acm Annals of Clinical Microbiology Ann Clin Microbiol 2288-0585 2288-6850 The Korean Society of Clinical Microbiology 10.5145/ACM.2013.16.3.120 acm-16-120 Original Article Loss of blaVIM-2 and blaIMP-1 during the Storage of Gram-Negative Bacilli, Antimicrobial Susceptibility of the Gene-Lost Strain, and Location of the Gene in the Cell LimYoungsik 1 LeeYangsoon 1 2 SeoYounghee 1 YumJong Hwa 3 YongDongeun 1 LeeKyungwon 1 ChongYunsop 1 Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea Department of Laboratory Medicine, National Health Insurance Corporation Ilsan Hospital, Goyang, Korea Department of Clinical Laboratory Science, Dong-eui University, Busan, Korea Correspondence: Kyungwon Lee, Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea. (Tel) 82-2-2228-2446, (Fax) 82-2-313-0908, (E-mail) leekcp@yuhs.ac 122013 5122013 163 120125 20022013 8032013 8032013 Copyright © 2013 The Korean Society of Clinical Microbiology 2013 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. Background

Gram-negative bacilli can be stored in cystine tryptic agar (CTA) at room temperature for over 1 year, but we experienced a loss of imipenem resistance among VIM-2-producing isolates. The aims of this study were to determine the frequency of loss of IMP-1 and VIM-2 genes during storage in CTA at room temperature and to document any change in the MIC of antimicrobial agents and the location of the gene.

 Methods

Bacteria were isolated from clinical specimens at Severance Hospital collected from 1995-2000. Modified Hodge and double disk synergy tests were performed for screening of MBL-production isolates, and blaIMP-1 and blaVIM-2 were detected by PCR. Loss of resistance was tested in CTA at room temperature. PFGE and hybridization using a blaVIM-2 probe were carried out to determine the location of the VIM-2 gene.

 Results

When VIM-2- and IMP-1-producing strains of eight P. aeruginosa and two Acinetobacter spp. were stored in CTA at room temperature, some isolates lost imipenem resistance after 3 days and 90% lost resistance after 15 weeks. Loss of resistance genes resulted in a decrease of the MIC of imipenem from 32-128 μ g/mL to 0.5-8 μ g/mL for P. aeruginosa, and from 32 μ g/mL to 0.25-4 μ g/mL for Acinetobacter spp. Hybridization of I-Ceu I and S1-digested and PFGE suggested that VIM-2 genes are located on approximately 50-100 kb or 400 kb plasmids.

 Conclusion

Isolates may lose resistance genes when stored in CTA at room temperature. Therefore, it is necessary for MBL-production tests including the Modified Hodge test and double disk synergy test and detection of MBL genes.

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Pulsed field gel electropho-resis of whole genomic DNA of P. aeruginosa isolates digested with I- Ceu I (A), and S1 nuclease (D). Sou-thern blot hybridization with 16S rRNA gene probes (B) and bla VIM-2 gene probe (C and E). Lanes 1 to 8, whole genomic DNA from isolates no. 1-3, 6-9 and 12 P. aeruginosa; lane M, lambda ladder (Bio-Rad) as a marker (kb).

Detection of cells with partial or complete loss of MBL gene during storage in CTA tubes at room temperature∗

Organism (No. of isolates tested) MBL gene　 No. of isolates which lost MBL gene
Partial† Complete Total
P. aeruginosa (54) bla VIM-2 15 4 19 (35.1)
P. putida (10) bla VIM-2 1 0 1 (10.0)
Acinetobacter spp. (16) bla VIM-2 1 0 1 (6.3)
Acinetobacter spp. (4) bla IMP-1 1 0 1 (25.0)
Total (84) 　 18 4 22 (26.1)

Storage time was approximately 4-12 months depending on iso-late;

Isolates which lost partially MBL gene showed heterogeneous growth within the zone of inhibition in disk diffusion test using imipenem.

Proportion of cells which lost imipenem resistance during storage in CTA tubes at room temperature

Isolate no. Species MBL type Colonies (%) with loss of resistance at∗
3 days 1 week 2 week 4 week 15 week
1 P. aeruginosa VIM-2 3 6 7 11 28
2 P. aeruginosa VIM-2 0 0 2 9 9
3 P. aeruginosa VIM-2 0 0 1 15 92
6 P. aeruginosa VIM-2 5 18 6 15 20
7 P. aeruginosa VIM-2 1 11 5 7 19
8 P. aeruginosa VIM-2 0 0 0 1 2
9 P. aeruginosa VIM-2 0 0 0 0 1
12 P. aeruginosa VIM-2 0 0 0 0 0
13 A. baumannii VIM-2 1 3 3 5 33
5 A. baumannii IMP-1 0 8 5 8 55

One hundred colonies were tested for each isolate.

Comparison of MICs of antimicrobial agents for MBL-gene positive isolates and MBL-gene lost counterpart strains

Antimicrobial agents MBL gene MIC (μ g/mL) for:
P. aeru ACI
1 2 3 6 7 8 9 12 13 5
IMP Positive 64 128 128 32 128 32 32 32 32 32
Negative 4 8 0.5 0.5 0.5 1 1 2 4 0.25
MER Positive 16 ＞128 ＞128 8 ＞128 32 32 8 16 32
Negative 0.5 NT 8 1 8 1 0.5 0.5 4 0.5
PIP Positive 128 ＞256 256 ＞256 ＞256 ＞256 256 64 256 256
Negative 2 NT ＞256 256 ＞256 16 8 64 256 32
CAZ Positive ＞128 128 ＞128 128 128 ＞128 128 64 64 128
Negative 2 NT 4 4 4 4 2 16 16 4
CAZ-CLV Positive ＞128 128 ＞128 128 128 ＞128 128 64 64 128
Negative 2 NT 4 4 4 4 4 16 8 4
CTX Positive ＞128 ＞128 ＞128 ＞128 ＞128 ＞128 ＞128 ＞128 ＞128 ＞128
Negative 16 NT 32 64 32 32 16 64 16 16
FEP Positive 64 64 64 64 32 64 64 32 16 128
Negative 8 NT 8 16 16 16 16 16 8 8
AZT Positive 16 32 16 16 16 16 8 32 8 4
Negative 4 NT 16 16 16 16 8 32 16 4

Abbreviations: ACI, Acinetobacter spp.; IMP, imipenem; MER, meropenem; PIP, pipracillin; CAZ, ceftazidime; CAZ-CLV, ceftazidime-clavulanic acid; CTX, cefotaxim; FEP, cefepim; AZT, aztreonam; NT, not tested.