## 2 Results and Discussion
### Investigation of Discriminating Features  
The plots in Figure 6 display the VOCs which presented statistically significant alterations in their responses (_p_ < 0.05) after bio-AgNPs addition. Four types of alterations were verified: volatiles that had their areas increased or decreased when medium was treated with the nanoparticles, as well as those that became absent or incident only in the headspace of cultures supplemented with nanoparticles. For _E. coli_, _E. faecalis_, _K. pneumoniae_ and _P. mirabilis_, the numbers of found altered features were 64, 36, 77 and 62, respectively. In general, the main observed effect was the absence of compounds previously found being emitted by the original cultures (disappearance of 23 VOCs in _E. coli_, 36 in _E. faecalis_, 56 in _K. pneumoniae_ and 40 in _P. mirabilis_). However, such a phenomenon appears mostly associated with the processing of cultures with the greatest concentration of agent (C3). Significant changes related to the use of intermediary and lowest concentrations of bio-AgNPs (C2 and C1) refer mostly to the modulation in the response of the detected VOCs, as well as the incidence of new compounds. In this sense, C3 configures as a threshold level that induces irreversible modifications in the pattern of emitted bacterial metabolites, as a possible result of substantial metabolism inhibition. General considerations can be drawn regarding the volatiles that became present only after supplementation with nanoparticles. C1 level led to the production of 3-methyl-1-butanol propanoate for _E. coli_; dimethyl disulfide for _E. coli_, _E. faecalis_ and _K. pneumoniae_; 3-methyl-1-butanol for _K. pneumoniae_; ethyl propanoate for _K. pneumoniae_ and _E. faecalis_; 4-methyldecane for _P. mirabilis_; and 2-decanol for _E. faecalis_. Dimethyl disulfide can have its origin in the degradation of methionine. The formed Strecker aldehyde (methanol) can be converted into methanethiol, and such species can auto-oxidize to produce sulfides [37]. A previous study described the stimulation of antibiotic resistance in _E. coli_ exposed to dimethyl disulfide, demonstrating that volatiles emitted by _Burkholderia ambifaria_ strains can influence the development of other organisms [38]. Moreover, 3-methyl-1-butanol is probably derived from enzymatic conversion of leucine-derived keto acid by Ehrlich pathway. The \(\alpha\)-keto acids produced during the processing of amino acids are transformed into the corresponding alcohols after action of the \(\alpha\)-ketoacid decarboxylase and alcohol dehydrogenase enzymes [39; 40; 41]. These \"fused alcohols\" can undergo esterification by activity of alcohol acyltransferase enzyme [42; 43]. In this sense, esters such as 3-methyl  
Figure 6: Plots showing alterations in VOCs’ response in treated samples in relation to untreated ones (expressed in terms of binary logarithm of fold change) for (**a**) _E. coli_, (**b**) _K. pneumoniae_, (**c**) _P. mirabilis_ and (**d**) _E. faecalis_. Thickness of the edges are proportional to the significance of the alteration (based on p value). Four main compound behaviors are outlined: incident (diamonds) or absent (circles) only after bio-AgNPs treatment, and those that had increased (triangles) or decreased (squares) responses after the supplementation. C1, C2 and C3 = supplementation with bio-AgNPs at 12.5, 25 and 50 \(\mathrm{\SIUnitSymbolMicro mL^{-1}}\), respectively. 1-butanol propanoate and ethyl propanoate, may be sourced by this pathway. Among other alcohols, 3-methyl-1-butanol (isoamyl alcohol) proved to promote biofilm formation in strains of _S. aureus_. The alcohol treatment triggered the transcription of genes related to biofilm build-up and promotion of antibiotic resistance [44]. Specifically, this same concentration level also modulates positively a series of compounds. Decanoic acid, undecanoic acid (increased in _E. coli_) and nonanoic acid (increased in _P. mirabilis_) can be derived from stimulation of fatty acid synthesis.