## 2 Results and Discussion  
Salforabane and 2-Phenethyl Isothiocyanate Impair the Growth of V. cholerae in a Dose-Dependent Manner  
The number of newly discovered or designed antimicrobials in recent years is dramatically low and even decreasing further. Thus, it can be expected that the imbalance between rising antimicrobial resistance among bacterial pathogens and the availability of treatment strategies will expand continuously [26; 27]. Therefore, in this study, we decided to employ two members of ITC group, naturally occurring in _Brassicaceae_ plants, SFN and PEITC, to test their antibacterial potency against _V. cholerae_. So far, their antimicrobial effect was demonstrated for such bacterial pathogens as _E. coli_, _E. faecalis_, _S. aureus_, _Salmonella Montevideo, and C. jejuni_[21; 24]. Hence, we assessed the antimicrobial activity of ITC by microdilution sensitivity assay to obtain the inhibitory concentration values for the tested compounds. As shown in Figure 1a, the minimal inhibitory concentrations (MIC) of SFN and PEITC were 0.5 mM and 2 mM, respectively, while their bactericidal potency was disclosed at 4 mM and 10 mM (SFN and PEITC, respectively). Chloramphenicol was used as a positive growth inhibition control in this study, and its MIC was assessed as 4 \(\mu\)g/mL and the MBC was 8 \(\mu\)g/mL. These results were confirmed by the disc diffusion assay where SFN and PEITC were used in the concentrations of 30 \(\mu\)g/disk resulting in a 23 \(\pm\) 2 and 13 \(\pm\) 1 mm diameter zone of inhibition. This is consistent with results observed for chloramphenicol (30 \(\mu\)g/disk) expressed as 21 \(\pm\) 2 mm diameter zone of inhibition. Next, we tested the assessed MIC and their fractions (1/2 to 1/16 MIC) to elucidate whether their action is dose-dependent. For this, the fresh cultures (optical density, OD\({}_{600}\) = 0.1) were treated with SFN or PEITC in a range of concentrations, and the growth was monitored spectrophotometrically during 3 h and compared to untreated control (Figure 1b,d). This experiment showed that the statistically significant growth impairment was observed for concentrations even lower than MIC, namely \(\frac{1}{2}\) and \(\frac{1}{4}\) MIC (for SFN and PEITC, respectively) effectively inhibited the growth of bacterial cultures in their log-phase. The antimicrobial action of ITC differs among bacterial species tested to date [21; 24] and still remains poorly understood in terms of the molecular targets which decide about nature of their bacteriostatic and bactericidal action. However, as it has been previously reported, the bactericidal activity of aromatic glucosinolate-derived compounds, such as 2-phenylethylly and benzyl isothiances, was higher in comparison with aliphatic glucosinolate derivatives, such as SFN, allyl- 3-butenyl, and 4-pentenyl isothiances [28]. Similarly, the bactericidal effect of isothi cyan derived from aromatic glucosionolates was more pronounced than the activity of those derived from aliphatic precursors [25; 29]. One of the proposed explanations of this phenomenon is the activation of oxidative stress response and subsequent changes in cellular redox homeostasis. It might be caused by interaction between isothiances and thiol groups of microbial proteins engaged in redox homeostasis, such as glutathione [30]. Moreover, it was proposed that aromatic glucosionolate-derived isothi cyan had higher reactivity through their ability to act as electron donors from a benzene ring [31; 32]. However, our examination of the level of reactive oxygen species (ROS) in _V. cholerae_ treated with SFN or PEITC in the 2,7-dichlororescein diacetate (DCFH-DA) assay did not show a significant elevation of oxidative stress (data not shown), which is in agreement with our previous analyzes performed using the _E. coli_ strain [25]. As the ROS level correlates with the mode of action of antimicrobials; therefore, the death curve upon ITC action was further analyzed (Figure 1c,e) to determine bacteriostatic/bactericidal activity against _V. cholerae_.