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

摘要

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 N-15 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 x 10(3) to 3.0 x 10(9) CFU/ml is attained in LB, while between 4.1 x 10(4)-2.7 x 10(9) CFU/ml and 1.9 x 10(3)-3.0 x 10(7) 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 x 10(3) CFU/ml, 4.1 x 10(4) CFU/ml and 1.9 x 10(3) 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. (C) 2014 Elsevier B.V. All rights reserved.