Bacteriophage-Based Rapid Bacteria Detection

Foodborne illness is a leading public health concern with a significant impact on food industries and society. Thus, detecting pathogens at various stages of the food supply chain is essential to reduce the risk of foodborne illness. Several new detection technologies have been developed to improve the detection of bacterial contamination in food. Nucleic acid-based and immunological-based methods are considered rapid detection methods; however, these approaches also have several limitations. These limitations include the need for expert users, the inability to distinguish the cells' viability, and the interference from food compositions and its impact on reducing the detection efficiency. Therefore, it is crucial to develop novel rapid bacteria detection methods with high sensitivity and specificity that can detect bacteria in complex food matrices with reduced resource requirements.This research focuses on using bacteriophage as a tool for rapid and specific bacteria detection in complex food matrices to address some of the current challenges in the food industry. The research evaluated the application of engineered bacteriophage and precipitate forming enzyme substrate to enhance the sensitivity of bacterial detection. The engineered phage induces overexpression of alkaline phosphatase in infected target bacterial cells. Detection of overexpression of alkaline phosphatase activity using a precipitate forming fluorescence substrate enhanced the detection sensitivity compared to conventional soluble substrates. With fluorescence imaging and quantitative image analysis, 100 CFU/g of Escherichia coli could be detected within less than 6 hours. Further extending this concept, the research results demonstrated that a combination of precipitate-forming colorimetric substrate and engineered phage infection could enable visual detection of bacteria. Using this approach, the results demonstrate a simple visual assay for detecting 10 CFU of E. coli in 1 ml of coconut water and 102 CFU of E. coli on 1 g of baby spinach leaves without isolation of bacteria from the food sample. Phage-bacterial interactions are known to induce lysis of bacteria cells and thus influence bacterial cell morphology. These changes in the morphology of bacterial cells induced by specific phage-induced-lysis were detected by fluorescence imaging, and a quantitative image analysis approach was developed to indicate the presence of target bacteria in food samples. This approach detected E. coli at 10 CFU/ml within 8 hours in both laboratory-based cell culture medium and complex matrices. Further building on this approach, we also focused on the detection of phage amplification as an indicator of the presence of target bacteria in food systems. Phage progenies generated after phage infection and lysis bacterial cell lysis were observed by fluorescence staining, and the number of phage particles was analyzed by image analysis. This approach enables the detection of 10 CFU/ml of E. coli in coconut water and simulated spinach wash water within 8 hours. Overall, this research demonstrates simple and rapid bacteriophage-based bacteria detection approaches that can be applied to detect the bacterial pathogen in complex matrices. With low-cost setup and easy-to-perform procedures, these detection methods can be beneficial in facilities with limited access to some advanced instruments. Further development of these approaches can lead to the invention of highly efficient rapid bacterial detection devices that can be applied to detect bacteria in real food samples and industrial food facilities