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 IMP-6r 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℃ and 32 cycles of 10 seconds at 95℃, 30 seconds at 50℃, and 20 seconds at 72℃. DNA fragments were analyzed by electrophoresis. We used IMP-1-producing E. coli (NR2406) and IMP-6-producing E. coli (NR2407) from Japanese clinical isolates as positive controls. The bands, 120- or 350-bp in size, were obtained within 90 minutes or less (Fig. 1).
Fig. 1. Representative multiplex ARMS PCR patterns of IMP-1- and IMP-6-producing strains.
Abbreviations: ARMS, amplification refractory mutation system; Lane M, 100-bp DNA ladder marker.
We performed this method on 350 carbapenemase-producing clinical isolates collected from general hospitals across Japan from 2012 to 2016 and archived in our laboratory [4,8]. We also determined the antibiotic susceptibility of the isolates by the agar dilution method in accordance with CLSI recommendations [9]. The multiplex ARMS PCR results and imipenem and meropenem minimum inhibitory concentration (MIC) ranges of the isolates are shown in Table 1. The IMP-1 and IMP-6 producers yielded PCR products of the expected size. We confirmed the accuracy of this method by DNA sequencing of the PCR products (120-bp and 350-bp bands) of three representative strains. Cross-reactivity for other types of carbapenemase producers was not detected, and false-positive or false-negative results were not observed (Table 1).
Table 1. Characteristics of carbapenemase-producing strains used in this study and the multiplex ARMS PCR results.
| Species | Strains (N) | β-lactamase | MIC range (μg/mL) | Specific band* (N) | |
|---|---|---|---|---|---|
| IPM | MEPM | ||||
| Klebsiella pneumoniae | 31 | IMP-1 | 0.125–64 | 0.125–64 | 120 bp (31) |
| Enterobacter cloacae | 18 | IMP-1 | 0.125–4 | ≤ 0.06–4 | 120 bp (18) |
| Klebsiella oxytoca | 9 | IMP-1 | 0.125–1 | 0.125–2 | 120 bp (9) |
| Escherichia coli | 5 | IMP-1 | 0.125–1 | 0.5–2 | 120 bp (5) |
| Citrobacter freundii | 2 | IMP-1 | 0.25–1 | 0.125–0.5 | 120 bp (2) |
| Escherichia coli | 170 | IMP-6 | ≤ 0.06–1 | 0.125–32 | 350 bp (170) |
| Klebsiella pneumoniae | 87 | IMP-6 | ≤ 0.06–0.5 | 0.25–16 | 350 bp (87) |
| Enterobacter cloacae | 1 | IMP-6 | 0.125 | 0.25 | 350 bp (1) |
| Klebsiella pneumoniae | 12 | KPC | 2–32 | 2–64 | None |
| Escherichia coli | 12 | NDM | 1–16 | 0.5–32 | None |
| Klebsiella pneumoniae | 2 | OXA-48 like | 0.5–1 | 0.5 | None |
| Citrobacter freundii | 1 | VIM | 1 | 1 | None |
*120-bp band, IMP-1-specific band; 350-bp band, IMP-6-specific band.
Abbreviations: MIC, minimum inhibitory concentration; IPM, imipenem; MEPM, meropenem; ARMS, amplification refractory mutation system.
This multiplex ARMS PCR assay successfully discriminated between blaIMP-1 and blaIMP-6. Thus, this method could serve as a specific, rapid, and simple alternative for the detection of IMP-1 or IMP-6 producers and could facilitate clinical treatment, infection control, and large-scale IMP producer screening.
Acknowledgment
This study was supported by JSPS KAKENHI (Grant no. 17K16228 and 17K10027).
Footnotes
Authors' Disclosures of Potential Conflicts of Interest: No potential conflicts of interest relevant to this article were reported.
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