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Dear Editor, Carbapenem-resistant Enterobacteriaceae have acquired carbapenemase genes [1], which differ substantially across countries [2]. Transferable carbapenemase IMP-type metallo-β-lactamases, particularly IMP-1 and IMP-6, are commonly identified in the clinical setting in Japan [3, 4] and exhibit different substrate specificity despite having a difference of only one amino acid (IMP-6: Ser214Gly). IMP-1 producers are more resistant to imipenem than to meropenem, whereas IMP-6 producers are more resistant to meropenem [5]. We previously found that the susceptibility rate of IMP-6-positive Escherichia coli was higher for imipenem than for meropenem [3]. Thus, IMP-6-producing isolates may be erroneously categorized as imipenem-susceptible, which could lead to treatment failure in patients. It is important to distinguish between the genes blaIMP-1 (encoding IMP-1) and blaIMP-6 (encoding IMP-6) because of the differences in substrate specificity; these variants can be identified by either DNA sequencing or amplification refractory mutation system (ARMS) PCR [6]. However, these methods are costly and time-consuming, and ARMS PCR requires multiple reaction tubes per sample. Therefore, we developed a simple, rapid multiplex ARMS PCR assay to discriminate between IMP-1 and IMP-6. We designed two sets of Multiplex ARMS PCR primers based on the nucleotide sequences of blaIMP-1 and blaIMP-6. Primers IMP-f and IMP-r were designed to recognize the conserved sequences of blaIMP-1 and blaIMP-6, while primers IMP-1f and IMP6r were designed to recognize specific nucleotide sequences of blaIMP-1 and blaIMP-6, respectively [7]. PCR amplification using the primer pair IMP-1f (5 ́-AAGGCAAAACTGGTTGTTCCTA-3 ́) and IMP-r (5 ́-CGACTTGTTAGAAATTTAGTTGC-3 ́) results in a 120-bp blaIMP-1 gene-specific fragment, and PCR amplification using the primer pair IMP-f (5 ́-CTCGATCTATCCCCACGTATG-3 ́) and IMP-6r (5 ́-TCGTCTCCAACTTCACTGTGAGC-3 ́) generates a 350-bp blaIMP-6 gene-specific fragment (underlined sequences indicate nucleotide alternations for allele-specific primers). The 3 ́ termini of the primers correspond to the specific nucleotide sequence at position 640 of IMP-1 (AGT, Ser) and IMP-6 (GGT, Gly), respectively. We performed multiplex ARMS PCR amplification using the Qiagen Multiplex PCR Master Mix (Qiagen, Venlo, The Netherlands) with the following conditions: denaturation for 15 minutes at 95°C and 32 cycles of 10 seconds at 95°C, 30 seconds at 50°C, and 20 seconds at 72°C. DNA fragments were analyzed by electrophoresis. We used IMP-1-producing E. coli (NR2406) and IMP6-producing E. coli (NR2407) from Japanese clinical isolates as positive controls. The bands, 120or 350-bp in size, were obtained

Rapid Identification of blaIMP-1 and blaIMP-6 by Multiplex Amplification Refractory Mutation System PCR

Rapid Identification of bla_IMP-1 and bla_IMP-6 by Multiplex Amplification Refractory Mutation System PCR