was developed for groups intending to apply for an evidence-based variance from this proposed rule.  This framework recommends use of a non-pathogenic bacterium with a stable detectable phenotype and history of use in field studies as a surrogate for a pathogen compared to strain(s) with only laboratory data.  

Purpose: The purpose of this study was to examine the longitudinal persistence of three non-pathogenic rifampicin-resistant Escherichia coli strains (TVS 353,354,355), originally isolated from agricultural environments, in various manure-amended soils to determine their suitability for use as non-pathogenic surrogates for E. coli O157:H7.   

Methods:  An inoculum containing equal populations of each of the three E. coli strains was applied to poultry litter-, dairy manure-, and horse manure-amended soils at two different sites (Maryland and Pennsylvania) in five separate seasons. E. coli were isolated from amended soils between 114 - 150 days post-inoculation. DNA was extracted and amplified by BOX-PCR to create unique banding patterns by gel electrophoresis to identify specific E. coli.

Results: Overall, 141 E. coli isolates were identified from five different field studies: 105 (74%) were TVS 355, 28 (20%) were TVS 354, and 8 (6%) were TVS 353.  In four of the five studies evaluated, TVS 355 was the E. coli isolate isolated most frequently on the last day of the field study.

Significance: The increased prevalence of TVS 355 compared to the other E. coli isolates evaluated displays its extended persistence in manure-amended soils, and indicates that TVS 355 may be an appropriate non-pathogenic E. coli isolate to use in variance request and other UTSA field /soil research studies.