## 2 Results and Discussion
### Morphostructural Analysis of Bacterial Cells  
To confirm our assumptions about the lack of degradation of _H. pylori_ cells exposed to concentrations of \(\frac{1}{4}\)\(\times\) MIC--MIC of dioscin, we performed morphostructural analysis of bacterial cells using scanning electron microscopy (SEM). The results obtained from the micrographs were unequivocal (Figure 6), and in all cases the bacterial cells were unchanged in size. _H. pylori_ cells treated with dioscin at \(\frac{1}{4}\)\(\times\) MIC, \(\frac{1}{2}\)\(\times\) MIC, and MIC concentrations showed normal morphology (Figure 6B-D), where they presented typically spiral/rod forms and non-numerous coccoids (both morphological forms were with intact cell surface). Similarly, images of the untreated control cells showed undeformed structure of their cell membrane (Figure 6A). It is noteworthy that the cells treated with a MIC value of dioscin displayed several apparent, distinguished signs showcasing a specific defense mechanism of _H. pylori_ expressed in secretion of membrane vesicles (Figure 6D). Figure 4: Representative photos obtained after 24 h of experiments determining anti-biofilm activity of dioscin under flow conditions against _H. pylori_ 199. The flow conditions were generated using the Bioflux 1000 system. The MIC, \(\frac{1}{2}\times\) MIC, and \(\frac{1}{4}\times\) MIC had values of 64 \(\mu\)g/mL, 32 \(\mu\)g/mL, and 16 \(\mu\)g/mL, respectively. Control samples were bacteria not exposed to any concentration of dioscin. The pictures on the right show cells stained with the LIVE/DEAD kit (green and red fluorescence showing live and dead cells, respectively) and viewed under a fluorescence microscope, while the pictures on the left show cells under a light microscope. Scale bars show 10 \(\mu\)m.  
Figure 5: Data quantifying anti-biofilm activity of dioscin against _H. pylori_ 199 after 24-h incubation under flow conditions. The flow conditions were generated using the Bioflux 1000 system. The MIC, \(\frac{1}{2}\times\) MIC, and \(\frac{1}{4}\times\) MIC had values of 64 \(\mu\)g/mL, 32 \(\mu\)g/mL, and 16 \(\mu\)g/mL, respectively. Control samples were bacteria not exposed to any concentration of dioscin. Viability was defined as the ratio of green to red fluorescence of bacterial cells stained with the LIVE/DEAD kit, while the development of biofilm was verified by determining the degree of capillary coverage (both parameters were determined using the Bioflux Montage software). The study was performed with triplicate biological replications (_n_ = 3). The _p_-value represented by ns, ***, and *** is equal to \(>\) 0.05, \(<\) 0.001, and \(<\) 0.0001, respectively. Membrane vesicles are nanometric structures that are secreted by both Gram-positive and Gram-negative bacteria [58; 59; 60]. Historically, they have been considered as evidence of microbial cell membrane instability and lysis. Nowadays, however, it has been proved that the increase in secretion of these structures is associated with adaptation to unfavorable environmental conditions, as they may promote cell-cell interactions and biofilm development [61]. For diverse groups of bacteria, an intensification in the production of membrane vesicles has been observed in response to exposure of bacteria to different classes of antibiotics and other antimicrobial compounds [62]. For _H. pylori_, also, it has been shown that treatment with antibiotics (including clarithromycin and levofloxacin) increases the amount of membrane vesicles with a positive effect on their survival in these conditions [63]. On this basis, we suspect that at MIC levels, dioscin may also induce a similar mechanism in _H. pylori_ cells. However, this type of research is beyond the main scope of this research article, and the hypothesis we presented will require verification in the future. Summarizing this part of the analysis, we did not observe any significant structural changes in cells treated with dioscin regardless of the applied concentrations.