## 3 Discussion  
In our study, we demonstrated the antibacterial activity of the Esc(1-18) and Esc(1-21) peptides against _E. coli_ O157:H7, with Esc(1-21) appearing to be the most efficient one, as indicated by its lower MIC and MBC values. In addition, both peptides turned out to be stronger than kanamycin. This effect correlated with the killing kinetics, showing a significant reduction (99%) in viable bacterial cells within a short time (30 min) for Esc(1-21) or within 60 min for Esc(1-18) at 4 \(\times\) Mic compared to 75 min for kanamycin. The results of the Styox Green assay indicated the rapid perturbation of the cytoplasmic membrane by both peptides, with a more marked effect elicited by Esc(1-21), which was in agreement with the results of the time-kill curves (Figure 2), proving a stronger and faster bactericidal activity for Esc(1-21) with respect to Esc(1-18). This can be ascribed to the higher cationicity of Esc(1-21) compared to the isoform Esc(1-18) due to the additional basic residue at its C-terminal portion (Lys 20), which should increase the peptide's interaction with the negatively charged phospholipids of the bacterial membrane. Previous circular dichroism experiments indicated an alpha-helical structure for both Esc(1-18) and Esc(1-21) in a membrane-mimicking environment [16; 17]. The distribution of basic residues along the entire sequence of the peptides is expected to hamper their aggregation that is required for the formation of transmembrane pores, as described by the barrel-stave mechanism. Therefore, it is more likely that both Esc(1-18) and Esc(1-21) bind to the membrane surface in a carpet-like manner, inducing an unfavorable tension that would result in the formation of local membrane breakages leading to bacterial death, which is in line with previous results for Esc(1-18) on non-pathogenic _E.coli_[17]. In addition, we found that both Esc(1-21) and Esc(1-18) have the ability to prevent biofilm formation at a sub-inhibitory concentration  
Figure 6: Effects of Esc(1-18) and Esc(1-21) on biofilm formation by _E. coli_ strains. SEM was used to examine biofilm cells grown on glass coverslips in modM9 in the presence or absence of peptides at the concentration of 1/2 MIC for 24 h (magnification 30,000\(\times\)). (1/2 MIC), with Esc(1-21) being more potent than Esc(1-18). Moreover, both peptides turned out to be stronger inhibitors of biofilm formation than of planktonic growth; in fact, the planktonic growth of bacterial cells was not affected at the peptide concentration used in the antibioofilm assay. To understand the role played by a series of genes related to biofilm formation/dispersal and to stress conditions, we examined their expression levels with real-time PCR and found an upregulation of all genes. In _E.coli_, motility is regulated by several global regulatory circuits that converge to modulate the overall expression of the master operon _flhDC_ (class I flagellar operon) [24], which regulates the flagellar gene expression [25] and is controlled by a variety of physiological and environmental cues. This operon, in turn, regulates the class III flagellar gene (_fliC_), which encodes the major structural protein of the _E.coli_ flagellum FliC. Flagella are known to have a crucial role in both early biofilm development and later biofilm dispersal. We demonstrated an increased expression of both the _flhDC_ and _fliC_ genes, indicating the genes that are involved in flagellar synthesis as targets of esculentins. Two proteins associated with the _flhDC_ operon, Hha and the global regulator CsrA, are involved in biofilm formation, and CsrA is also involved in biofilm dispersal. In _E.coli_, CsrA increases the expression of _flhDC_ by directly binding to the mRNA to stabilize the transcript [24; 26]. [PERSON] and colleagues [27] reported the positive interaction of Hha with the promoter of _flhDC_, demonstrating an Hha control of biofilm formation in _E.coli_ O157:H7.