Antimicrobial Susceptibility Testing Protocol

The antimicrobial susceptibility testing was done using the Kirby-Bauer disc diffusion method on Mueller Hinton Agar (Becton, Dickinson and Company, MD, USA) based on the Clinical Laboratory Standard Institute (CLSI) guidelines [18 ]. The antibiotic discs (Becton, Dickinson and Company, MD, USA) used included ampicillin (10 μg), sulfamethoxazole/trimethoprim (1.25/23.75 μg), streptomycin (300 μg), ciprofloxacin (5 μg), tetracycline (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), chloramphenicol (30 μg), ceftazidime (30 μg), norfloxacin (10 μg), and cefotaxime (30 μg). The phenotypic confirmation of ESBL isolates was done by the combination of disc approximation method using either ceftazidime (30 μg) or cefotaxime (30 μg) alone followed by overnight incubation at 37°C for 18–24 hrs. Interpretation of susceptibility patterns on other antimicrobial discs was done using guidelines laid down in the CLSI, which provides break points corresponding to zone of inhibition diameter. An increase in antibiotic zone diameter (5–12 mm) for either ceftazidime or cefotaxime indicated ESBL production [10 (link), 18 ]. Quality control standard laboratory procedures were strictly adhered to, to avoid contamination. Escherichia coli ATCC 25922 was used as a quality control organism.