Application of Engineered Bacteriophage T7 in the Detection of Bacteria in Food Matrices

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.

Molecular Targets for Foodborne Pathogenic Bacteria Detection

“…Indeed, a variety of methods have been developed on the basis of the interactions between phages and host cells [ 142 ]. Detection of these interactions is usually achieved through genetic modification of the phage to overexpress β-galactosidase, alkaline phosphatase or the lux gene, coupled with subsequent colorimetric detection, in solid or liquid media [ 143 , 144 , 145 , 146 ]. However, its application in food is limited by the interference of food matrix constituents in signal detection [ 147 ].…”

Development of an HPV Genotype Detection Platform Based on Aggregation-Induced Emission (AIE) and Flow-Through Hybridization Technologies

“…The colorimetric method is employed currently in the signal read-out approach of Hybribio’s platform. Enzyme-sensitive chromogenic substrates, such as the mixture of 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (BCIP) and nitro-blue tetrazolium chloride (NBT) could form intense, insoluble black-purple precipitates after reacting with alkaline phosphatase (ALP) [ 26 ]. In the presence of ALP labels, the dye will form colorimetric signals on the boxes to detect disease-related genes in the patient sample.…”

Quantitative Imaging of Bacteriophage Amplification for Rapid Detection of Bacteria in Model Foods

“…For E. coli detection, the T7 phage is a well-established model system due to its ability to infect a wide range of E. coli hosts (Studier, 1969). With genetically modified T7 phages, previous studies have illustrated detection of 10-100 CFU/ml of target bacteria in water and beverage samples within 6-8 h without isolation of nucleic acids or extensive sample preparation steps (Chen et al, 2015(Chen et al, , 2017Wisuthiphaet et al, 2019Wisuthiphaet et al, , 2021. However, the application of this approach requires genetic modification of phages.…”