Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF Mass spectrometry Roger Stephan a, ⁎, Nicole Cernela a , Dominik Ziegler b , Valentin Pflüger b , Mauro Tonolla c , Damiana Ravasi c , Maria Fredriksson-Ahomaa d , Herbert Hächler a a Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Zurich, Switzerland b Mabritec AG, Riehen, Switzerland c Microbial Ecology, Microbiology Unit, Plant Biol. Dept., University of Geneva c/o Institute of Microbiology, Via Mirasole 22A, 6500 Bellinzona, Switzerland d Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland abstract article info Article history: Received 21 June 2011 Received in revised form 19 August 2011 Accepted 26 August 2011 Available online 3 September 2011 Keywords: Yersinia enterocolitica Identification subtyping MALDI-TOF-MS Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the dif- ferentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We eval- uated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic bio- types 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF- MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Yersinia enterocolitica, a Gram-negative, oxidase-negative, and facultative anaerobic species, is an important foodborne pathogen in Europe as well as in USA (Anonymous, 2011; Scallan et al., 2011). Typical symptoms are fever, abdominal pain and diarrhea, most com- monly in young children (Bottone, 1997). Rapid and reliable identification of strains of the species Y. enteroco- litica and the differentiation of the pathogenic from the non-pathogenic biotype is important for diagnostic purposes, surveillance, prevention and control of food-borne diseases. A differentiation within the species Y. enterocolitica can be done by a combination of biotyping and serotyp- ing since strains belonging to bioserotypes 2/O:9, 2/O:5,27, and 4/O:3 are commonly associated with human infections. However, identifica- tion and further differentiation of Y. enterocolitica by biochemical tests is laborious, time-consuming and not always reliable. In recent years, several reports have shown the feasibility of using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) to identify microorganisms [for a review see Sauer and Kliem, 2010]. The detection of protein mass patterns has become a convenient tool for the rapid analysis of bacteria (Freiwald and Sauer, 2009). The method analyses the profiles of proteins that are extracted from whole bacteria. A MALDI mass spectrometer can efficiently detect numerous molecules simultaneously. Protein mass patterns can be used for identification of bacteria at the genus, the species and in some cases the subspecies level. The mass pattern de- tection method has been validated in several studies for different gram-negative foodborne pathogens (Dieckmann et al., 2008; Hazen et al., 2009; Alispahic et al., 2010; Stephan et al., 2010). Recently, the application of the MALDI-TOF mass spectrometry was also shown for the identification of Yersinia pestis as well as other Yersinia species (Ayyadurai et al., 2010; Lasch et al., 2010). The aim of the present study was to develop a reference MS data- base library of biomarker protein mass patterns (SARAMIS Super- Spectrum™) to identify and subtype Y. enterocolitica strains, and to perform thereafter a blind-test using Y. enterocolitica strains isolated from human clinical cases to validate the MALDI-TOF based approach. Journal of Microbiological Methods 87 (2011) 150–153 ⁎ Corresponding author at: Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Winterthurerstr. 272, CH-8057 Zurich, Switzerland. Tel.: + 41 44 6358651; fax +41 44 6358908. E-mail address: stephanr@fsafety.uzh.ch (R. Stephan). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.08.016 Contents lists available at SciVerse ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth