Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-beta-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp

Abstract

Gram-negative bacilli with acquired metallo-beta-lactamase (MBL) production have been increasingly reported in some countries, necessitating their detection. The aim of this study was to evaluate the performance of the Hodge test and those of the imipenem (IPM)-EDTA, ceftazidime (CAZ)-mercaptopropionic acid (MPA), and CAZ-sodium mercaptoacetic acid (SMA) double-disk synergy tests (DDSTs). The efficiencies of testing CAZ-resistant and IPM-nonsusceptible isolates were also compared. Strains used for the evaluation were known IMP-1 and VIM-2 MBL-producing isolates and consecutive and CAZ-nonsusceptible isolates of pseudomonads and acinetobacters. The performance of the Hodge test was improved by addition of zinc sulfate (140 microg/disk) to an IPM disk. In DDSTs, EDTA (ca. 1,900 microg) disks were better at detecting MBL-producing strains among pseudomonads, while MPA (3 microl) and SMA (3 mg) disks performed better for acinetobacters. EDTA (ca. 750 microg)-plus-SMA (ca. 2 mg) disks performed better than EDTA, MPA, or SMA disks with both organisms. CAZ-SMA DDSTs failed to detect 22 of 80 (28%) MBL-producing acinetobacters. In conclusion, use of an IPM disk and an EDTA (750 microg)-plus-SMA (2 mg) disk improves performance, and testing IPM-nonsusceptible isolates rather than CAZ-resistant isolates could reduce screening work. Further evaluation of the test is required for the detection of other types of MBL-producing gram-negative bacilli.

FIG. 1.

Performances of DDSTs using disks of IPM or CAZ and disks of EDTA (1,900 μg) or MPA. Panels A and B show results for two different P. aeruginosa isolates. An IPM disk and an EDTA disk produced large synergistic inhibition zones for both isolates (A, top, and B, top). A CAZ disk and a 2-μl MPA disk failed to produce a synergistic zone (A, bottom), but a CAZ disk and a 3-μl MPA disk produced a small synergistic zone (B, bottom).

FIG. 2.

Performances of DDSTs with different β-lactams and chelating agents. (A) Synergistic inhibition zones tended to be larger with EDTA (1,900 μg) disks for P. aeruginosa (a and b), while they tended to be larger with SMA (3 mg) disks for Acinetobacter spp. (e and f). EDTA disks occasionally produced small, suspect synergistic zones with MBL-nonproducing isolates (c and d), but SMA (3 mg) disks alone did not inhibit growth (a to f). (B) The synergistic inhibition zones produced by an IPM disk and an EDTA (750 μg) + SMA (2 mg) disk were large for all MBL-producing isolates (h, l, n, and p), but with an IPM disk and an SMA (3 mg) disk, a small zone was produced for an isolate of P. aeruginosa (g). The MBL-nonproducing isolate which showed an equivocal zone (c) was not inhibited by an SMA (3 mg) or an EDTA + SMA disk (i and j, respectively). CAZ disks failed to show synergistic zones for an isolate of A. baumannii (k and l). An isolate with VIM β-lactamase showed an arrowhead-shaped synergistic zone (q), and an isolate with an IMP enzyme showed an oval synergistic zone (r).

FIG. 2.

Performances of DDSTs with different β-lactams and chelating agents. (A) Synergistic inhibition zones tended to be larger with EDTA (1,900 μg) disks for P. aeruginosa (a and b), while they tended to be larger with SMA (3 mg) disks for Acinetobacter spp. (e and f). EDTA disks occasionally produced small, suspect synergistic zones with MBL-nonproducing isolates (c and d), but SMA (3 mg) disks alone did not inhibit growth (a to f). (B) The synergistic inhibition zones produced by an IPM disk and an EDTA (750 μg) + SMA (2 mg) disk were large for all MBL-producing isolates (h, l, n, and p), but with an IPM disk and an SMA (3 mg) disk, a small zone was produced for an isolate of P. aeruginosa (g). The MBL-nonproducing isolate which showed an equivocal zone (c) was not inhibited by an SMA (3 mg) or an EDTA + SMA disk (i and j, respectively). CAZ disks failed to show synergistic zones for an isolate of A. baumannii (k and l). An isolate with VIM β-lactamase showed an arrowhead-shaped synergistic zone (q), and an isolate with an IMP enzyme showed an oval synergistic zone (r).

FIG. 3.

Proposed scheme for detection of MBL-producing gram-negative bacilli. IPM-nonsusceptible isolates are tested by the Hodge test using an IPM disk to which 140 μg of zinc sulfate has been added. Hodge test-positive isolates are then tested to confirm MBL production by a DDST using an IPM disk and a disk containing 750 μg of EDTA plus 2 mg of SMA. Synergy-positive isolates are then further tested by PCR to detect the bla_IMP or bla_VIM allele.