Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Abstract

The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria. This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0 x 10(6) cells/mL. In this paper, we describe a bacterial detection method that integrates immunomagnetic separation with bacteriophage amplification prior to MALDI-MS analysis. The developed method consists of three main stages: (1) isolation of a target bacterium by immunomagnetic separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein. With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of approximately 5.0 x 10(4) cells/mL.

Mass spectrometry and tandem mass spectrometry characterization of protein patterns, protein markers and whole proteomes for pathogenic bacteria

“…In one technique, a specific bacteriophage is amplified by multiplication within an infected bacterial host and the phage proteins are targeted for detection. (Madonna et al, 2003). Another technique consists of incubating exotoxin-producing bacteria with a synthetic peptide substrate, which upon enzymatic cleavage generates peptide products.…”

Mass Spectrometry: Identification and Biodetection, Lessons Learned and Future Developments

“…Nevertheless, proteomics approaches are time-consuming and not suitable for real-time biodetection. Recognizing the importance of cleanup, efforts have been made to develop simpilified cleanup methods using affinity chromatography (including both lectins 38 or antibodies 39 prior to MS analysis. The difficulty here is how to apply such approaches for universal detection, since the appropriate affinity reagent must be selected for each ANALYSIS OF PROTEINS BY MALDI-TOF MS organism of interest.…”

“…Phages specific to the organism of interest have also been employed. 39 After bacterial infection the phages are amplified by intracellular growth generating high concentrations of phage-specific proteins. 39 However, unlike PCR (see above) where amplification reagents (primers) can be simply designed, chemically synthesized and purchased commercially, phages have to be individually selected (e.g., for specificity) and tested for each organism of interest.…”

A Review of Antibody Capture and Bacteriophage Amplification in Connection with the Direct Analysis of Whole‐Cell Bacteria by MALDI‐TOF MS

“…33 In the initial step bacteriophage is added to a bead-E. coli complex at a titer below the MALDI-MS detection level. Following room temperature incubation, which can be as short as 30 minutes, a one microliter aliquot is removed and spotted onto the MALDI plate.…”