## 4 Discussion
### Microbiology  
On the basis of the presented antimicrobial activity assays in vitro, it can be stated that in some cases, the antibacterial activities of tested hydrazide-hydrazones **2-16** were better or equal to those of commonly used antibacterial agents, which we employed as reference substances (nitrofuranotin, cefuroxime or ampicillin).  
Acylhydrazone **16** showed four times greater activity (MIC = 1.95 \(\upmu\)g/mL) than nitrofuranotin (MIC = 7.81 \(\upmu\)g/mL) towards methicillin-resistant _Staphylococcus aureus_ ATCC 43300 strain. The same compound again displayed the same activity as nitrofuranotin against _Staphylococcus epidermidis_ ATCC 12228 (MIC = 3.91 \(\upmu\)g/mL). Additionally, its activity against these bacterial stains was bactericidal (MBC/MIC = 1).  
The highest activity against _Staphylococcus aureus_ ATCC 6538 (MSSA strain), a methicillin-susceptible strain was shown for compounds **11**, **13** and **16**. The minimal inhibitory concentration value towards this strain for compound **11** (MIC = 15.62 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/mL) was equal to the activity of nitrofurantoin (MIC = 15.62 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/mL). Then, compounds **13** (MIC = 1.95 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/mL) and **16** (MIC = 7.81 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/mL) showed an activity level that was, respectively, eight times and two times higher against this bacterium than the activity of nitrofurantoin (MIC = 15.62 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/mL).  
It is also worth underlining the activity of the tested hydrazide-hydrazones towards _Microoccus luteus_ ATCC 10240. The antibacterial activity of compounds **6**, **11** and **13** (MIC = 31.25 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/mL) was two times higher than the activity of nitrofurantoin (MIC = 62.5 \(\mathrm{\SIUnitSymbolMicro\text{g}}\)/mL).  
It can be stated that the highest antibacterial activity was manifested by hydrazide-hydrazones of phenylacetic acid, which were substituted with 3-bromo-5-chloro-2-hydroxyphenyl, 3-ethoxy-2-hydroxyphenyl, 2-hydroxy-5-nitrophenyl and 2,3,4-trihydroxy phenyl substituents from the hydrazone part of the compound.  
The antibacterial activity towards Gram-positive bacteria falls when beside the hydroxyl group in position two in the phenyl ring two identical halogen atoms (compounds **3** and **5**) are also present. When only one halogen atom is present, the antibacterial activity rises (acylhydrazones **2** and **4**). Where two halogen atoms are present in the chemical structure of hydrazide-hydrazone, the activity rises (substance **6**), but this fact is not observed when one of these atoms is iodine (compound **7**).  
The presence of the OH group in position two in the phenyl ring in the acylhydrazone of phenylacetic acid is crucial for the antibacterial activity. When this group is placed in a different position, the activity against Gram-positive bacteria is much lower (compound **10**). The significant rise in the activity is especially seen when more than one hydroxyl group (compound **16**) is present in the structure of tested acylhydrazones **2-16**. The presence of more than one OH group is also crucial to the activity against Gram-negative bacterial strains (acylhydrazone **16**).  
In the case of the nitro group in the structure of tested acylhydrazones (**12**, **13**, **14**), its presence increases the antibacterial activity against Gram-positive bacteria. The introduction of two NO\({}_{2}\) groups lowers the activity (substance **15**).  
In general, the highest antibacterial activity was shown by hydrazide-hydrazones with more than one hydroxyl group (**16**) and without halogen atoms (**11**). A lower activity was displayed by the compounds with one NO\({}_{2}\) group (**12**, **13**, **14**) and two NO\({}_{2}\) groups. Finally, the lowest activity was shown by acylhydrazones with one or two halogen atoms (**2-5**, **7**).