Modified CLSI Extended-Spectrum β-Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases

The worldwide dissemination of Enterobacteriaceae producing AmpC β-lactamases and carbapenemases makes difficult the phenotypic detection of extended-spectrum β-lactamases (ESBLs), as they may be masked by these additional enzymes. A modification of the CLSI ESBL confirmatory test was developed and evaluated in a comparative study for its ability to successfully detect ESBLs among Enterobacteriaceae producing various carbapenemases (Klebsiella pneumoniae carbapenemase KPC], VIM, NDM, and OXA-48) and plasmidic or derepressed AmpCs. The modified CLSI ESBL confirmatory test was performed with cefotaxime and ceftazidime disks with and without clavulanate, on which both boronic acid (BA) and EDTA were dispensed. A total of 162 genotypically confirmed ESBL-positive Enterobacteriaceae isolates (83 carbapenemase/ESBL producers, 25 AmpC/ESBL producers, and 54 ESBL-only producers) were examined. For comparison, 139 genotypically confirmed ESBL-negative Enterobacteriaceae isolates (94 of them possessed carbapenemases and 20 possessed AmpCs) were also tested. The standard CLSI ESBL confirmatory test was positive for 106 of the 162 ESBL producers (sensitivity, 65.4%) and showed false-positive results for 4 of the 139 non-ESBL producers (specificity, 97.1%). The modified CLSI ESBL confirmatory test detected 158 of 162 ESBL producers (sensitivity, 97.5%) and showed no false-positive results for non-ESBL producers (specificity, 100%). The findings of the study demonstrate that the modified CLSI ESBL confirmatory test using antibiotic disks containing both BA and EDTA accurately detects ESBLs in Enterobacteriaceae regardless of the coexistence of additional β-lactam resistance mechanisms.