Linezolid

Antibiotic susceptibility testing was performed by the Kirby–Bauer disk-diffusion method on Mueller–Hinton agar supplemented with 5% sheep blood for the following antimicrobial agents: penicillin (10 U), ampicillin (10 µg), erythromycin (15 µg), clindamycin (2 µg), ofloxacin (5 µg), cefotaxime (30 µg), linezolid (30 µg), and vancomycin (30 µg) (HiMedia Laboratories, Mumbai). The inoculum for susceptibility testing and interpretation was performed as per the Clinical and Laboratory Standards Institute (CLSI) guidelines [14 ].

Initially, the minimum inhibitory concentration (MIC) of test antibiotics was assessed using microbroth dilution assay (CLSI, 2018 ) as described earlier. MIC is determined as minimum concentration that displayed complete visible growth inhibition of MRSE following 24 h of incubation. Then, the influence of UMB on MIC of test antibiotics was examined by Kirby–Bauer agar diffusion method. Preliminarily, the stock solutions of gentamycin, rifampicin, vancomycin, and linezolid (Hi-Media Laboratories, India) were prepared. Then, overnight culture adjusted to 0.5 McFarland was swabbed uniformly on TSA plates supplemented with and without UMB (at 500 μg/ml) using sterile cotton swabs. Further, wells of 3 mm diameter were punched at the center of the agar plates and test antibiotics at their respective MIC were added to the wells. In parallel, UMB at 500 μg/ml was also loaded in the well of a plain TSA plate, in order to evaluate its individual antibacterial effect. After incubation for 24 h at 37°C, the zone diameter of antibacterial activity in control and treated plates was measured using HiAntibiotic zone scale (Hi-Media Laboratories, India) and subsequently, the plates were documented using high resolution CCD camera (GelDoc XR+, Bio-Rad).

MRCoNS isolates were subjected to an in-vitro antimicrobial susceptibility test by the disc diffusion method with the following antibiotics: gentamicin (10 µg), erythromycin (15 µg), ciprofloxacin (5 µg), tetracycline (30 µg), clindamycin (2 µg), co-trimoxazole (25 mcg), linezolid (30 µg) and chloramphenicol (50 µg) (HiMedia, India). The results were interpreted following the Clinical and Laboratory Standard Institute guidelines M100 (CLSI 2019). The minimum inhibitory concentration (MIC) of oxacillin (0.125 to 64 mg/L) to the bacterial isolates was determined by agar dilution method. Briefly, colonies isolated from the overnight growth of each organism were selected to prepare direct suspensions in tryptic soy broth. The suspensions were adjusted to a 0.5 McFarland standard. Control Mueller–Hinton agar and oxacillin Mueller–Hinton agar plates were inoculated with the final suspensions of bacteria. Plates were incubated at 35 °C for 24 h. The MIC was determined as the lowest concentration of oxacillin that completely inhibited growth. erythromycin-resistant and clindamycin-susceptible isolates were also tested for inducible clindamycin resistance by D zone test [9 ].

Antibiotics discs containing amikacin (30 μg), amoxicillin-clavulanic acid (30 μg), aztreonam (30 μg), ampicillin (10 μg), azithromycin (30 μg), cefepime (30 μg), Cefoperazone/Sulbactam (75/30 μg), ceftriaxone (30 μg), cefotaxime (30 μg), cefuroxime (30 μg), cephalexin (30 μg), ciprofloxacin (1 μg), clindamycin (2 μg), cloxacillin (30 μg), trimethoprim/sulfamethoxazole (25 μg), ertapenem (10 μg), erythromycin (15 μg), gatifloxacin (5 μg), gentamicin (10 μg), imipenem (10 μg), levofloxacin (5 μg), linezolid (30 μg), meropenem (10 μg), netilmicin (30 μg), norfloxacin (10 μg), ofloxacin (5 μg), piperacillin-tazobactam (100/10 μg), teicoplanin (30 μg), tetracycline (30 μg), and vancomycin (30 μg) were obtained from Himedia Laboratories (Mumbai, India) and used as per manufacturer's instructions.

Antimicrobial susceptibility testing of all isolates of S. aureus was performed by modified Kirby–Bauer disc diffusion method on Mueller–Hinton Agar (MHA) (HiMedia Pvt. Ltd. India), as recommended by CLSI guidelines [13 ]. Isolates were tested for susceptibility against amikacin (30 µg), gentamicin (10 µg), ofloxacin (5 µg), clindamycin (2 µg), erythromycin (15 µg), ceftazidime (30 µg), linezolid (30 µg), levofloxacin (5 µg), ampicillin (10 µg), cefoxitin (30 µg), cotrimoxazole (25 µg), ampicillin-clavulanate (10 µg) and vancomycin (30 µg) (HiMedia Pvt. Ltd. India). The results were interpreted as per the guidelines of the CLSI zone size interpretive 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)].