An Engineered Reporter Phage for the Fluorometric Detection of Escherichia coli in Ground Beef

Anqi Chen,Danhui Wang,Sam R Nugen,Juhong Chen

doi:10.3390/microorganisms9020436

Anqi Chen, Danhui Wang + Show 2 more

Open Access

https://doi.org/10.3390/microorganisms9020436

Copy DOI

Abstract

Despite enhanced sanitation implementations, foodborne bacterial pathogens still remain a major threat to public health and generate high costs for the food industry. Reporter bacteriophage (phage) systems have been regarded as a powerful technology for diagnostic assays for their extraordinary specificity to target cells and cost-effectiveness. Our study introduced an enzyme-based fluorescent assay for detecting the presence of E. coli using the T7 phage engineered with the lacZ operon which encodes beta-galactosidase (β-gal). Both endogenous and overexpressed β-gal expression was monitored using a fluorescent-based method with 4-methylumbelliferyl β-d-galactopyranoside (MUG) as the substrate. The infection of E. coli with engineered phages resulted in a detection limit of 10 CFU/mL in ground beef juice after 7 h of incubation. In this study, we demonstrated that the overexpression of β-gal coupled with a fluorogenic substrate can provide a straightforward and sensitive approach to detect the potential biological contamination in food samples. The results also suggested that this system can be applied to detect E. coli strains isolated from environmental samples, indicating a broader range of bacterial detection.

Highlights

A BioTek spectrophotometer (Winooski, VT, USA) with a Gen5TM Microplate Reader was used to determine the E. coli concentrations at OD600 and the fluorophore synthesis level was monitored at an emission/excitation wavelength of 365/460 nm since the cleavage of 4-methylumbelliferyl β-Dgalactopyranoside (MUG) by the β-gal enzyme yields the fluorescent molecule 4-MU that emits light at 460 nm when excited by 365 nm light [23]
The evaluation of β-gal production was based on the conversion of the substrate MUG, a non-fluorescent galactosidase analogue, into the highly fluorescent molecule MU
Our assay revealed that wild-type phages that can be genetically manipulated to carry a reporter gene can be expressed only when phage–host infection occurs, resulting in a higher signal than that generated from the assays using unmodified phages

Summary

Introduction

Foodborne pathogens are important food safety issues in both developed and developing countries [1]. In the United States, close to 10 million food-related illnesses have been reported each year, resulting in excessive burdens on public health and major impediments to socio-economic growth [2,3]. Since bacterial pathogen infection has been the key cause of foodborne diseases, the identification of these microbes in food samples is of importance in ensuring food safety. Escherichia coli (E. coli) has been considered one of the most common causes of hundreds of reported foodborne outbreaks in the United States [4]. According to the Centers for Disease Control and Prevention (CDC), E. coli contamination has been found in a wide range of food and environmental samples including raw and pasteurized fluid milk, cheese, and drinking water [5]. Developing improved methods that are practical for the food industry for implementation is necessary

Objectives

Methods

Results

Discussion

Conclusion