Whole-cell MALDI-TOF MS Research Articles

Rapid Identification of Vibrio parahaemolyticus by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.

Species identification of Campylobacter jejuni ssp. jejuni and C. coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains--C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination.

Whole‐cell matrix‐assisted laser desorption/ionization mass spectrometry for rapid identification of bacteriocin/lantibiotic‐producing bacteria

The potential of whole-cell matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for the discovery of microorganisms that produce lantibiotics and/or bacteriocins in the mass range 3-5 kDa is demonstrated. Cells of the antibiotic-producing model strain Bacillus subtilis ATCC 6633 were grown on Landy agar plates, and prepared with a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid (DHBs) as matrix using the bottom-layer method. Time-course analyses showed that cells grown for 1-2 days on agar plates gave the best results in terms of intensity of the bacteriocin/lantibiotic signals. Even after storage of matrix-crystal-embedded cells for several days high-resolution spectra (>10,000) could be obtained. To verify the applicability of this technique, different Bacillus wild-type and gene deletion mutant cells were analyzed. In most of the cases the known antibiotic phenotype of the producer could be corroborated by whole-cell MALDI-TOFMS, particularly for the lantibiotics subtilin and ericin. The preparation recipe for whole-cell analysis by MALDI-TOF was also highly sensitive for hydrophobic lipopeptide antibiotics like surfactin, mycosubtilin, fengycin, and bacillomycin in the lower mass range (1-1.5 kDa).