Penicillin

Antibiotic susceptibility was tested using the Kirby–Bauer disc diffusion method with some modifications [33 (link)]. Susceptibilities to ten different antibiotic discs (ampicillin (10 µg), penicillin G (10 IU), kanamycin (30 µg), gentamicin (10 µg), streptomycin (10 µg), tetracycline (30 µg), erythromycin (15 µg), vancomycin (30 µg), chloramphenicol (30 µg), and methicillin (5 µg) (Liofilchem, Teramo, Italy)) were evaluated. Each bacterial suspension was adjusted to a turbidity of McFarland 0.5 and inoculated to BHI plates. After antibiotic discs were carefully placed on BHI plates, plates were incubated at 37 °C for 24 h. The diameter of a clear zone was measured and interpreted according to the manufacturer’s instructions based on CLSI guidelines.

The pellet was dissolved in sterile physiological saline (Polpharma) to obtain a suspension with an optical density of 0.5 McFarland. The prepared suspension was plated on Mueller Hinton Agar with 5.0% horse blood and β-NAD (MHF, Graso) and then gradient strips with antibiotics, i.e., penicillin (0.016-256 μg/ml) (Liofilchem), ampicillin (0.016-256 μg/ml) (Liofilchem), meropenem (0.002-32 μg/ml) (Liofilchem), erythromycin (0.016-256 μg/ml) (Liofilchem) and trimethoprim*/sulfamethoxazole (1/19) (co-trimoxazole) (0.002-32* μg/ml) (Liofilchem) were applied. After 20-h incubation at 35 °C MICs (based on the eclipse-shaped inhibition zone) were determined. The results were interpreted in accordance with EUCAST v. 13.0 recommendations [33 ].