Trimethoprim sulfamethoxazole

The antimicrobial susceptibility testing of MRSA was detected using
the modified Kirby-Bauer disk diffusion method according to the clinical
laboratory standard institute (CLSI) guidelines (CLSI 2013 ). The following antimicrobials were used in
their respective concentration: Ciprofloxacin (5 μg),
Trimethoprim-Sulfamethoxazole (1.25/23.75 μg), Erythromycin (15 μg), Clindamycin
(2 μg), and Amikacin (30 μg) (Hi-Media, India). These antimicrobials were selected
based on the local usage to treat MRSA and considering the recommended
antimicrobial agents for MRSA infections.

Kirby-Bauer disk diffusion method was also performed using 10-cm diameter MHA plates (HiMedia Laboratories, India) and ampicillin (10 µg), amoxicillin-clavulanate (20/10 µg), piperacillin-tazobactam (100/10 µg), cefazolin (30 µg), ceftriaxone (30 µg), ceftazidime (30 µg), cefepime (30 µg), imipenem (10 µg), meropenem (10 µg), gentamicin (10 µg), amikacin (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), and trimethoprim-sulfamethoxazole (1.25/23.75 µg) disks (HiMedia Laboratories, India) with the same bacterial inoculum from protocol B and incubated at 35°C ± 2°C in ambient air for 16–18 hours. AST results were interpreted as per the performance standards for AST by CLSI.
The workflow of standard inoculation and direct inoculation protocols of AST is depicted in Fig. 1.

Antimicrobial susceptibility testing of the E. coli and Salmonella isolates was performed by the Kirby-Bauer disc diffusion method using Mueller-Hinton agar (Merck Co., Darmstadt, Germany) according to the Clinical and Laboratory Standards Institute (CLSI) [13 ]. The following used antibiotics were tested: Ampicillin (10 µg), amoxicillin/clavulanic acid (30 µg), gentamycin (10 µg), tetracycline (30 µg), trimethoprim/sulfamethoxazole (10 µg), ceftazidime (30 µg), ciprofloxacin (10 µg), nitrofurantoin (300 µg), norfloxacin (30 µg), kanamycin (30 µg), ceftriaxone (30 µg), and chloramphenicol (30 µg) (HiMedia Laboratories Pvt. Ltd, Mumbai, India). Briefly, a bacterial suspension with equivalent turbidity to 0.5 McFarland standard (1.5 × 10⁸ CFU/mL) was prepared in sterile phosphate buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4). The sterile swab stick was dipped into bacterial suspension and then on the surface of agar was uniformly inoculated. Afterward, antibiotic disks were placed for each plate and incubated at 35°C for 24 h. Inhibition zones on agar plate were measured, and the results were recorded in accordance with interpretive criteria provided by CLSI.

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.

Disk diffusion testing directly from the flagged positive blood culture bottles was performed using the CLSI-described method. Four to five drops of blood culture broths were dispensed onto MHA plates after thoroughly mixing by inverting the blood culture bottles 5–10 times. The blood culture broths were then spread on the entire surface of MHA using a sterile cotton swab (the procedure was repeated twice more by rotating the plate at 60°C to ensure even distribution of inoculum). The inoculated plate was kept lid ajar for 5 minutes before ampicillin (10 µg), ceftriaxone (30 µg), ceftazidime (30 µg), meropenem (10 µg), ciprofloxacin (5 µg), and trimethoprim-sulfamethoxazole (1.25/23.75 µg) disks (HiMedia Laboratories, India) were placed on the agar surface. Plates were incubated for 16–18 hours at 35°C ± 2°C in ambient air. Results are interpreted as per the zone diameter breakpoints for disk diffusion directly from flagged positive blood as per CLSI.

The antibiotic resistance pattern of isolates was determined by using the disk diffusion method according to the Clinical and Laboratory Standard Institute (CLSI) guidelines (10 ). The antibiotics tested were ceftizoxime (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cephalexin (30 μg), amoxicillin (30 μg), imipenem (10 μg), cefepime (30 μg), cefoxitin (30 μg), gentamycin (30 μg), tetracycline (30 μg), trimethoprim/sulfamethoxazole (30 μg), nalidixic acid and ciprofloxacin (30 μg) (Himedia, India). E. coli ATCC 25922 and K. pneumoniae 700603 were used as quality control strains for antimicrobial susceptibility testing.