3.4. Antimicrobial Screening Assays

The microorganisms used in this study were obtained from the American Type Culture Collection (ATCC). They included five strains Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Saccharomyces cerevisiae ATCC 2601, Staphylococcus aureus. CIP were obtained from the CIP 106760, and the yeast strain Candida albicans ATCC 10231.

The antimicrobial activity of each obtained extract was evaluated against two Gram-positive bacteria (E. faecalis and S. aureus), two Gram-negative bacteria (E. coli and P. aeruginosa), and two yeasts (S. cerevisiae and C. albicans), according to Rijo et al. [41]. The extracts were diluted in DMSO from 10 mg/mL to a final concentration of 1 mg/mL. Stock solutions of reference antibiotics (vancomycin, norfloxacin, and nystatin) were also prepared at 1 mg/mL in DMSO.

In aseptic conditions, Petri dishes containing 20 mL of solid Mueller–Hinton for bacteria, or Sabouraud Dextrose Agar culture medium (from Biokar Diagnostics), for yeasts, were inoculated with 0.1 mL of bacterial suspension matching a 0.5 McFarland standard solution and uniformly spread on the medium surface using a sterile swab. Wells of approximately 5 mm in diameter were made in the medium, using a sterile glass Pasteur pipette, and 50 μL of each extract were added into the wells. A positive control of vancomycin for Gram-positive bacteria, norfloxacin for Gram-negative bacteria, and nystatin for yeasts, and a negative control of DMSO, were used in the assay. Plates were incubated at 37 °C for 24 h. The antimicrobial activity was evaluated by measuring the diameter (mm) of the inhibition zone formed around the wells and compared to controls.

The MIC and MBC/MFC were determined using the microdilution technique proposed by National Committee for Clinical Laboratory Standards (NCCLS) [42]. Briefly, 100 mL of Mueller–Hilton broth for bacteria and Sabouraud for yeasts was placed into each well of a 96 microplate, under aseptic conditions. Each extracted sample (100 μL), the appropriate positive control of each microorganism, and negative controls at a concentration of 1 mg/mL, were added to the first well. Using a multichannel micropipette, a 1:2 microdilution series was made. A standardized bacterial suspension (10 μL), corresponding to 0.5 McFarland of each microorganism, was then placed in all wells. Finally, the plates were incubated at 37 °C for 24 h. The MIC was determined when no growth was detected in the well of the microplate. Each measurement was performed in triplicate using 96 well microtiter plates with enrichment. A total of 10 µL was withdrawn from the microplate and sown in a Petri dish to verify the MBC/MFC, which was determined when there was no visible microbial growth on the plates [43].