Evaluation of GFP Reporter-labeled Control Strains for Shiga Toxin-producing Escherichia coli (STEC) Assays

Introduction: Growing concern over bacterial food contamination has led to increased examination of food testing protocols in today’s industry. Currently, the use of bacterial strains as positive controls in testing protocols is not widely practiced for fear of cross-contaminating samples. Due to ongoing scrutiny of food testing methodology and growing regulations under the Food and Drug Administration (FDA) Food Safety Modernization Act, it is imperative to have control strains with unique, easily detectable traits that distinguish positive control strains from actual food contaminants, diminishing the fear of cross-contamination and improving current practices. We have created green fluorescent protein (GFP) reporter-labeled Escherichia colistrains, including Shiga toxin-producing O157, for use as controls in QC testing.

Purpose: To evaluate the use of E. colilabeled with GFP as a positive control in media and food safety testing.  

Methods: A plasmid encoding the synthetic Dasher GFP (DNA 2.0) was transformed into toxigenic E. coli O157:H7 and different serogroups of the “Big Six” non-O157 E. coli (ATCC® MP-9™) strains. The stability of the reporter was determined through unselective serial passage and by plating on tryptic soy agar, following the USDA FSIS MLG Chapter 5.05 protocol. Additionally, the growth rate and chromogenic properties of reporter-labeled and native strains were compared. 

Results: The GFP-transformed STEC colonies were readily detectable with the naked eye when exposed to a 310 nm UV light source. Reporter-labeled strains containing GFP were compared with their native strains to identify phenotypic changes on Rainbow Agar™ (Biolog), a chromogenic media commonly used to assist in the identification of E. coli serotypes. Minimal color variations between the reporter versus native strains were observed. To determine the effect of GFP expression on the growth and viability of the labeled E. coli, growth studies were performed using BioScreen C™ (Growth Curves, USA). Minimal optical density variation was detected. To determine GFP reporter stability in these engineered strains, they were passaged once every 24 hours under temperature stress at 42°C. The percentage of GFP positive colonies was 60-100% after two days (percentage variation was strain dependent), permitting their use as controls in the intended qualitative food testing workflows. 

Significance: This study demonstrates that these GFP-labeled E. coli strains can be routinely used as positive controls for media testing and in the detection of microbial pathogens in food.