Extended-spectrum-β-lactamase production in E. coli and Klebsiella species was detected following CLSI guidelines [10].

Screening Test. For ESBL screening, bacterial isolates were examined for their susceptibility to third-generation cephalosporins using ceftazidime (30 μg) and cefotaxime (30 μg) disks. If the ZOI was ≤22 mm for ceftazidime and/or ≤27 mm for cefotaxime, the isolate was considered a potential ESBL-producer [10].

Confirmatory Test. Confirmation of ESBL production was carried out using the combination disk test (CDT). In this test, ceftazidime (30 μg) disk alone and in combination with clavulanic acid (30/10 μg) or cefotaxime (30 μg) disk alone and in combination with clavulanic acid (30/10 μg) were applied onto a plate of MHA previously inoculated with the test strain. The plate was incubated in ambient air for 16–18 hours at 37°C. Isolates showing an increase of ≥5 mm in the zone of inhibition (ZOI) for either antimicrobial agent tested in combination with clavulanic acid versus the zone diameter of the agent when tested alone were considered positive for ESBL production. For standardization of the ESBL detection test, K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as positive and negative controls, respectively [10].

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