Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis

ABSTRACT Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt, a bacterial disease transmitted by the corn flea beetle mainly to sweet corn (Zea mays). In many countries, it is classified as a quarantine organism and must be differentiated from other yellow enteric bacteria frequently occurring with corn. We have created novel primers from the pstS-glmS region of P. stewartii for use in conventional PCR (cPCR) and quantitative PCR (qPCR). To facilitate rapid diagnosis, we applied matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using whole-cell protein extracts, profiles were generated with a Bruker microflex machine, and the bacteria classified. P. stewartii strains were clearly distinguished from strains of Pantoea agglomerans, Pantoea dispersa, and Pantoea ananatis. Dendrogram analysis of the protein profiles confirmed the score values and showed the formation of separate clades for each species. The identification achieved by MALDI-TOF MS analysis agrees with the diagnosis by specific PCR primers. The combination of both methods allows a rapid and simple identification of the corn pathogen. P. stewartii subsp. stewartii and P. stewartii subsp. indologenes are highly related and can be distinguished not only by virulence assays and indole tests but also by a characteristic pattern in the nucleotide sequence of recA.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01171-09 ◽

2009 ◽

Vol 75 (21) ◽

pp. 6745-6756 ◽

Cited By ~ 51

Author(s):

Tracy H. Hazen ◽

Robert J. Martinez ◽

Yanfeng Chen ◽

Patricia C. Lafon ◽

Nancy M. Garrett ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Laser Desorption ◽

Rapid Identification ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Whole Cell ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Tof Ms

ABSTRACT 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.

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