2.6. Antibiotic Susceptibility Testing

Antibiotic susceptibility testing was performed with the agar disk diffusion test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [42], except for kanamycin, for which the recommendations of the Clinical and Laboratory Standards Institute (CLSI) were followed [43]. For this purpose, the isolates were grown on TSA plates (Oxoid) at 37 °C for 24 h. Afterward, a colony was transferred to 3 mL tubes of sterile saline solution (0.9%, w/v) and seeded on Mueller Hinton agar (Oxoid). Disks containing known amounts of each antibiotic were placed on the surface of the agar plates, which were then incubated at 37 °C for 16 h. For this test, a total of 14 antibiotics were used: cefoxitin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), erythromycin (15 μg), fusidic acid (10 μg), gentamycin (10 μg), kanamycin (30 μg), linezolid (10 μg), mupirocin (200 μg), penicillin (1 U), tetracycline (30 μg), tobramycin (10 μg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg). The susceptibility of CoNS to vancomycin was determined using a microdilution method in order to determine the minimum inhibitory concentration (MIC) following the guidelines provided by EUCAST (2023) [42]. Briefly, individual colonies were suspended in a sterile glass tube containing 10 mL saline solution (0.85% NaCl) to a turbidity of 0.5 on the McFarland scale, and then, the bacterial suspensions were diluted 1000-fold in Mueller Hinton broth (Oxoid). A volume of 50 μL of the diluted bacterial suspensions was added to each microplate well containing 50 μL of Mueller Hinton broth with vancomycin (1–64 µg/mL). After incubation at 37 °C for 18 h, the MIC for vancomycin was established as the lowest antibiotic concentration inhibiting bacterial growth and interpreted according to MIC breakpoints established for CoNS by EUCAST. S. aureus ATCC25923 was used as the quality control.