Abstract
Detection of pathogens in a food matrix is challenging due to various constraints including complexity and the cost of sample preparation for microbial analysis from food samples, time period for the detection of pathogens, and high cost and specialized resources required for advanced molecular assays. To address some of these key challenges, this study illustrates a simple and rapid colorimetric detection of target bacteria in distinct food matrices, including fresh produce, without prior isolation of bacteria from a food matrix. This approach combines bacteriophage-induced expression of an exogenous enzyme, alkaline phosphatase, the specific colorimetric substrate that generates insoluble color products, and a simple filtration method to localize the generation of colored signal. Using this approach, this study demonstrates the specific detection of inoculated Escherichia coli in coconut water and baby spinach leaves. Without isolating bacteria from the selected food matrices and using a food sample size that is representative of industrial samples, the inoculated samples were added to the enrichment broth for a short period (5 h) and incubated with an engineered bacteriophage T7 with a phoA gene. The incubation period with the engineered bacteriophage was 30 min for liquid samples and 2 h for fresh produce samples. The samples were then filtered through a 0.2-micron polycarbonate membrane and incubated with a colorimetric substrate, i.e., nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). This substrate forms a dark purple precipitate upon interactions with the released enzyme on a filter membrane. This approach successfully detected 10 CFU/ml of E. coli in coconut water and 102 CFU/g of E. coli on baby spinach leaves with 5 h of enrichment. Success of this approach illustrates potential for detecting target bacteria in food systems using a simple visual assay and/or quantitative colorimetric measurements.
Highlights
Human illness due to foodborne pathogens is a major cause of morbidity and mortality
One of the most challenging aspects in the detection of bacteria from a complex food system is the presence of food components and non-pathogenic microbes associated with the food
The food samples, coconut water, and baby spinach leaves were mixed with the tryptic soy broth (TSB) before enrichment and used for the detection of bacteria using a simple colorimetric approach
Summary
Human illness due to foodborne pathogens is a major cause of morbidity and mortality. Even within the United States, which has advanced sanitation systems, an estimated 48 million people experience foodborne illness each year (Centers for Disease Control and Prevention, 2018). The leading cause of foodborne illnesses has been the contamination of food, such as fresh. Bacteria Detection Using Engineered Bacteriophage produce, dairy and meat products with microbial pathogens, for example, among those pathogens of most concern are Shiga toxin-producing Escherichia coli (STEC), which are reported as a cause for an estimated 265,000 illnesses and 3,600 hospitalization annually in the United States (Scallan et al, 2011). Rapid and efficient detection of the microbial contaminants plays an important role in mitigating the risk of food borne outbreaks and improving sanitary controls in the food supply chain. Rapid and sensitive detection of bacteria, especially in complex food matrices, continues to be a challenge
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