Ann Clin Microbiol.  2013 Sep;16(3):120-125. 10.5145/ACM.2013.16.3.120.

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

• Lim Y
• Lee Y
• Seo Y
• Yum JH
• Yong D
• Lee K
• Chong Y

• KMID: 1494328
• DOI: http://doi.org/10.5145/ACM.2013.16.3.120

Abstract

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 mug/mL to 0.5-8 mug/mL for P. aeruginosa, and from 32 mug/mL to 0.25-4 mug/mL for Acinetobacter spp. Hybridization of I-CeuI 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.

Keyword

Carbapenems; Metallo-beta-lactamase; Resistance loss

MeSH Terms

Acinetobacter
Agar
Anti-Infective Agents
Bacteria
Carbapenems
Chimera
Cystine
Imipenem
Mass Screening
Polymerase Chain Reaction
Sprains and Strains
Agar
Anti-Infective Agents
Carbapenems
Cystine
Imipenem

Figure

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  Fig. 1. 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).

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