Korean J Lab Med.  2009 Jun;29(3):204-211. 10.3343/kjlm.2009.29.3.204.

Evaluation of Dipicolinic Acid-Based Mueller Hinton Agar Biplate for Detection of IMP-1 and VIM-2 type Metallo-beta-Lactamase in Imipenem Non-susceptible Gram Negative Bacilli

• Shin KS
• Son BR
• Koo SH
• Lee SH
• Ahn JB
• Park SH
• Hwang SY

• KMID: 1096971
• DOI: http://doi.org/10.3343/kjlm.2009.29.3.204

Abstract

BACKGROUND: Since metallo-beta-lactamase (MBL)-producing isolates can hydrolyze carbapenem and also easily transfer the resistance genes to other bacteria, a rapid and accurate detection of MBL has become very important. We evaluated the utility of Mueller Hinton agar (MHA) biplate containing dipicolinic acid (DPA) as a screening method to detect IMP-1 and VIM-2 type MBL-producing isolates.
METHODS
Based on our preliminary tests using various concentrations of DPA, 200 and 300 microg/mL concentration of DPA were chosen for further study. Bacterial lawns were grown on MHA biplate, one half of which contained DPA while the other did not. The inhibition zone around the imipenem (IPM) disk on both sides of this plate was compared. The stability of DPA in the stored DPA-MHA biplate was also evaluated during three months using two MBL- and one non-MBL-producing isolates.
RESULTS
When the criterion of a > or =7 mm increase of inhibition zone around the IPM disk on the MHA containing DPA compared to MHA without DPA was used, the sensitivities and specificities were 94.7% and 97.6% for 200 microg/mL DPA-MHA biplate, and 98.2% and 97.6% for 300 microg/mL DPA-MHA biplate, respectively. The activity of the DPA in this biplate was stable for three months.
CONCLUSIONS
Assays using DPA 300-MHA biplate were highly sensitive and specific for the detection of IMP-1 and VIM-2 type MBL-producing bacteria. In addition, it is easy to perform; so, it may be useful to apply this method for detection of IMP-1 and VIM-2 type MBL in clinical laboratories.

Keyword

Dipicolinic acid; DPA-MHA biplate; Metallo-beta-lactamase

MeSH Terms

Agar
Anti-Bacterial Agents/*pharmacology
Bacteriological Techniques
Chelating Agents/chemistry/*pharmacology
Drug Resistance, Bacterial
Gram-Negative Bacteria/drug effects/enzymology/*isolation & purification
Imipenem/*pharmacology
Picolinic Acids/chemistry/*pharmacology
Reagent Kits, Diagnostic
Sensitivity and Specificity
beta-Lactamases/*analysis/biosynthesis

Figure

• 

  Fig. 1. DPA-based MHA biplate for the detection of MBL in IPM non-susceptible clinical isolates. (A) IMP-1 type Acinetobacter spp., (B) VIM-2 type Pseudomonas aeruginosa, (C) VIM-2 Achromobacter xylosoxidans, and (D) non-MBL producing Acinetobacter spp. The lower part of each biplates contained 300 μg/mL DPA. MBL producing isolates (A-C) showed increased inhibition zones (≥7 mm) around IPM disks in the presence of DPA as compared to disks that lacked DPA. The non-MBL-producing isolate (D) showed no significant difference in inhibition zone size. Abbreviations: IPM, imipenem; MHA, Mueller Hinton agar; DPA, dipicolinic acid.

• 

  Fig. 2. Increase of inhibition zone size around the imipenem disk in MHA with DPA as compared with inhibition zones produced by imipenem disk in MHA without DPA for 57 MBL positive and 41 MBL negative clinical isolates. Abbreviations: DPA, dipicolinic acid; MHA, Mueller Hinton agar; MBL, metallo-β-lactamase; N-MBL, non-MBL.

• 

  Fig. 3. DPA-based MHA biplate for the detection of three type β-lactamase from Providencia rettgerii carrying ESBL, AmpC β-lactamase and MBL. When boronic acid (BA) was add to cefotaxime (CTX) and cefotaxime/clavulanic acid (CTX/CLA) disk, a ≥5 mm increase of inhibition zone around CTX/CLA disk in comparison to CTX disk suggested the co-production of ESBL and AmpC β-lactamase (A→B), and the increase of inhibition zone around CTX/CLA disks on DPA-containing MHA relative to MHA at both A and B suggested the concurrent production of MBL (upper→lower). Abbreviations: DPA, dipicolinic acid; MHA, Mueller Hinton agar; ESBL, extended-spectrum β-lactamase; MBL, metallo-β-lactamase.

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