Immunomagnetic Isolation of Enterohemorrhagic Escherichia coli O157:H7 from Ground Beef and Identification by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Database Searches

摘要

A rapid (25 min) and facile method was developed for the isolation and identification of the enterohemorrhagic Escherichia coli (serotype O157:H7) in ground beef. The isolation method employed microscopic magnetic beads coated with antibodies covalently bonded to the surface that were specific to antigens of serotype O157. This selective preconcentration step was necessary because direct matrix-assisted laser desorption/ionization (MALDI) MS analysis of bacteria was not amenable, serving to isolate the bacteria from meat components and other nontarget bacteria. The immunomagnetic separation increased the sensitivity of the method and permitted the detection of bacteria in meat. MALDI time-of-flight MS furnished bacterial mass spectra that were useful for organism identification. Molecular weight database searches using the Expert Protein Analysis System proved useful for confirmation of the organism's identity. Bacterial biomarkers from direct MALDI analysis of pure bacterial suspensions were consistently present in bacterial suspensions of buffer/tryptic soy broth (positive controls) and meat extract samples. The detection limits were 2 X 10~(6) cells/mL for the experimental approach used herein. Cross-reactivity studies performed on three nontarget bacterial strains revealed that the immunomagnetic beads are specific only to E. coli strain serotype O157:H7, and there is no cross-reactivity with the other relatively innocuous strains studied.