## 3 Discussion  
One of the most striking issues in the worldwide pig industry is represented by PWD. Despite recent advances, ranging from improved breeding conditions to new strategies to control infectious diseases, PWD still constitutes a significant challenge. Antibiotics represent a major strategy to manage infections by _E. coli_ K88, identified as the main etiological agent for PWD [1, 18]. In this study, an _E. coli_ K88 field strain was tested against a panel of several antibiotics widely used to manage livestock infections. The strain was resistant to the vast majority of them, with colistin and doxycycline not only being the only two effective molecules in inhibiting the growth of _E. coli_, but also in repressing virulence gene expression, especially for genes encoding bacterial LT and ST toxins, primarily responsible for the onset of diarrhea symptoms in pigs. In an extensive analysis by [PERSON] et al., more than 200 _E. coli_ strains across eight European countries were assessed for their resistance against dozens of antibiotics: results showed a considerable resistance towards antimicrobial compounds, including amoxicillin [19]. Other studies reported similar results, with MIC values for different _E. coli_ strains equal to or greater than 64 mg/L for neomycin, ampicillin, and tetracyclines, proving an established or increasing spread of resistance genes [20, 21, 22, 23]. _E. coli_ resistance to penicillin G and limomycin is also reported [20]. Colistin is one of the few antibiotics employed both in human and veterinary medicine and it is considered as a last resort for infections by multi-drug resistant Gram-negative species [24]. Its use in the pig industry allows the control of _E. coli_ infections with a certain degree of effectiveness [25]. However, the appearance of the _mcr-1_ colistin resistance gene is a serious threat to the efficacy of this primary importance antibiotic [26], therefore European authorities encouraged member states to support initiatives oriented towards a significant reduction in its use [27]. Our study showed that colistin was effective at an MIC of 4 mg/L, a value considered to be a sign of antimicrobial resistance, according to EUCAST breakpoints [28]. The spread of multi-resistant bacteria caused by antimicrobials use and abuse, together with the difficulties in finding new antibacterial drugs [29], dictates that novel strategies to manage _E. coli_ K88 infections are required. OA and NIC have gained attention because of their recognized antimicrobial properties [13, 14] and could therefore represent a valid approach to help manage bacterial infections as stand-alone treatments or as antibiotic adjuvarants [9, 30]. In this study, a large number of NIC and OA was tested against _E. coli_ K88, proving the efficacy of three phenol derivatives of terpen--i.e., eugenol, thymol, and its isomer carvacrol--and the growth-inhibiting activity of sorbic acid, benzoic acid, and hexanoic acid. Thymol, carvacrol, and eugenol were also tested (individually or as essential oils) in many previous studies which reported the positive effects of these bioactives against several _E. coli_ strains both in vivo and in vitro [15, 31, 32, 33]. Moreover, several in vivo studies demonstrated the usefulness of OA in controlling PWD in orally challenged piglets [34, 35, 36, 37]. The target of _E. coli_ K88 is represented by intestinal mucosa cells: the bacterium needs close contact to the epithelial cells to exert its pathogenicity [38, 39]. For this reason, the flagellar movement is vital for _E. coli_ colonization of the hindgut and can also help the F4 fimbrial adhesin to bind cellular targets [40]. Our study revealed that selected antibiotics, NIC, and benzoic acid downregulate the expression of _motA_ and _faeG_ genes crucial for bacterial motility and cellular adhesion. The same trend was registered on _E. coli_ O157:H7: an important downregulation of genes related to flagella was shown for bacteria conditioned with sub-MIC doses of thymol and carvacrol [41].