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Shin, Son, Koo, Lee, Ahn, Park, and Hwang: Evaluation of Dipicolinic Acid-Based Mueller Hinton Agar Biplate for Detection of IMP-1 and VIM-2 type Metallo-β-Lactamase in Imipenem Non-susceptible Gram Negative Bacilli
Original Article

Korean J Leg Med 2009;29(3):204-211.

Published online: 14 January 2009

DOI: https://doi.org/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-β-Lactamase in Imipenem Non-susceptible Gram Negative Bacilli

Kyeong Seob Shin, M.D.1, Bo Ra Son, M.D.1, Sun Hoe Koo, M.D.2, Sang Hwa Lee, Ph.D.3, Jun Bae Ahn, Ph.D.4, Seock Hwan Park, Ph.D.5, Seock Yeon Hwang, Ph.D.6

1Department of Laboratory Medicine, College of Medicine, Chungbuk National University, Cheongju, Korea

2Department of Laboratory Medicine, College of Medicine, Chungnam National University, Daejeon, Korea

3Departments of Food and Nutrition, Food Service Industry, Daejeon, Korea

4 Department of Environment, Civil Engineering and Information Systems, Seowon University, Cheongju, Korea

5Department of Biomedical Laboratory Science, Daejeon, Korea

6 College of Applied Science and Industry, Daejeon University, Daejeon, Korea

Corresponding author: Kyeong Seob Shin, M.D. Department of Laboratory Medicine, College of Medicine, Chungbuk National University, San 62 Gaeshin-dong, Heungdeok-gu, Cheongju 361-711, Korea Tel: +82-43-269-6240, Fax: +82-43-271-5243 E-mail: ksshin@chungbuk.ac.kr

∗ This study was supported by Bio Organic Material & Food Center at Seowon University in 2008, a part of Regional Innovation Center (RIC) program of the Ministry of Knowledge Economy in Republic of Korea.

      Revised 13 October 20082 February 2009       Accepted 2 February 2009

Copyright © 2009 Korean Society for Legal Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background:

Since metallo-β-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 μg/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 ≥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 μg/mL DPA-MHA biplate, and 98.2% and 97.6% for 300 μg/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.

Keywords: Dipicolinic acid, DPA-MHA biplate, Metallo-β-lactamase

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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.
kjlm-29-204f1.tif
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.
kjlm-29-204f2.tif
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.
kjlm-29-204f3.tif
Table 1.
Results of dipicolinic acid-containing Mueller Hinton agar biplate for detection of metallo-β-lactamase in imipenem non-susceptible gram-negative bacilli
Species and allele gene of MBL genes (N of isolates) DPA 200-MHA biplate DPA 300-MHA biplate
Positive† Negative Positive Negative
Pseudomonas aeruginosa (46)        
IMP-1 type MBL (8) 8 0 8 0
VIM-2 type MBL (17) 17 0 17 0
N-MBL (21) 1 20 1 20
Acinetobacter spp. (45)        
IMP-1 type MBL (13) 12 1 13 0
VIM-2 type MBL (12) 11 1‡ 11 1‡
N-MBL (20) 0 20 0 20
Other isolates∗ (7)        
VIM-2 type MBL (7) 6 1 7 0
Total isolates (98)        
MBL (57) 54 3 56 1‡
N-MBL (41) 1 40 1 40
Sensitivity (%) 94.7 98.2
Specificity (%) 97.6 97.6

∗ Pseudomonas fluorescens (3), Achromobacter xylosoxidans (3) and Providencia rettgerii(1);

† The criterion of a ≥7 mm increase in inhibition zone size around the imipenem disk in DPA-containing MHA compared to MHA without DPA was used as a positive indication of MBL;

‡ Isolates co-producing MBL and OXA-51 type carbapenemase.

Abbreviations: DPA, dipicolinic acid; MHA, Mueller Hinton agar; MBL, metallo-β-lactamase; N-MBL, non-MBL.

Table 2.
Stability of dipicolinic acid activity in DPA 300-Mueller Hinton agar biplate during 12 weeks
Inhibition zone around IPM 10 μg disk (mm)
Storage (weeks) IMP-1 Acinetobacter spp.   VIM-2 P. aeruginosa   P. aeruginosa ATCC 27853  
  MHA DPA 300 μg MHA DPA 300 μg MHA DPA 300 μg
Immediate 13 28 (15)∗ 7 32 (25) 23 27 (4)
1 13 28 (15) 9 33 (24) 23 26 (3)
2 14 28 (14) 9 33 (24) 23 27 (4)
4 15 28 (13) 9 34 (25) 25 29 (4)
6 15 28 (13) 9 34 (25) 23 26 (3)
8 15 28 (13) 9 34 (25) 25 28 (3)
10 14 28 (14) 8 32 (24) 25 28 (3)
12 14 28 (14) 9 33 (24) 24 27 (3)

∗ The difference of inhibition zone size around IPM 10 μg disks between 300 μg/mL DPA-containing MH agar and MH agar without DPA.

Abbreviations: IPM, imipenem; MHA, Mueller Hinton agar; DPA, dipicolinic acid.

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