*2.3. Evaluation of Antimicrobial Activity*

## 2.3.1. Qualitative Assessment of Antimicrobial Activity

The agar diffusion assay showed that compounds **7a**, **8a**, and **8b** did not interfere with microbial growth (Table 2). *Enterococcus faecium* E5 was the most susceptible species to compounds **5** and **6** with a growth inhibition zone diameter of 17 and 15 mm, respectively. Compounds **6** and **7b** also inhibited the growth of *Staphylococcus aureus* ATCC 6538 and *Bacillus subtilis* ATCC 6683, respectively with diameters of growth inhibition zones of 8 mm and of 10 mm.

**Table 2.** Results of the qualitative analysis of the antimicrobial effect of compounds **5**–**8** tested at 5000 μg/mL, using an adapted disk diffusion method (diameters of growth inhibition zones were measured in mm).


\* -: not tested.

#### 2.3.2. Effects of Compounds on Antibiotic Susceptibility Profile

For compounds **5** and **6** only, their influence on the *E. faecium* E5 and *S. aureus* ATCC 6538 strains' susceptibility to different antibiotics was evaluated. Although compound **7b** was active against *B. subtilis* ATCC 6683, the influence on this strain's susceptibility to antibiotics was not investigated, as there are no specific guidelines regarding the susceptibility breakpoints of this species.

No significant changes were noticed in the *E. faecium* E5 strain's susceptibility to current antibiotics after culture in the presence of compound **5** tested at subinhibitory concentration (Table 3), suggesting both a low selective pressure for resistance occurrence and a different mechanism of action. Regarding the *E. faecium* E5 strain cultured in the presence of compound **6**, it was observed that it determined an increase in the antimicrobial effect of the tested antibiotics (ampicillin, penicillin, linezolid, and vancomycin). The diameters of growth inhibition zones (in mm) are shown in Table 3.

**Table 3.** Antibiotic susceptibility testing of *E. faecium* E5 strain cultured in the presence of compounds **5** and **6** tested at a subinhibitory concentration of 250 μg/mL and of dimethyl sulfoxide (DMSO) (growth inhibition zone diameters were measured in mm).


\* liquid culture medium inoculated with standardized suspension of *E. faecium* E5.

With respect to the *S. aureus* ATCC 6538 strain cultured in the presence of compound **6**, a slight increase in the diameter of growth inhibition zone was observed in the case of cefoxitin (Table 4). Regarding other studied antibiotics, namely vancomycin, linezolid, clindamycin, and rifampicin, the susceptibility was reduced, suggesting a phenotypic change in the tested strain exposed to compound **6** at subinhibitory concentration. There were no changes in the growth inhibition zone diameters compared with those of the microbial growth control in the case of the studied antibiotics: azithromycin, penicillin, and trimethoprim-sulfamethoxazole.

**Table 4.** Antibiotic susceptibility testing of *S. aureus* ATCC 6538 strain cultured in the presence of compound **6** tested at 250 μg/mL and of DMSO control (growth inhibition zone diameters were measured in mm).


\* liquid culture medium inoculated with standardized suspension of *S. aureus* ATCC 6538.

#### 2.3.3. Quantitative Testing of Antimicrobial Activity

The results of the quantitative antimicrobial testing by the standard broth microdilution method are presented in Table 5. The majority of the tested compounds exhibited MIC values equal to or higher than 500 μg/mL. The most active proved to be compound **6** which was found to have a moderate antimicrobial activity (MIC value of 250 μg/mL) against the *S. aureus* ATCC 6538 reference strain.

**Table 5.** MIC (minimal inhibitory concentration) and MBIC (minimal biofilm inhibitory concentration) values in μg/mL determined for compounds **5**–**8** tested at concentrations between 500 and 0.97 μg/mL.


\* -: not tested.
