Phenotypic detection of carbapenem-susceptible metallo-beta-lactamase-producing gram-negative bacilli in the clinical laboratory

Abstract

Rapid detection of metallo-beta-lactamase (MBL)-producing gram-negative pathogens is critical to prevent their widespread dissemination. Thus far, no standardized phenotypic method is available, and previously reported techniques have poor sensitivity for detecting carbapenem-susceptible MBL-carrying isolates, an increasingly described phenomenon. We developed a phenotypic detection method using both a double-disk synergy test and a combined-disk test with imipenem and 292 microg EDTA on one agar plate. Genotypic confirmation was used for validation. Of the 134 clinical isolates, 84 were confirmed to carry an MBL. Of these, 51 (61%) were susceptible to at least one carbapenem, and 22 (26%) were isolated from blood. The phenotypic method correctly differentiated all MBL-producing isolates (sensitivity, 100%). Fifty-one of the 52 MBL-negative isolates were correctly differentiated (specificity, 98%). This study reports the validation of a simple and accurate MBL detection method that can be easily incorporated into the daily routine of a clinical laboratory. Early detection of MBL-carrying organisms, including those with susceptibility to carbapenems, is of paramount clinical importance, as it allows rapid initiation of strict infection control practices as well as therapeutic guidance for confirmed infection.

FIG. 1.

The phenotypic appearance of an MBL-producing Serratia marcescens isolate carrying the bla_IMP-4 gene. (A) Combined-disk test, using two imipenem (10 μg) disks, one with 292 μg EDTA, showing an increase in zone inhibition of >4 mm around the disk with EDTA. (B) Double-disk synergy test, using an IPM (10 μg) disk placed 20 mm (center to center) from a blank filter disk containing 292 μg EDTA. (C) Aztreonam (30 μg) disk with a >30-mm zone of inhibition.

FIG. 2.

Increase (in millimeters) in zone of inhibition around the imipenem-EDTA disk compared with the imipenem disk alone for 52 MBL-negative (MBL -ve) and 54 MBL-positive (MBL +ve) clinical isolates.

FIG. 3.

Inhibition zone diameters (in millimeters) around the aztreonam disk (30 μg) for the 84 metallo-β-lactamase-carrying isolates. Asterisks indicate Enterobacteriaceae isolates (n = 8) found to produce an AmpC-type or extended-spectrum β-lactamase.