An increase in the frequency of V. parahaemolyticus
-associated infections has heightened the need for a method of rapid and reliable identification of V. parahaemolyticus
). Current methods for identification of V. parahaemolyticus
involve biochemical tests (15
); serotyping, PCR screening, and DNA hybridization analysis of housekeeping genes or disease-associated genes (3
); MLSA (9
); and PFGE (22
). Often, distinction of V. parahaemolyticus
from other closely related vibrios requires the use of multiple techniques.
In the current study, we demonstrate the development of a high-throughput whole-cell MALDI-TOF MS technique for the identification of V. parahaemolyticus
clinical and environmental strains, which takes less than 2 minutes for each sample spot. In this study, we demonstrate that whole-cell MALDI-TOF MS analysis of V. parahaemolyticus
and other Vibrio
spp. is a highly reproducible method that is effective for distinguishing between related species as previously demonstrated for other bacteria (37
). Comparison of MALDI-TOF MS fingerprints and cluster analysis of the peaks can be used to distinguish V. parahaemolyticus
from other vibrios including the closely related V. alginolyticus
, V. harveyi
, and V. campbellii
. Often, these three species are nearly indistinguishable from each other by 16S rRNA gene analysis, requiring analysis of multiple housekeeping genes for accurate species identification (39
). We identified a total of 30 peaks that were present only in the spectra of the V. parahaemolyticus
strains examined in this study. These peaks could be potential V. parahaemolyticus
biomarkers, though further investigation will be needed to validate their use as biomarkers for the rapid detection of V. parahaemolyticus
by whole-cell MALDI-TOF MS. In the present study, we normalized the spectra to a threshold that excluded most of the peaks from the several thousand that were generated for each strain, reducing the number of analyzable peaks to a manageable set of approximately 30 to 60 per spectra. Analysis of a larger number of strains and exclusion of outlier peaks would increase the level of similarity and better resolve the relationships among the V. parahaemolyticus
strains in the cluster analysis.
In addition, we show that isolates of the V. parahaemolyticus
pandemic clone exhibited spectra that had detectable and reproducible differences in the numbers and positions of peaks. This was especially true when comparing more recently isolated strains to older strains. For example, the more recently isolated clonal O3:K6 and O4:K12 strains exhibited MALDI-TOF MS fingerprints different from those of strains isolated in 1996 and 1997 (when the pandemic clones were first detected). In addition, there was one peak at m/z
10,430 that was detected only for the O4:K12 strains analyzed. Analysis of additional clonal O4:K12 strains will be required to ascertain whether this peak and additional peaks can be used as biomarkers for identification of the O4:K12 strains that are frequently associated with disease outbreaks in the United States (8
Furthermore, differences in the whole-cell MALDI-TOF MS spectra of some of the V. parahaemolyticus
strains examined may reflect strain adaptation to a particular geographic location. For example, the MALDI-TOF MS spectra of the V. parahaemolyticus
O3:K6 strains RIMD2210633 and ATCC 17802, which were isolated from Japan, exhibited more similarities than the spectra of O3:K6 strains isolated from the United States. In addition, the spectra of the V. parahaemolyticus
environmental strains SG176 and SG258, both of which were isolated from coastal Georgia, exhibited many similarities. The analysis of additional V. parahaemolyticus
strains isolated from distinct geographical regions and development of biomarker peaks are required to support the use of MALDI-TOF MS for identification of the geographical origin of certain strain types. In this regard, MALDI-TOF MS could be a powerful tool to monitor the emergence and spread of clonal groups as they become more frequent causative agents of disease outbreaks, such as the emergence of the O3:K6 clonal pandemic strain in India in 1995 (31
). Overall, the detectable differences among the isolates of the pandemic clone and the closely related O4:K12 strains suggest that MALDI-TOF MS may be a valuable tool to distinguish between isolates of the pandemic clone that were associated with outbreaks during different years and possibly biogeographic regions. Further analysis of a larger number of the V. parahaemolyticus
isolates of the pandemic clone would be required in order to develop a database of fingerprints and to validate the usefulness of MALDI-TOF MS for rapidly tracking the emergence and distribution of the disease-associated strains. In addition, the development of a whole-cell MALDI-TOF MS fingerprint database for the rapid identification of diverse vibrios would require the use of a standardized protocol that exhibits interlaboratory reproducibility and consists of an optimized medium type and growth conditions for each species.
In this study, we demonstrate that whole-cell MALDI-TOF MS analysis is sensitive enough to detect only a few genetic changes such as inactivation of a single regulatory gene or DNA repair pathway. Whole-cell MALDI-TOF MS analysis showed variation in the MALDI-TOF MS fingerprints of a single strain following deletion of the quorum sensing regulator opaR
or the mismatch repair gene mutS
, which results in an increased accumulation of mutations in V. parahaemolyticus
). Quorum sensing has been shown to regulate type III secretion of V. parahaemolyticus
), which was shown to translocate effector proteins, resulting in cytotoxicity to eukaryotic cells (33
). The ability to monitor changes in global regulatory systems such as quorum sensing that are involved in the pathogenicity mechanism of vibrios would provide additional information on the nature of disease-causing strains.
In this study, we report the first use of whole-cell MALDI-TOF MS analysis as a powerful tool for identification of V. parahaemolyticus strains. Based on the findings of our study, the rapid and reliable generation of whole-cell MALDI-TOF MS fingerprints would be an important tool for the initial identification of V. parahaemolyticus and other Vibrio spp. In addition, we identified potential peaks that could be further developed into biomarkers for detection of V. parahaemolyticus and analysis of differences among disease-causing strains. Further application of this method would involve the construction of a database of whole-cell MALDI-TOF MS fingerprints from V. parahaemolyticus and related disease-causing vibrios that could be referenced for identification of the causative agents of disease outbreaks. Research is ongoing to determine whether the whole-cell MALDI-TOF MS approach described in this study may be used to detect multiple disease-causing vibrios present in food or environmental samples.