We incorporated the profiles that we obtained for 39 different Yersinia selleck compound isolates representative of 12 Yersinia species, including 13 Y. pestis strains, into this database. Every Yersina strain profile obtained in this study was also copied to a separate folder to form a new database in addition to the MALDI BioTyper™ database. The profiles were matched with the existing MALDI BioTyper™ database, and identification of the bacteria was carried out using MALDI BioTyper™ version 2.0. MALDI-TOF-MS identification A total of 13 Yersinia isolates including 2 environmental Y. pestis Orientalis biotype isolates

and 11 clinical isolates of Y. enterocolitica collected from feces were inactivated Erastin chemical structure and blindly analyzed by MALDI-TOF-MS against the local updated database as described above. Identification scores were assigned using the following scoring parameters [13]: a score ≥ 1.9 indicated species identification; a score of 1.7-1.9 indicated genus identification; and

a score < 1.7 indicated no identification. An isolate was considered to be correctly identified by MALDI-TOF when two of two spectra had a score ≥ 1.9. For organisms identified as Y. pestis, we further separated the protein profiles into three folders corresponding to each of the three biotypes. Using ClinPro Tools software, we analyzed the specific protein MLN0128 datasheet profile pattern for each biotype. ClinPro Tools software in-build, quick classifier and genetic algorithm analyses were used to differentiate the three Y. pestis biotypes. Quick classifier compares the average sprectum of the differentes classes in order to find the specific different peaks. The genetic algorithm

creates a random peak list, changes the list (“”mutation”") and compares the discriminating capacity until obtaining the best list for discriminating classes. Reproducibility of MALDI-TOF-MS identification In order to assess the reproducibility of MALDI-TOF-MS identification, every strain studied was tested in triplicate (i.e., on three different MALDI-TOF plates run on three different days from three different batches of culture). For every condition, 4 different spots were loaded on the MALDI-TOF plate, giving a total of 12 MALDI-TOF-MS protein profiles that were derived from each strain. Results Constituting a MALDI-TOF-MS Yersinia database Progesterone Accurate identification at the species level was confirmed for every isolate by partial sequencing of the rpoB gene. In addition, the presence of Y. pestis was confirmed by sequencing specific targets in each plasmid for each of the Y. pestis isolates used in this study. MST analysis discriminated the 13 Y. pestis isolates into 3 biotypes (Antiqua, Midievalis and Orientalis) with smaller variation in the number of alleles than previously reported [21]. The MST profile for the Y. pestis JHUPRI strain was most closely related to the Antiqua biotype but was atypical in that it contained spacer sequences from each of the three biotypes.