Oxacillin

All the identified S. aureus isolates were subjected to an in vitro antimicrobial susceptibility test by employing the modified Kirby–Bauer disc diffusion method as recommended by Clinical and Laboratory Standard Institute (CLSI) guidelines [19 ]. The antimicrobial disks used were ampicillin (30 μg), cefoxitin (30 μg), chloramphenicol (30 μg), ciprofloxacin (25 μg), clindamycin (31 μg), cotrimoxazole (25 μg), erythromycin (26 μg), gentamycin (28 μg), linezolid (30 μg), oxacillin (10 μg), tetracycline (20 μg), and penicillin (10 μg) from HiMedia, India.

Raw materials of antibiotics of doripenem, tigecycline, and tetracycline were provided by Cayman Chemical Company (Michigan, USA) and cefepime was provided by Demo S.A. Pharmaceutical (Votanikos, Greece). doripenem and cefepime were suspended in saline while tigecycline and tetracycline were suspended in distilled water. The antibiotic disks of tigecycline (15 µg), doripenem (10 µg), oxacillin (1 µg), and cefoxitin (30 µg) were provided by Oxoid (Hampshire, UK), while cefepime (30 µg) and tetracycline (30 µg) were provided by Bioanalyse (Ankara, Turkey). The antibiotic E-strips of tigecycline (0.016-256 µg/ml), tetracycline (0.016-256 µg/ml), cefepime (0.016-256 µg/ml), oxacillin (0.016-256 µg/ml), cefoxitin (0.016-256 µg/ml), and doripenem (0.002-32 µg/ml) were provided by HiMedia (Mumbai, India).

The antibiotic susceptibility test was conducted using the Kirby–Bauer disk diffusion method [37 (link)]. The standard antibiotic discs were procured from ‘HiMedia, Mumbai, India, which include polymyxin-B (300 µg), amoxiclav (30 µg), rifampicin (5 µg), tetracycline (30 µg), oxacillin (5 µg), amikacin (30 µg), cefoxitin (30 µg), cefepime (30 µg), ceftazidime (30 µg), cefotaxime (30 µg), chloramphenicol (30 µg), cefdinir (5 µg), penicillin g (10 µg), moxifloxacin (5 µg), ampicillin (10 µg), vancomycin (30 µg), ceftriaxone (30 µg), neomycin (10 µg), ofloxacin (5 µg), norfloxacin (10 µg), kanamycin (30 µg), bacitracin (10 µg), co-trimoxazole (25 µg), methicillin (10 µg), streptomycin (10 µg), levofloxacin (5 µg), erythromycin (15 µg), clindamycin (2 µg), gentamycin (120 µg), and sterile disc (control). The antibiotic discs were placed onto the freshly prepared lawns of each isolate on Mueller–Hinton agar (MHA) plates. The plates were incubated at 40 °C for 24–48 h, and the diameter of the zone of inhibition was measured in millimetres. The strains were classified in accordance with the Clinical and Laboratory Standards Institute [38 ], following the standard antibiotic disc chart.

Antibiotic susceptibility was tested using a disk-diffusion method. Briefly, the bacterial culture was calibrated to a turbidity of McFarland Standard No. 0.5 (about 1 × 10⁸ colony-forming units [CFU]/mL) and swabbed onto Mueller–Hinton agar (MHA) (HiMedia Laboratories). The plates were allowed to dry for 3 min, and an antibiotic disk was placed on the swabbed surface of the agar. The following antibiotics were tested: oxacillin(30μg), penicillin (10 U), cefoxitin (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), ofloxacin (5 μg), tetracycline (30 μg), erythromycin (15 μg), clindamycin (2 μg), linezolid (30 μg), and gentamicin (10 μg) (HiMedia Laboratories).
The methicillin sensitive S. aureus subsp. aureus Rosenbach (Seattle 1945) from the American Type Culture Collection (ATCC) No. 25923 was used as a quality control strain. Experiments were conducted and interpreted following Clinical and Laboratory Standards Institute (CLSI) guidelines [14 ]. The antibiotic resistance of each isolate from the disk-diffusion test was recorded independently and multiple-antibiotic resistance (MAR) indices were calculated. The MAR index is defined as “a/b, where a represents the number of antibiotics to which the isolate was resistant, and b represents the number of antibiotics to which the isolate was exposed” [15 (link)].