## 3 Results and Discussion
### SFN Reduces the Toxicity of S. dysenteriae In Vivo  
Our cell lines experiments described above provided further proof for potential therapeutic use of ITC in treating _Shigella_ infections. However, more important are studies performed in living organisms, not only in isolated cell cultures. Thus, we decided to employ an animal model. The _Shigella_ infections have been studied in many models, including primates, rodents and piglets [18]; nevertheless, the use of these animals is expensive, requires a specific facility and raises significant ethical issues. Thus, a more convenient model was introduced, namely, the larvae of _Galleria melonella_, the greater wax moth. This surrogate model is widely used for novel antibiotics' efficacy studies, pathogen virulence and resistance screening, and its use in monitoring _Shigella_ infections has been described in detail [27]. Briefly, these insects are easy to maintain and handle, their size allows for injection of pathogenic bacteria and simple-to-monitor steps of infection were described. Furthermore, the insect immune system, although evolutionarily distant, presents significant similarities to vertebrates, which makes it useful to make conclusions about general response to pathogens. In particular, specific larvae  
Figure 3: SFN and PEITC impair expression of the Shiga toxin genes and induce stringent response in \(S\). _dysenterae_. (**a**) Synthesis of the stringent response alarmones, ppGpp and ppPGpp, was assessed by culturing bacteria with the addition of [\({}^{32}\)P]orthophosphoric acid (150 uCi/mL) followed by cell lysis and nucleotide separation by thin-layer chromatography. ITC or SHX were added at time zero. Samples were withdrawn at 15 and 30 min after the compound addition. The positions of ppGpp and ppPGpp are indicated at the right side of the panel. (**b**) Relative cellular level of (p)ppGpp was assessed densitometrical, where treated samples were compared to the nontreated negative control (at the 30 min endpoint) using Student’s \(t\)-test. Statistical significance is marked with asterisks: *** \(p<0.001\); (**c**) Relative expression of \(stx1\) in \(S\). _dysenteriae_ SZ2855/97 Japan was determined by qRT-PCR and is presented using \(2^{-\Delta\text{ACT}}\) quantification normalized to the _recA_ gene expression level. cells, hemocytes, provide a line of defense against pathogenic bacteria due to phagocytosis [39]. In addition, the activation of hemocytes provides a signal for melanization in larvae [40]. Thus, the development of the infection can be monitored by melanization, with an easy-to-determine scale [27]. Therefore, we took advantage of the _G. mellonella_ model and assessed the effect of SFN on the progress of infection. First, the survival of the larvae was monitored after injection of various c.f.u. of the three tested _S. dysenteriae_ isolates. As shown in Figure 4, the most virulent effect was caused by the _S. dysenteriae_ SZ2855/97 Japan strain, with the lowest survival level (down to 20% after 96 h) at a dose of \(10^{8}\) per ml of bacteria. Thus, for the ITC test, this inoculum was chosen. The larvae were then infected with bacteria and then injected with SFN and/or azithromycin (typically used antibiotic for treatment of bacterial gastroenteritis). For control, PBS injection was used. While the antibiotic alone has reduced insect mortality, a similar effect was observed after injection with SFN. In particular, when both azithromycin and SFN were used, mortality was the lowest, not exceeding 20% even after 96 h (Figure 5a). These results indicates that SFN alone has a significant anti-pathogenic activity, and in combination with azithromycin, this effect is even more pronounced. In the checkerboard tests conducted by us, SFN and azithromycin in combination showed an additive effect (FICI \(\geq\) 1, data not shown). Interestingly, the ability of SFN to protect the larvae from developing a potentially lethal infection is related to activation of hemocytes.