## 3 Discussion
Moreover, in this study, effective antibiotics, selected NIC, and benzoic acid demonstrated a significant ability to downregulate the expression of genes encoding for LT, STa, and STb enterotoxins, the true heart of ETEC pathogenicity. To the best of our knowledge, this was the first study that not only proved the efficacy of thymol, carvacrol, eugenol, and benzoic acid against bacterial proliferation,but also demonstrated the capacity of these compounds to downregulate the expression of the three _E. coli_ K88 major enterotoxin genes. The same molecules also affected _E. coli_ genetic machinery involved in quorum sensing (QS) and biofilm formation via downregulation of _luxS_, a gene encoding for a lyase that synthesizes a precursor of the QS autoinducer-2 [42], but also _bssS_ and _tnaA_, two genes involved in the regulation of biofilm formation [43; 44; 45]. Taken all together, these data suggest a diversified mode of action of these molecules that seems to be exploited by a complex series of reactions, rearrangements, and influences of the genetic apparatus of _E. coli_. Globally, while NIC seem to share a common pathway in downregulating _E. coli_ K88 virulence genes, sub-lethal concentrations of OA evidenced three differential behaviours on transcription modulation, despite their comparable ability to inhibit bacterial growth at higher doses. While thymol, carvacrol, and eugenol all have a common chemical structure--as they all share traits of aromaticity and hydrophobicity--organic acids, except for benzoic, have different chemical properties and represent a more diverse group of compounds. Thymol, carvacrol, eugenol, and benzoic acid all share an aromatic benzene ring with polar bioactive groups. These similarities in structure, as well as their high hydrophobicity, would suggest that their mode of action might be connected to their capability of diffusing through the cell membrane and causing impairment to normal membrane functions [46; 47]. Moreover, their aromatic rings, naturally able to delocalize electrons, can contribute to the reduction of membrane proton gradient, thus resulting in a collapse of the proton motive force and the depletion of ATP [47; 48], with a potential consequence even on virulence gene expression levels because of an extensive effect on several bacterial enzymes [15], finally resulting in an impairment of virulence functions such as the gene expression of bacterial toxins like LT, STa, and STb. This would also suggest a mechanism of action of these compounds similar to certain antibiotics like colistin, doxycycline, and bacitracin, for example, that would implicate a possible synergy between these categories of molecules as a new frontier of therapeutic approach against enterotoxin-producing pathogens [49; 50; 51; 52; 53; 54]. On the other side, hexanoic and sorbic acid were not equally effective as benzoic in downregulating _E. coli_ gene expression despite their inhibitory activity in MIC and MBC tests. Hexanoic acid was ineffective concerning modulating the virulence genes, as its mechanism of action is mostly directed against Gram-positive bacteria [55; 56], whereas sub-MIC concentrations of sorbic acid, which is equally defined as a medium chain fatty acid with six atoms of carbons, increased virulence gene expression despite its relatively high bactericidal activity: mild acid-stress conditions generated by sorbic acid in _Bacillus subtilis_ can activate the ppGpp-dependent stringent response and an acid shock response mediated by RelA and Fur regulons [57; 58], conditions that also strengthen virulence gene expression in _E. coli_[59; 60]. This evidence further suggests that the antimicrobial action and the ability to modulate _E. coli_ K88 virulence genes are two independent properties of OA, and are likely not related to each other.