Characterization of Yersinia Using MALDI-TOF Mass Spectrometry and Chemometrics

Abstract

Yersinia are Gram-negative, rod-shaped facultative anaerobes, and some of them, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis, are pathogenic in humans. Rapid and accurate identification of Yersinia strains is essential for appropriate therapeutic management and timely intervention for infection control. In the past decade matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) in combination with computer-aided pattern recognition has evolved as a rapid, objective, and reliable technique for microbial identification. In this comprehensive study a total of 146 strains of all currently known Yersinia species complemented by 35 strains of other relevant genera of the Enterobacteriaceae family were investigated by MALDI-TOF MS and chemometrics. Bacterial sample preparation included microbial inactivation according to a recently developed mass spectrometry compatible inactivation protocol. The mass spectral profiles were evaluated by supervised feature selection methods to identify family-, genus-, and speciesspecific biomarker proteins and-for classification purposes-by pattern recognition techniques. Unsupervised hierarchical cluster analysis revealed a high degree of correlation between bacterial taxonomy and subproteome-based MALDI-TOF MS classification. Furthermore, classification analysis by supervised artificial neural networks allowed identification of strains of Y. pestis with an accuracy of 100%. In-depth analysis of proteomic data demonstrated the existence of Yersinia-specific biomarkers at m/z 4350 and 6046. In addition, we could also identify species-specific biomarkers of Y. enterocolitica at m/z 7262, 9238, and 9608. For Y. pseudotuberculosis a combination of biomarkers at m/z 6474, 7274, and 9268 turned out to be specific, while a peak combination at m/z 3065, 6637, and 9659 was characteristic for strains of Y. pestis. Bioinformatic approaches and tandem mass spectrometry were employed to reveal the molecular identity of biomarker ions. In this way, the Y. pestis-specific biomarker at m/z 3065 could be identified as a fragment of the plasmid-encoded plasminogen activator, one of the major virulence factors in plague infections. The genus Yersinia belongs to the family Enterobacteriaceae and presently comprises 13 validly described species.(1-3) Three of them, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis, are important pathogens with relevance to animal and human health which have been studied extensively by various methods. The other species, Yersinia aldovae, Yersinia aleksicae, Yersinia bercovieri, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia massiliensis, Yersinia molaretii, Yersinia rohdei, Yersinia ruckeri, and Yersinia similis, are less intensively characterized possibly because of their lower clinical importance.(4, 5) Pathogenic Yersinia are the cause of serious diseases in animals and humans; Y. enterocolitica, for example, is a foodborne enteropathogen which can pro...

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

“…The species Y. pestis and Y. pseudotuberculosis are highly related and have recently been proposed to represent two lineages of a single species rather than two different species. The ability of MALDI-TOF MS to discriminate between these species was investigated previously (181), with the study concluding that the technology was able to accurately identify members of each genus. Wittwer et al conducted similar experiments using a collection of 61 well-characterized Yersinia species isolates to generate a collection of spectra using the SARAMIS software package for analysis.…”

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

“…Identification approach C. In the third analysis approach, the Matlabbased software solution MicrobeMS version 0.72 (24,(36)(37)(38)(39) developed at RKI was used (Matlab; The Mathworks, Inc., Natick, MA). MicrobeMS is publicly available as Matlab p-code (free of charge) and provides direct access to Bruker's raw spectral data and to spectra acquired by the Vitek MS workflow (formerly SARAMIS, bioMérieux/Shimadzu) via the mzXML data format (40).…”

“…Both species share genomic sequences and have identical 16S rRNA genes (53). As a consequence, their differentiation by MALDI-TOF MS is challenging; it has been found that differentiation can be carried out only on the basis of a single mass peak at m/z 3,065 (36,38). This peak has been assigned to a fragment of the plasmid-encoded (pPCP1) Pla protein.…”

First Report: Application of MALDI-TOF MS within an External Quality Assurance Exercise for the Discrimination of Highly Pathogenic Bacteria from Contaminant Flora

“…thuringiensis or Y. pestis and Y. pseudotuberculosis frequently exceeds the discriminatory power of the rather straightforward data analytical approaches utilized by the commercial systems. As a consequence, three of the five laboratories that routinely use MALDI-TOF ICMS rely on customized in-house data analysis platforms for RG3 diagnostics, which allow the application of more sophisticated statistical algorithms (Lasch et al, 2008;Lasch et al, 2010;Wittwer et al, 2011).…”