Reporter phage assays are a promising alternative to culture-based assays for rapidly detecting viable bacteria. The reporter systems used in phage-based detection are typically enzymes and their corresponding substrates that provide a signal following infection and expression. While several reporter systems have been developed, comparing reporter systems based on reported bacteria detection limits from literature can be challenging due to factors other than the reporter system that influence detection capabilities. To advance the development of phage-based assays, a systematic comparison and understanding of the components are necessary. The objective of this study was to directly compare two common enzyme-mediated luminescence reporter systems, NanoLuc/Nano-Glo and alkaline phosphatase (ALP*)/DynaLight, for phage-based detection of bacteria. The detection limits of the purified enzymes were determined, as well as the expression levels and bacteria detection capabilities following engineering of the coding genes into T7 phage and infection of E. coli BL21. When comparing the sensitivity of the purified enzymes, NLuc/Nano-Glo enzyme/substrate system demonstrated a lower detection limit than ALP*/DynaLight. In addition, the expression of the NLuc reporter following phage infection of E. coli was greater than ALP*. The lower detection limit combined with the higher expression resulted in a greater than 100-fold increase in sensitivity for the NLuc/Nano-Glo® reporter system compared to ALP*/DynaLight when used for the detection of E. coli in a model system. These findings provide a comparative analysis of two common reporter systems used for phage-based detection of bacteria and a foundational understanding of these systems for engineering future reporter phage assays.

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Rapid and specific detection of pathogenic bacteria in food and water sources can be crucial to disease diagnosis and prevention. Genetically modified bacteriophage-based assays are a promising alternative over standard overnight culture-based assays as they can provide comparatively rapid detection. Bacteriophage (phage) viruses specifically infect live bacterial cells for the rapid replication of their viral genome. Scientists exploit this in-built molecular amplification system by genetically modifying phage genes to express certain reporter proteins during an infection. The expression of reporter proteins is confirmed through enzymatic and/or sensory assays, indicating the presence of the pathogenic bacteria. The sensitivity of the reporter phage assays depend on the ability of the genetically engineered phage to successfully express its reporter protein with conserved activity. In this study, we compared two enzymes, alkaline phosphatase (ALP) and nanoluc luciferase (NLuc) as reporter proteins in the context of the above criteria. We genetically modified T7 phage genome to overexpress these enzymes upon infecting BL21 E. coli cells. The reporter proteins were quantified and detected by measuring its luminescence activities. NLuc phage was significantly better at its gene expression in comparison to ALP phage, averaging at 9.8 × 105 molecules of protein/CFU of E. coli and providing a limit of detection at 107 CFU of E. coli/mL. On the other hand, ALP phage was only able to produce 8.6 × 104 molecules of protein/CFU of E. coli and provide a limit of detection at 109 CFU of E. coli/mL. These findings will allow researchers to select their choice of reporter proteins to improve phage-based assays and continue their progress in the field of pathogen detection.