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 bla_IMP-1 and bla_VIM-2 were detected by PCR. Loss of resistance was tested in CTA at room temperature. PFGE and hybridization using a bla_VIM-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 μg/mL to 0.5-8 μg/mL for P. aeruginosa, and from 32 μg/mL to 0.25-4 μg/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. (Ann Clin Microbiol 2013;16: 120-125)