Rapid quantification of Escherichia coli in food and media using bacteriophage T7 amplification and liquid chromatography-multiple reaction monitoring tandem mass spectrometry

Conventional microbiological assays have been a valuable tool for specific enumeration of indicative bacteria of relevance to food and public health, but these culture-based methods are time-consuming and require tedious biochemical and morphological identification. In this work, we exploit the ability of bacteriophage T7 to specifically infect Escherichia coli and amplify nearly a 100-fold in 1-2 h. Bacteriophage amplification is integrated with liquid chromatography-multiple reaction monitoring tandem mass spectrometry (LC-MRM-MS/MS) for quantitation of phage-specific peptides. Heavy isotopic 15N labeled T7 is introduced as the inoculum phage and internal standard. Quantification is performed by determining the ratio of phage-specific peptides over the internal standard which value is proportional to E. coli numbers. A broad dynamic range of 6-log orders ranging from 3.0 × 103 to 3.0 × 109 CFU/ml is attained in LB, while between 4.1 × 104-2.7 × 109 CFU/ml and 1.9 × 103-3.0 × 107 CFU/ml was enumerated respectively in coconut water and apple juice. With this method, viable E. coli are quantified in 4 h with a detection limit of 3.0 × 103 CFU/ml, 4.1 × 104 CFU/ml and 1.9 × 103 CFU/ml in LB, coconut water and apple juice, respectively. This method has potential as a rapid tool for detection of fecal contamination during food bioprocessing and distribution to safeguard public health.