Quantitative Fluorometric Detection of Escherichia coli in Ground Beef Using Genetically Engineered Bacteriophages

Introduction: Conventional methods carried out for Escherichia coli detection, one of the major pathogens, require long time for bacterial growth followed by a series of time-consuming and complicated procedures. Fortunately, determination of β-galactosidase (β-gal), an intercellular enzyme released by E. coli, is an ideal alternative for E. coli detection. The release of β-gal can be achieved by introducing bacteriophage (phage) to infect and lyse the E. coli bacterial cells.

Purpose: This study investigated detection of E. coli in drinking water using genetically modified T7 bacteriophages with 4-Methylumbelliferyl-β-D-galactopyranoside (MUG) as substrate. The product from MUG cleaved by β-gal is fluorescent, therefore detectable by fluorescence spectroscopy.

Methods: Four factors were considered to optimize E. coli detection: MUG concentration, growth media (PBS buffer vs. LB broth), control vs. engineered phage, and temperature (25°C vs. 37°C). Under optimized conditions, T7 genetically engineered phage and MUG were added to E. coli cultures with the following concentrations (10⁵, 10⁴,10³,10², and 10¹ ,CFU/mL) plus the negative control group. The fluorescence intensity was measured every hour through 8 hours for the determination of the E. coli detection limit.

Results: The optimized condition included 2µM MUG, PBS buffer, engineered phage, and 37°C. It was found that the concentration of E. coli cells is critical to the fluorescence response: higher E. coli concentration required less detection time. Escherichia coli cells at the concentration of 10⁵, 10⁴,10³,10², 10¹ CFU/mL were detectable after total detection times of 5,6,7, and 8 hours, respectively. The fluorescence intensity data was represented as the average of three independent experiments.

Significance: This is a rapid and facile approach for examining the presence of E. coli in drinking water when compared to conventional methods. It can also be applied to the food industry for microbiological testing to ensure food safety.