4.3. Phenotypic Confirmatory Test for ESBLs in E. coli

According to EUCAST recommendations [57], the combination disc test (CDT) was chosen for the confirmation of ESBL. The CDT was performed in accordance with the Clinical and Laboratory Standards Institute guidelines [58]. A standardized inoculum (0,5McF) of the presumptive ESBL-producing E. coli isolate was swabbed on the surface of Muller Hinton agar II (Biolife, IT). Cefotaxime 30 μg (CTX-30, BD BBL) and ceftazidime 30 μg (CAZ-30, BD BBL) discs alone and in combination with clavulanic acid 10 μg (CTX CLA and CAZ CLA, Biolife) were placed on the plate. The results were obtained after 18 h incubation at 36 °C. ESBL-producing E. coli was confirmed as positive if there was a ≥5 mm increase in the inhibition zone. This was detected by clavulanic acid around either the cefotaxime or the ceftazidime disc, compared to the diameter around the disc containing cefotaxime or ceftazidime alone. E. coli ATCC 25922 (Bioscience, Botolph Claydon, United Kingdom) was used for the quality control when the ESBL confirmatory test was carried out.