M eller hinton agar

Determinations were carried out using the diffusion disk method on Müeller-Hinton agar (Merck, Germany) against 25 isolated strains selected, using inocula equivalent to 0.5 McFarland scale (Pro-Lab Diagnostics). The inhibition zone was interpreted according to the Clinical and Laboratory Standards Institute (CLSI, 2012 ) guidelines. Zones of inhibition were measured and compared to standardized tables usually published in the laboratory manual or provided with the antibiotic disks. The antibiotics were selected in line with the recommendation of the European Committee on Antimicrobial Susceptibility Testing (EUCAST)² and included clindamycin (2 μg/disc), vancomycin (30 μg/disc), erythromycin (15 μg/disc), gentamicin (120 μg/disc), oxacillin (1 μg/disc), sulfamethoxazole-trimethoprim (23.75–1.25 μg/disc), amoxicillin/clavulanic acid (20/10 μg/disc), and ciprofloxacin (5 μg/disc). The isolates were classified as susceptible, intermediate resistant, or resistant according to the size of the inhibition zones after growth of the indicator strain used, pursuant to the suppliers' criteria. The phenotypic resistance to vancomycin was confirmed in triplicate in 96-well plates using the broth microdilution method according to CLSI recommendations.

The resistance of staphylococcal isolates to methicillin was tested via disk diffusion assay using Müeller-Hinton agar as recommended by the guidelines of the National Committee for Clinical Laboratory Standards (CLSI 2010)23 . Methicillin-resistant isolates were recognized via the Kirby-Bauer disk diffusion method8 (link) using cefoxitin-impregnated (MAST, UK) disks on Müeller-Hinton agar (Merck, Darmstadt, Germany) and commercially available disks (MAST, UK) supplemented with 2% NaCl for MRSA isolates. The following antibiotic disks were tested: penicillin, oxacillin, ampicillin, gentamicin, ciprofloxacin, erythromycin, clindamycin, chloramphenicol, vancomycin, tetracycline, and rifampin. Suspensions equivalent to 0.5 McFarlands were cultured on MHA and incubated at 35 °C for 18–24 h. Inhibition zones were measured and interpreted as recommended by the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2010)23 .

Assays for bioprospecting of antimicrobial compounds from metagenomic libraries were performed on Müeller Hinton agar (Merck), pH 7,2 (±0,2), with 0.2 mL of the standardized inoculum of microorganisms: S. aureus (CCMB 262), Salmonella choleraesius (CCMB 281), and Bacillus subtilis (INCQS 00002). The clones contained in the 96-well plates were inoculated with a replicator, and the plates were incubated at 37°C for 16 hours.