Bacterial Detection Using Unlabeled Phage Amplification and Mass Spectrometry through Structural and Nonstructural Phage Markers

According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. <i>E. coli</i> at 1 × 10⁵, 5 × 10⁵, and 1 × 10⁶ CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria–Bertani broth, orange juice, and French bean stew (“cassoulet”) matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection