Broth microdilution methods using B-lactamase inhibitors for the identification of Klebsiella pneumoniae carbapenemases and metallo-B-lactamases in gram-negative bacilli

This study was designed to evaluate the performance of the broth microdilution (BMD) test in detecting the production of Klebsiella pneumoniae carbapenemases (KPCs) and metallo-beta-lactamases (MBLs) in clinical isolates of gram-negative bacilli by using various beta-lactamase inhibitors. A carbapenemase detection test, comprised of Mueller-Hinton broth containing serial twofold dilutions of imipenem or meropenem with and without aminophenylboronic acid (APB), phenylboronic acid (PB), cloxacillin (CLX), dipicolinic acid (DPA), or EDTA, was evaluated against 31 Klebsiella pneumoniae strains with KPC, 21 MBL-producers (3 Enterobacteriaceae and 18 non-fermenters), and 26 carbapenemase-non-producing Enterobacteriaceae strains. A threefold or greater decrease in the MIC of imipenem or meropenem with beta-lactamase inhibitors, when compared with the imipenem or meropenem alone, was considered a positive result. Imipenem with and without PB had the most comparable sensitivity (93.5%) and specificity (93.6%) for the detection of K. pneumoniae with KPC enzymes if the additional criterion of a negative CLX result was included. Both DPA and EDTA had excellent sensitivity (imipenem with and without DPA vs. EDTA, 90.5% vs. 95.2%) and specificity (imipenem with and without DPA vs. EDTA, 98.2% vs. 100%) for the detection of MBL-producing gram-negative bacilli. The comparative study showed that, by using imipenem with and without PB, CLX, and EDTA, the BMD test was effective in detecting KPC and MBL enzymes. The BMD test could be applied for routine use in commercially available semiautomated systems for the detection of KPCs and MBLs in gram-negative bacilli.