Antimicrobial Evaluation of Novel Compounds against Staphylococcus aureus

The antimicrobial activities were assessed on a panel of both reference and clinically isolated Staphylococcus aureus strains, as follows: S. aureus ATCC^® 6538™, S. aureus ATCC^® 6538p™, methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), β-lactamase-producing S. aureus (β-LPSA), macrolide-lincosamide-streptogramin B-resistant S. aureus (MLSB), quinolone-resistant S. aureus (QRSA), vancomycin-resistant S. aureus (VRSA) [28 (link)]. First, each strain was plated on Luria-Bertani (Lennox) (CondaLab, Madrid, Spain) agar (LBA) plates and incubated overnight at 37 °C. Then, 2–3 fresh colonies were transferred to 3 mL of Muller Hinton (MH) broth (HiMedia, Einhausen, Germany) and incubated in agitation overnight at 37 °C. The day after, the bacterial suspensions were transferred to fresh MH medium and further incubated until they reached the turbidity of 0.5 McFarland. Finally, serial dilutions of the inoculum were performed to obtain a final bacterial concentration of about 5 × 10⁵ CFU/mL. Stock solutions of the samples were prepared by dissolving M15RL, MDRL, and DDRL in dimethylsulfoxide (DMSO) at 10 mg/mL. The tests were conducted with the broth microdilution method to evaluate the antimicrobial potential of these mixtures. Briefly, 4 µL of each sample was dispensed into 200 µL of MH medium in a microtiter plate and twofold serially diluted. Then 100 µL of bacterial suspension was inoculated into the broth (~5 × 10⁴ CFU/well) and incubated statically for 18h at 37 °C. Vancomycin and DMSO (2% v/v) represented the positive and negative controls, respectively.