# Genus, Species, and Subspecies Classification of _Salmonella_ Isolates by Proteomics
## 3 Results and Discussion
The data showed that all tested _Salmonella_ isolates were correctly identified at the serovar level. Further, for isolates that have been sequenced and annotated (SARA2, SARA23, SARA31, SARA39, SE8a, SARC5, SARC6, and SARC12), the highest match was the correct strain, suggesting that our approach can detect strain-level differences. Figure 3: The number of peptides identified in the _S. typhimurium_ SARA2 isolate present in each reference proteome. See Table S1 for the detailed reference proteome information. In addition to identification, the degree of similarity between the tested isolates and the reference proteomes was shown to correlate with taxonomic relatedness, viz. the degree of similarity decreases as the taxonomic distance increases. Given the small serovar-to-servor genomic variation (99% ANI), it is expected that the degree of similarity to the correct reference strain is only marginally better than from another strain of the same serovar or a strain from a closely related serovar (Figure 4). In contrast, isolates are clearly distinguished from reference proteomes at further taxonomic positions, e.g., different subspecies, species, and genera, on the right-hand side of the heatmap. Interestingly, reference proteomes \(\mathrm{D_{SS2}}\) and \(\mathrm{D_{SP1}}\) do not appear to be suitable surrogate databases for their serovars. Although \(\mathrm{D_{SS1}}\) was the best match for SARA23, the other reference proteome of \(S\). Saintpaul, \(\mathrm{D_{SS2}}\), was the ninth in rank (\(\mathrm{D_{SS1}}>\mathrm{D_{ST1}}>\mathrm{D_{ST2}}>\mathrm{D_{SH2}}>\mathrm{D_ {SP2}}>\mathrm{D_{SM2}}>\mathrm{D_{SH1}}>\mathrm{D_{SS2}}\), row 4, Figure 4). It has been shown that gene contents can sometimes differ more within a serovar than between serovars [35]. For example, the degree of genome homology between \(S\). Saintpaul SARA23 and SARA29 was shown to be relatively low [36]. \(\mathrm{D_{SS2}}\) is translated from the genome of \(S\). _saintpaul_ SARA26 and is likely less representative of SARA23 than expected. Notably, reference proteome \(\mathrm{D_{SP1}}\) (derived from \(S\). Paratyphi B SARA42) is surprisingly dissimilar to all 12 of the \(S\). _enterica_ subsp. _enterica_ isolates and was an outlier across all 19 isolates shown on the heatmap (column 7, Figure 4). This is likely due to a poorly annotated or poorly sequenced genome and suggests that visualization of the similarity of peptide identifications to a reference proteome can be used to assess the quality of a genomic assembly. Figure 4: Heatmap representing the degree of similarity in sequences between proteomic data and reference proteomes, with the similarity decreasing from red (100%) to blue (48%). The match to other foodborne bacteria is less than 40% and not shown here. The detailed data are provided in Table S2. Figure 5 summarizes the degree of proteome similarity of the fifteen tested _Salmonella_ isolates to the reference proteomes. An average of 96 \(\pm\) 1.3% similarity was observed for a _Salmonella_ isolate to its strain-specific reference proteome, 94 \(\pm\) 2.4% to different strains within the same serovar, and 92 \(\pm\) 1.8% to different serovars with the same subspecies. While the overall trend suggests that a reference proteome at a closer taxonomic position yields a higher degree of similarity, finding a clear threshold score to define the taxonomic relationship to a specific _Salmonella_ serovar might not be possible. For example, a degree of similarity of 94% could be the correct strain, a different strain of the same serovar, or a different serovar of the same subspecies. The data are consistent with the high degree of sequence homology between serovars, and similarly presents a challenge for conventional _Salmonella_ subtyping tools such as pulsed-field gel electrophoresis (PFGE) and PCR.