Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system

Reporter phage systems have emerged as a promising technology for the detection of bacteria in foods and water. However, the sensitivity of these assays is often limited by the concentration of the expressed reporter as well as matrix interferences associated with the sample. In this study, bacteriophage T7 was engineered to overexpress mutated alkaline phosphatase fused to a carbohydrate-binding module (ALP*-CBM) following infection of E. coli to enable colorimetric detection in a model system. Magnetic cellulose particles were employed to separate and concentrate the overexpressed ALP*-CBM in bacterial lysate. Infection of E. coli with the engineered phage resulted in a limit of quantitation of 1.2 × 10⁵ CFU, equating to 1.2 × 10³ CFU/mL in 3.5 h when using a colorimetric assay and 100 mL sample volume. When employing an enrichment step, < 10¹ CFU/mL could be visually detected from a 100 mL sample volume within 8 h. These results suggest that affinity tag modified enzymes coupled with a material support can provide a simple and effective means to improve signal sensitivity of phage-based assays. Graphical abstract.