## 3 Results and Discussion
Figure 3: On the left, the data processing flow-chart, from the generation of the data matrix after the alignment to the feature’s reduction after KW test (\(p\leq 0.05\)). On the right, the PCA of seven mycobacteria species + medium plotting the 607 volatile significant features. Figure 2: [PERSON]’s correlation coefficients plot obtained for each mycobacterium specie. Abs—M. _abscssus_; Avi—M. _avium_; BCG—M. _bovis_; Bol—M. _boletiti_; Chim—M. _chimera_; Int—M. _intracellular_; Mas—M. _massiliense_; Med—growth media. The number following the abbreviation stands for the replicate number. An outlier—The sample circled in red (Mas1, top right) resulted as an outlier and was excluded from further data elaboration. The samples were visualized again using a PCA and heatmap (HM) but limiting the data matrix to the selected 13 features (Figure 4). The variance explained by the 2D-PCA, considering both the first and the second principal components, was improved from 38% to 62%. All the different mycobacteria species were well discriminated, resolving most of the misclassifications present in the PCA built with the panel of 607 features (example the misclassification of sample Int5, Figure 3). Moreover, proximity-based on the complex they belong to, namely MTB, MAB and MAC were also depicted both on the PCA and heatmap. On the right side of the heatmap and on the bottom of the PCA, we can observe the proximity of Int, Avi and Chim, belonging to the MAC complex. On the left side, Abs and Mas are clustering together, as both belong to the MAB complex. While Bol, still part of the MAB complex, is clustered in a separate branch in proximity to BCG, which is the only member of the MTB complex. The taxonomic of the MAB complex has been the subject of intense investigation since a clear classification is still not reach, in fact, [PERSON] et al. suggested to combine the species MAS and ABS [28]. However, it has been clearly demonstrated that the two groups are not completely homogeneous, especially in terms of susceptibility to macrolides [29]. Our results confirmed this difficulty, although discrimination can be observed between Mas and Abs. Moreover, these two species can be clearly differentiating from BOL (Figure 4). However, it is not clear why Bol was in such proximity with BCG, although perfectly separated, at a higher distance compared to the other species belonging to the same complex. The panel of the most discriminatory features (\(n=13\)) is reported in Table 1. The table contains the original number of features (FT), the name of the VOC, experimental LRI and the LRI reported in the literature along with the MS similarity match with the library. The compounds were putatively identified based on the combination of a dual filter: the MS similarity with the NIST17 library (\(\geq\)80%) and the experimental linear retention index (LRI) within a \(\pm\)5 range compared to the literature on the same or equivalent column phase. Compounds that did not match with the previous filters were assigned as unknown. Considering the combination of the filters used for the identification of the discriminatory features (MS similarity + LRI), it was possible to name 8 out of 13 volatile molecules (Table 1). Figure 5 shows the box-plot of each discriminatory features (FT) and how they were discriminatory among and within the three mycobacteria complex. FT1559 (Furan, 2-methyl-3-(methylthio)-) and FT0867 (Furan, 2-butyl-) were discriminant among each _Mycobacterium_ complex (MAB, MAC, and MTB), while FT0792 (Phenylacetaldehyde), FT1866 (unknown) and FT1521 (unknown) were discriminant within either the MAB complex (resolving the overlapping between MAS and ABS), or the MAC complex, well separating the classes of the mycobacteria belonging to these complexes individually.