The statistical analysis software package ClinProTools was applied in this study. Reproducibility of the data was assured by applying two independently generated datasets of the same strains to ClinProTools

analysis. The software automatically processes, recalibrates and compares the loaded spectra using an internal algorithm [47]. The processed peaks are then sorted according to their statistical separation strength [48]. Using this method, we were able to detect differentiating peaks for the serovars used in this study namely L. interrogans serovar Pomona and Copenhageni, L. kirschneri serovar Grippotyphosa and L. borgpetersenii serovar Saxkoebing and Acalabrutinib order Tarassovi (Table 4 and Table 5). Minor discrepancies in the protein profiles were present for Lazertinib manufacturer the serovars Australis and Icterohaemorragiae. Based on the statistical method PCA, one additional leptospiral strain, L. borgpetersenii serovar Sejroe, Epigenetics inhibitor formed a distant cluster with regard to the other strains (Figure 3). A L. borgpetersenii serovar Sejroe specific peak at 6,003 Da was also detected by applying ClinProTools analysis in one of the two datasets. Since it could not be verified by the second dataset, it has not been further considered for identification. No differentiation was observed for the genomospecies

L. kirschneri. Our findings lead to the conclusion that it is possible to discriminate our applied leptospiral strains on the basis of differences in their protein peak patterns, but we cannot claim this for other serovars or strains. Strain-specific differentiation using MALDI-TOF MS analysis has previously been shown by different studies [49–51] and discrimination of different serovars

of Salmonella enterica has been postulated before [46, 52]. This supports the hypothesis that MALDI-TOF MS is an important and useful technology for the identification and subtyping of bacterial isolates. Serovars of leptospiral CYTH4 strains are determined by antigenic variations in the LPS [15]. MALDI-TOF MS, however, mainly detects ribosomal proteins [45]. Consequently, we cannot claim conclusively that we identified universal serovar-specific peaks since we used a selected panel of serovars in this study. We suppose that the observed peak differences for some strains indicate serovar affiliation. To confirm this finding a larger panel of strains and serovars needs to be tested. The results of gene sequencing confirmed the MALDI-TOF MS-based species identification of all Leptospiral strains. The dendrogram of the reference spectra matched the phylogenetic trees constructed, using16S rRNA sequences and MLST data (Figures 4 and 5). Minimal discrepancies that occurred within single clades can be explained on the basis of the used target genes, since MALDI-TOF MS mainly detects ribosomal proteins [45]. That is why MSPs dendrograms are closely comparable to phylogentic trees based on 16S rRNA sequencing [23, 26, 35].