Evaluation of phenotypic tests for detection of metallo-b-lactamases in clinical isolates of Pseudomonas aeruginosa from Srinagarind Hospital

Abstract

Carbapenem resistance mediated by acquired carbapenemase genes has been increasingly reported in Pseudomonas aeruginosa. Class B carbapenemases or metallo-b-lactamases (MBLs) are the most clinical concern. Currently, there is no recommendation available from the Clinical Laboratory Standards Institute (CLSI) for MBL detection. In this study, we attempted to evaluate the performance for phenotypic detection of MBLs in imipenem-nonsusceptible clinical isolates of P. aeruginosa from Srinagarind Hospital. Five MBL-positive control strains, each producing IMP-1, IMP-4, IMP-9, VIM-1, or VIM-2 enzyme, and 74 clinical isolates of P. aeruginosa were screened for the presence of MBLs by combined-disk test (CDT) and double-disk synergy test (DDST) on a single agar plate using imipenem (10 µg IPM) and meropenem (10 µg MEM) disks as substrates and 292 µg of EDTA as an MBL inhibitor. Multiplex PCR for detection of MBL genes was used as a gold standard. Genotypic confirmation revealed that 9 of the 74 clinical isolates (12.2%) carried MBL genes, bla_VIM (6 isolates) and bla_IMP (3 isolates). Comparison of the MBL phenotypic tests to the multiplex PCR revealed that the CDT using IPM+EDTA/IPM correctly differentiated all MBL-producing isolates (sensitivity of 100%) but gave three false-positive isolates (specificity of 95.4%), whereas that with MEM+EDTA/MEM showed sensitivity and specificity of 92.9% and 92.3% respectively. In addition, the DDST using either IPM or MEM with EDTA gave the same result for all isolates with sensitivity and specificity of 92.9% and 96.9% respectively. These methods are simple and accurate for detection of MBLs in imipenem-nonsusceptible P. aeruginosa isolates from this hospital. Early detection of MBL producers and strict infection control will contribute to prevent further spread of these resistant strains.