Antimicrobial susceptibility testing

A modified Kirby-Bauer disk diffusion technique was used for AST of bacterial isolates according to the Clinical Laboratory Standards Institute (CLSI) 2021 guideline [44]. The suspension of confirmed pure isolates was done by taking 3 to 5 colonies and emulsifying them with 3 to 4 mL of normal saline, and adjusting them to 0.5% McFarland standard [43]. Using a sterile cotton swab, sufficient inoculum was taken and distributed on Muller-Hinton agar plates (Oxoid Ltd., Basingstoke, UK) (supplemented with 5% sheep blood if fastidious) with lawn culture technique. After 3 minutes, a set of standard antimicrobial disks was aseptically placed on the inoculated plates and allowed to stand at room temperature for 15 minutes. All inoculated media were incubated aerobically at 37°C for 24 hours. Zones of inhibition were measured using a ruler. Finally, the results were interpreted as susceptible, intermediate, or resistant. Inducible clindamycin resistance was detected among S.aureus isolates with a simple disk approximation test, commonly referred to as the D-test [45]. The following routinely used antimicrobial disks of different classes were checked: penicillins (penicillin (10 μg), ampicillin (10 μg), oxacillin (1 μg), and piperacillin (100 μg)), beta-lactamase inhibitor combination (amoxicillin-clavulanic acid (20/10 μg)), cephalosporins (ceftriaxone (30 μg), ceftazidime (30 μg), and cefoxitin (30 μg)), tetracyclines (tetracycline (30 μg) and doxycycline (30 μg)), macrolides (azithromycin (15 μg) and erythromycin (15 μg)), fluoroquinolone (ciprofloxacin (5 μg)), folate pathway antagonist (trimethoprim-sulfamethoxazole (1.25 + 23.75 μg) (co-trimoxazole)), aminoglycoside (gentamicin (10 μg)), lincosamide (clindamycin (2 μg)), and phenicol (chloramphenicol (30 μg)). All antibiotics were obtained from Abtek Biologicals, Ltd., Liverpool, UK [44].