Comparative Evaluation of Combined-Disk Tests Using Different Boronic Acid Compounds for Detection of Klebsiella pneumoniae Carbapenemase-Producing Enterobacteriaceae Clinical Isolates

The accurate phenotypic detection of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae is an increasing necessity worldwide. We evaluated the performance of boronic acid combined-disk tests using as substrate imipenem or meropenem and as inhibitor of KPC production 300 mu g aminophenylboronic acid (APBA), 600 mu g APBA, or 400 mu g phenylboronic acid (PBA). Tests were considered positive when an increase in the growth-inhibitory zone around a carbapenem disk with KPC inhibitor was 5 mm or greater of the growth-inhibitory zone diameter around the disk containing carbapenem alone. The comparison of the combined-disk tests was performed with 112 genotypically confirmed KPC-possessing Enterobacteriaceae isolates. To measure the specificity of the tests, 127 genotypically confirmed KPC-negative Enterobacteriaceae isolates that were nonsusceptible to at least one carbapenem were chosen for testing. Using disks containing imipenem without and with 300 mu g APBA, 600 mu g APBA, or 400 mu g PBA, 72, 92, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 64.3%, 82.1%, and 100%, respectively). Using disks containing meropenem without and with 300 mu g APBA, 600 mu g APBA, or 400 mu g PBA, 87, 108, and 112 of the KPC producers, respectively, gave positive results (sensitivities, 77.7%, 96.4%, and 100%, respectively). Among KPC producers, the disk potentiation tests using meropenem and PBA demonstrated the largest differences in inhibition zones (P < 0.001). All combined-disk tests correctly identified 124 of the 127 non-KPC producers (specificity, 97.6%). This comparative study showed that PBA is the most effective inhibitor of KPC enzymes, and its use in combined-disk tests with meropenem may give the most easily interpreted results.