## 3 Discussion
We obtained a significant induction of _csrA_ and _Iha_ in cells treated with Esc(1-21) and Esc(1-18), suggesting that, in the _E.coli_ O157:H7 strain, esculentin-1 derivatives influence the _flhDC_ operon through the induction of the _csrA_ and _Iha_ genes. This suggests the plausible involvement of these genes in the peptide-mediated regulation of biofilm formation and dispersal. It is known that a downregulation of the _flhDC_ operon and _Iha_ gene is induced by the stringent response, an ubiquitous stress response found in microorganisms [28]. It is a response to nutritional stress, such as growth in a minimal medium, stimulating and inhibiting several biosynthetic pathways. It is characterized by the production of the signal molecules ppGpp and ppGpp--together referred to as (p)ppGpp--which regulate the expression of a plethora of genes promoting biofilm formation [29]. In bacteria, including _E.coli_, (p)ppGpp metabolism is driven by two genes: _relA_ and _spo_T. RelA produces the (p)ppGpp signaling molecules, as it possesses a synthetase functional domain, while SpoT regulates the degradation and production of (p)ppGpp, as it has both synthetase and hydrolase functional domains, with a strong hydrolase activity and a weak synthetase activity [29]. The (p)ppGpp molecule binds to the RNA polymerase, decreases transcription, and causes a global rewiring of the gene expression profile, leading to slow growth for most cells [30]. [PERSON] and colleagues demonstrated the ability of Esc(1-21) and its diastereomer Esc(1-21)-1c to interact with (p)ppGpp [19], while, in another work [31], a specific interaction of a cationic peptide with (p)ppGpp was demonstrated, leading to the degradation of the nucleotide and the reduction of the available pool. More precisely, [PERSON] and coworkers hypothesized that AMPs blocked the widespread stringent response, thus reducing the ability of _E. coli_ cells to form biofilm. In our study, we obtained an induction of _spo_T, but not of _relA_ (unchanged with respect to the control), and due to the prevalent hydrolase activity of SpoT, we hypothesized that the peptides lowered the level of the (p)ppGpp pool through its hydrolysis with the consequent inhibition of the stringent response and upregulation of the _flhDC_ operon. It is known that in _E.coli_, flagella are important for reaching a suitable surface for adhesion. Once the surface is reached, other superficial cellular structures are important for adhesion, such as type I pili and curli [32; 33], and flagella appear to be required neither for initial adhesion nor for further biofilm development. Motility and adhesion are two different and mutually exclusive bacterial characteristics, and both are under the control of regulatory cascades, each with a master regulator at the top. [PERSON] and coworkers [34] provided evidence that in _E.coli_, the motility-to-adhesion switch requires a precise downregulation of motility. They found that the continued expression of the flagellar master regulator FlhDC strongly inhibits curliexpression. The induced overexpression of the flagellar system highlighted in our study could be responsible for the inhibition of the cascade control of the synthesis of curli and the consequent inhibition of the adhesion and development of biofilm. This effect was also visible through electron microscopy. Indeed, as shown in Figure 6, the untreated bacterial cells showed the presence of different types of fimbiae connecting the cells in the biofilm structure. On the contrary, peptide-treated cells were not able to form such communities, as indicated by the limited number of cells on the glass microscope slide and by the absence of a polymeric extracellular matrix, which was likely due to the impaired bacterial ability to adhere to surfaces, in line with both crystal violet (CV) staining (see Section 4 for antibiofilm activity) and gene expression analysis.