## 2 Results and Discussion
### ITC Reduces the Toxicity of V. cholera In Vitro and In Vivo  
Cholera infection symptoms strictly correspond to effective CT production in the host organism. Thus, downregulation of toxin synthesis together with growth inhibition by antibiotic compounds could provide higher efficiency than the traditional treatment. To evaluate the ITC effectiveness as anti-cholera agents, we checked their activity in human and simian cell lines, HeLa and Vero. These cell lines are suitable to test _V. cholerae_ pathogenicity because of their sensitivity to CT, and they are also routinely used to test the cytotoxicity of different compounds [53; 54]. We showed a significant reduction of the toxic effect of bacterial lysates treated prior with any of the compounds, SFN and PEITC (Figure 5). Observed results were similar in both cell lines. The slight differences in the activity of SFN and PEITC between cell lines could be related to the variation of the glycolipid receptor (GM1) expression on the cell surface, which may impact their sensitivity to CT [55; 56]. These findings are in line with results showing toxin expression impairment presented above. Moreover, we show that ITC is not toxic by itself for eukaryotic cell lines in doses employed in this study. Further, in order to determine if the inhibition of virulence assessed in vitro could also affect in vivo infection, we tested the ability of SFN to inhibit virulence of _V. cholerae_ in a surrogate host of _Galleria mellonella_ larvae. This model organism was described as a suitable alternative for vertebrates in selecting new antimicrobials as well as for _V. cholerae_ infections approaches [57; 58]. We first established the bacterial load appropriate for survival experiments (Figure 52). The \(5\times 10^{2}\) bacteria per larvae were chosen for further analyzes as they resulted in a 50% death rate at 24 h after infection. SFN safety (at the 25, 10, and 5 mg/kg doses) was assessed, and the tested concentrations were found to be non-toxic, and no symptoms of melanization-associated with the worsening of the insect condition were observed after the treatment (data not shown). This dose corresponds with results obtained in the rodent model, assessed by others [59; 60]. We demonstrate that the pretreatment of larvae with 25 mg/kg SFN administrated 3 h before infection improved the survival of _G. mellonella_ larvae by 60% after 48 h and by 30% after 72 h post infection (Figure 6). Additionally, we analyzed the potency of SFN to enhance azithromycin (20 mg/kg) activity against _V. cholerae_. Azithromycin is considered an antibiotic of choice for diarrheal treatment in children [61] and is now under clinical trial investigation. The observed larvae survival rate after a single dose of azithromycin treatment was surprisingly similar to the level observed in the objects which received SFN alone. Interestingly, administration of two  
Figure 5: SFN and PEITC reduce the toxicity of _V. cholerae_ against HeLa and Vero cell cultures. Bacterial lysates were obtained after centrifugation of \(10^{11}\) cells from culture supplemented with ITC and culture without treatment (negative control). HeLa and Vero cells (\(2\times 10^{3}\)) were grown in DMEM medium. The viability of HeLa (**a**) and Vero (**b**) cells after treatment with ITC and bacterial lysate was measured using the MTT assay. Cytotoxicity control of the vehicle (DMSO), SFN, and PEITC were assessed as indicated on the graphs. The results presented are mean values from 3 independent experiments. The significance of differences between the results and the positive control (bacterial lysate with CT) was tested using the Student’s \(t\)-test. Statistical significance is marked with asterisks: ***\(p<0.001\); ***\(p<0.01\). compounds together decreased mortality in _V. cholerae_ challenged larvae to 20% after 96 h as compared with infected control. The SFN is known for its anti-inflammatory and antioxidant properties but is also described as an immune response stimulator [62]. [PERSON] et al. [63] showed improved clearance of _E.