## 4 Conclusions  
A comprehensive investigation of VOCs extracted from the headspace of cultures of _E. coli_, _E. faecalis_, _K. pneumoniae_ and _P. mirabilis_ treated with bio-AgNPs was carried out. Firstly, the profile of released volatile compounds appear to be characteristic for each of the studied pathogens, serving as a fingerprint of individual metabolic systems and the with potential to be applied for discrimination between bacterial species. Used data analysis methods also allowed us to unravel the biochemical meaning of altered patterns of VOCs. Each of the tested concentrations of agents induced different metabolic responses on bacteria. The application of nanoparticles at 12.5 \(\mu\)g mL\({}^{-1}\) demonstrated that it triggers the biosynthesis of several bioactive volatiles, which appear to be connected with signaling, enhancement of virulence, biofilm formation and bacterial resistance. Besides that, alternative processes of energy obtaining are elicited--fermentation and \(\beta\)-oxidation of fatty acids. Increasing concentrations of nanoparticles evidenced interaction between ROS and fatty acids which compose the bacterial cell membrane, indicating the progressive damage of plasma membrane, and, therefore, successful incorporation of bio-AgNPs. Such a process was accompanied by the inhibition of pathways mainly related to the metabolism of amino acids and fatty acid biosynthesis and catabolism, resulting in the abrupt decrease in the variety of produced volatile metabolites. VOC profiles from supplemented cultures of _E. faecalis_ were the most preserved in comparison to the untreated ones, suggesting their lower susceptibility to the bio-AgNPs. On the other side, it is estimated that _P. mirabilis_ lost 80% of its metabolic diversity, while the response of three potential oxidative stress indicators (2-decenal, 2,4-decadienal and nonanoic acid) increased together with the added concentrations of agent. Evaluation of VOCs emitted by bacterial cultures proved to be an efficient tool to monitor metabolic changes and molecular reactions induced by antimicrobial agents, enabling us to elucidate the particularities of its action over different bacterial species. In addition, such an approach can be useful to foresee and track resistance mechanisms or aspects of agent effectiveness.  
Conceptualization, F.M., V.R.-P., M.M.-M. and P.P.; methodology, F.M., V.R.-P. and M.M.-M.; software, F.M.; validation, F.M.; formal analysis, F.M. and V.R.-P.; investigation, F.M.; resources, M.M.-M. and P.P.; data curation, F.M. and P.P.; writing--original draft preparation, F.M.; writing--review and editing, F.M., V.R.-P., P.P., M.M.-M. and B.B.; visualization, F.M.; supervision, B.B.; project administration, B.B.; funding acquisition, B.B. All authors have read and agreed to the published version of the manuscript.  
This work was supported by the \"Advanced biocomposites for tomorrow's economy BIOG-NET\" project (FNP POIR.04.04.00-00-1792/18-00) that is carried out within the TEAM-NET program of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. [PERSON] and P.P. are members of Torun Center of Excellence \"Towards Personalized Medicine\", operating under Excellence Initiative-Research University.  
**Institutional Review Board Statement**: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Nicolaus Copernicus University in Torun (Decision number KB 93/2020, 25 February 2020).  
**Informed Consent Statement**: Informed consent was obtained from all subjects involved in the study.  
**Conflicts of Interest**: The authors declare no conflict of interest.