Antibiotic disks

Recovered isolates were screened for presumptive presence of ESBLs using cefotaxime (30 μg), ceftazidime (30 μg) and cefpodoxime (10 μg) antibiotic disks (MAST Group Ltd., Bootle, UK) according to the CLSI guidelines [20 ]. Antibiotic disk diffusion tests were performed using 0.5 McFarland standard inoculums on Mueller Hinton agar (BioMerieux) and incubated overnight at 37°C. Using CLSI screening guidelines, bacterial isolates with zone inhibition diameters ≤ 27 mm for cefotaxime and ≤ 22 mm for ceftazidime were reported as positive for ESBL screening. As recommended by CLSI, cefpodoxime disks were included in the ESBL screening with breakpoints of ≤17 mm for study isolates. Study isolates resistant at these breakpoints were reported as positive for ESBL screening. All isolates with a positive result in the ESBL screening test with at least one of the three screening agents were selected for ESBL confirmation.

Antimicrobial susceptibility of E. coli and K. pneumoniae isolates was carried out using the Kirby–Bauer disk diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines [15 ]. Commercially available antibiotic disks (Mast Co., United Kingdom) used in this study included ampicillin, (10 µg), piperacillin (100 µg), piperacillin-tazobactam (100/10 µg), cefoxitin (30 µg), ceftriaxone (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefepime (30 µg), cefixime (30 µg), imipenem (10 µg), ertapenem (10 µg), meropenem (10 µg), aztreonam (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), ofloxacin (5 µg), moxifloxacin (5 µg), gatifloxacin (5 µg), gentamicin (10 µg), amikacin (30 µg), kanamycin (30 µg), tobramycin (10 µg), tetracycline (30 µg), and trimethoprim/sulfamethoxazole (1.25/23.75 µg).

Resistance or sensitivity of the strains toward eight antibiotics was interpreted by using several antibiotic diffusion disks compared with MIC for strains, which have determined previously toward various antimicrobial agents. Therefore, at first 1% (approximately 10⁷ cells) overnight strains were added to MRS and nutrient broth with concentration 0.5 MacFarlane, and a certain volume of the mediums divided into plates, pour plate method. By solidifying mediums, antibiotic disks (Mast Co, UK) applied the surface. Antibiotics were used in following concentration: 10 µg ampicillin, 10 µg penicillin, 10 µg gentamicin, 30 µg chloramphenicol, 30 µg novobiocin, 30 µg erythromycin, 30 µg nalidixic acid, 130 µg bacitracin. The plates were incubated at 30 °C under aerobic condition for Bacillus sp. and anaerobic for Lactobacillus sp., after passing 24 h, inhibition zone measured. In general, strains were considered as resistance if the inhibition zone diameter was ≤10 for ampicillin, penicillin and gentamicin, ≤15 mm for chloramphenicol, novobiocin, erythromycin and nalidixic acid, ≤18 mm for bacitracin. Results were interpreted according to the cut-off levels published in CA-SFM (2011).