## 2 Results and Discussion
The ITC was added to fresh culture adjusted to \(\sim\)10\({}^{7}\) c.f.u/mL, then samples were collected, and viable cells were enumerated at indicated time points up to 24 h. The results of this assay led us to the conclusion that antimicrobial action of both ITCs (in a range of concentrations from MIC to 5 \(\times\) MIC) is bacteriostatic as observed c.f.u. could not drop below the starting culture yield by 99.9% (\(\geq\)3 log\({}_{10}\)), which is an arbitrary threshold for bactericides. Just a relatively high concentration of PEITC (10 \(\times\) MIC) showed the bactericidal effect, but it could be difficult to consider it for in vivo use due to its cytotoxic effect. However, to date, bacteriostatic agents such as doxycycline, macrolides, or azithromycin are in routine use and recommended in cholera infection treatment (WHO). The biofilm formation of _V. cholerae_ plays a very important role in the survival of bacteria and their resistance to antimicrobials, as well as virulence level; thus, controlling biofilm by antimicrobial compounds is of crucial importance to prevent and combat infections. Figure 1: Antimicrobial activity of PEITC and SFN against _V. cholerae_. The _V. cholerae_ susceptibility tests evaluated as minimal inhibitory (MIC) and bactericidal (MRC) concentration and zone inhibition assays. (**a**) The growth inhibition of _V. cholerae_ treated with various concentrations of SFN (**b**) and PEITC (**d**). The killing curves of bacteria treated with SFN (**c**) and PEITC (**e**) in doses equal or greater than MIC. The results presented are mean values from 3 independent experiments with SD indicated. The reduction/inactivation of formed biofilm was assessed by crystal violet staining (**f**) and MTT assay (**g**). The significance of differences between treated samples and the controls was tested using the Student’s _t_-test. Statistical significance is marked with asterisks: \(\mathbf{{}^{***}}\)\(p<0.001\); \(\mathbf{{}^{*}}\)\(p<0.01\). Therefore, the ability of ITC to affect biofilm was assessed in this study. Biofilms already formed by _V. cholerae_ were exposed to the 1 or 5 \(\times\) MIC of SFN or PEITC and the biofilm mass was assessed by crystal violet staining (Figure 1f). Both tested ITCs reduced the biofilm significantly, and the effectiveness of this action was already exhibited for 1 \(\times\) MIC. In line with this observation, the viability of the biofilm-forming bacteria was also considerably reduced, as shown in the MTT assay (Figure 1g). Importantly, these effects were similar for PEITC and SFN. The ITCs were also tested for their ability to prevent biofilm formation. For this, compounds were added to the bacteria before the biofilm could be formed. This experiment showed that ITC can also reduce the transition of bacteria from sessile to biofilm state (Figure S1). SFN exhibited a stronger effect in this assay; however, PEITC at concentrations of 1 and 5 \(\times\) MIC also affected biofilm formation. To date, the antib biofilm activity of ITC has been scarcely described in the literature, with some exceptions [33; 34; 35]; therefore, the molecular basis of its nature is poorly understood. [PERSON] et al. [23] showed that PEITC is effective in inhibiting _P. aeruginosa_ biofilm formation even in concentrations significantly lower than the MIC (\(\leq\)29 mg/mL). The authors indicated a good ability of the tested compound to penetrate the complexed biofilm matrix. In another study [36] herein, ITC from horseradish, was proposed as a quorum sensing (QS) modulator that specifically blocked expression of QS-regulated genes in _P. aeruginosa_. These observations indicate that ITC can be used not only to inhibit bacterial exponential growth but, more importantly, also to diminish their biofilm formation and eradicate already formed biofilms, which highlights the important preventive role of ITC against pathologies induced by _V. cholerae_. Moreover, except for water contamination, seafood is most commonly associated with cholera [37].