Ann Lab Med.  2015 Mar;35(2):212-219. 10.3343/alm.2015.35.2.212.

Combined Use of the Modified Hodge Test and Carbapenemase Inhibition Test for Detection of Carbapenemase-Producing Enterobacteriaceae and Metallo-beta-Lactamase-Producing Pseudomonas spp.

• Song W
• Hong SG
• Yong D
• Jeong SH
• Kim HS
• Kim HS
• Kim JS
• Bae IK

• KMID: 2363181
• DOI: http://doi.org/10.3343/alm.2015.35.2.212

Abstract

BACKGROUND
We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-beta-lactamase (MBL)-producing Pseudomonas spp.
METHODS
A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum beta-lactamase [GES]-5, 9 New Delhi metallo-beta-lactamase [NDM]-1, 5 Verona integron-encoded metallo-beta-lactamase [VIM]-2, 3 imipenem-hydrolyzing beta-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain.
RESULTS
Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively.
CONCLUSIONS
Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.

Keyword

Modified Hodge test; Carbapenemase inhibition test; Phenylboronic acid; EDTA; Enterobacteriaceae; Pseudomonas spp.

MeSH Terms

Bacterial Proteins/antagonists & inhibitors/*metabolism
Boronic Acids/chemistry/pharmacology
Disk Diffusion Antimicrobial Tests/*methods
Edetic Acid/chemistry/pharmacology
Enterobacteriaceae/drug effects/*enzymology
Enterobacteriaceae Infections/diagnosis
Humans
Pseudomonas/drug effects/*enzymology
Pseudomonas Infections/diagnosis
Sensitivity and Specificity
beta-Lactamases/chemistry/*metabolism
Bacterial Proteins
Boronic Acids
Edetic Acid
beta-Lactamases

Figure

• 

  Fig. 1 Representative CIT results for the KPC-2-producing K. pneumoniae isolate (A) and IMP-6-producing P. aeruginosa isolate (B) are shown. Three horizontal lines of disks containing 3 ETP, 3 IPM, and 3 MEM were placed on a MHA plate seeded with the test strain. Then, 10 µL of EDTA (30 mg/mL) and PBA (30 mg/mL) were added along first and third vertical lines, respectively. (A) The difference in zone size in the presence and absence of PBA was ≥5 mm for ETP, IPM, and MEM, suggesting KPC production. (B) The difference in zone size in the presence and absence of EDTA was ≥5 mm for IPM, suggesting MBL production.Abbreviations: CIT, carbapenemase inhibition test; ETP, ertapenem; IPM, imipenem; MEM, meropenem; PBA, phenylboronic acid; KPC, K. pneumoniae carbapenemase; MBL, metallo-β-lactamase.

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