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Abstract

Antibiotic-resistant strains of bacteria continue to emerge, increasing the need for their fast and accurate identification. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), has become a prominent technique in biological mass spectrometry. We report the application of MALDI-TOF-MS for the identification of intact Gram-negative and Gram-positive microorganisms taken directly from culture. Analysis of bacteria from a single colony is possible, allowing the screening of mixed cultures. Sample preparation is simple and the analysis automated, providing spectra within minutes. The spectra obtained allow identification of microorganisms from different genera, different species, and from different strains of the same species. The procedure provides a unique mass spectral fingerprint of the microorganism, produced from desorbed components of the cell wall. Consistent data were obtained from subcultures grown for 3-day and 6-day periods, from the same cultures 1 day later and from fresh subcultures 2 months later.

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Most cited references 8

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The envelope of mycobacteria.

P Brennan, H Nikaido (1995)

Mycobacteria, members of which cause tuberculosis and leprosy, produce cell walls of unusually low permeability, which contribute to their resistance to therapeutic agents. Their cell walls contain large amounts of C60-C90 fatty acids, mycolic acids, that are covalently linked to arabinogalactan. Recent studies clarified the unusual structures of arabinogalactan as well as of extractable cell wall lipids, such as trehalose-based lipooligosaccharides, phenolic glycolipids, and glycopeptidolipids. Most of the hydrocarbon chains of these lipids assemble to produce an asymmetric bilayer of exceptional thickness. Structural considerations suggest that the fluidity is exceptionally low in the innermost part of bilayer, gradually increasing toward the outer surface. Differences in mycolic acid structure may affect the fluidity and permeability of the bilayer, and may explain the different sensitivity levels of various mycobacterial species to lipophilic inhibitors. Hydrophilic nutrients and inhibitors, in contrast, traverse the cell wall presumably through channels of recently discovered porins.

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Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry.

R. Holland, J Wilkes, F Rafii … (1996)

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was investigated as a method for the rapid identification of whole bacteria, either by comparison with archived reference spectra or by co-analysis with cultures of known bacteria. Bacteria were sampled from colonies on an agar plate, mixed with the matrix, air-dried, and introduced in batches into the mass spectrometer for analysis. In the first experiment, both bacterial strains that had been previously analyzed to obtain reference spectra and other strains that had not been analyzed were blind-numbered and their spectra were obtained. Those strains that matched reference spectra were found to be correctly identified. A second experiment involved co-analysis of reference strains and bind-numbered strains under identical conditions; species-specific identification was demonstrated by comparison of spectra of the blind-numbered strains with those of the standards. In all of the spectra obtained in these experiments, each bacterial strain showed a few characteristic high-mass ions which are thought to be derived from bacterial proteins. This work represents the first reported instance of successful bacterial chemotaxonomy by MALDI-TOFMS analysis of whole cells. For the strains tested, the method is rapid and simple.