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IMP metallo-β-lactamases hydrolyze virtually all β-lactams and are insensitive to clinically available inhibitors. IMP-1 has been reported repeatedly since 1991 in Japan (8), where it is now scattered in Pseudomonas aeruginosa and Serratia marcescens and has been found in Klebsiella pneumoniae (H. Kurkawa, Y. Tetsuya, N. Shibata, K. Shibayama, and Y. Arakawa, Letter, Lancet 354:955, 1999). Carriage of blaIMP-1 has not been confirmed outside Japan, but related enzymes—IMP-2 and -4—have been found in acinetobacters in Italy (9) and Hong Kong (1); IMP-4 was also found in Citrobacter youngae in Guangzhou, China (2).  In 1999, we reported a carbapenem-resistant K. pneumoniae isolate (DB96) from blood cultures of a leukemic patient at Singapore General Hospital (T. H. Koh, G. S. Babini, N. Woodford, L.-H. Sng, L. M. C. Hall, and D. M. Livermore, Letter, Lancet 353:2162, 1999). The isolate had a pI 9.0 carbapenemase and gave a PCR product with primers to blaIMP. Carbapenemase production was conjugatively transmissible to Escherichia coli in association with a 150-kb plasmid. We now report the sequence for the carbapenemase gene and show that other factors codetermined imipenem resistance.  When isolate DB96 was examined with E-tests (AB Biodisk, Solna, Sweden), confluent growth occurred up to an imipenem concentration of 3 μg/ml, but isolated colonies grew to the maximum drug concentration on the strip (32 μg/ml). One highly resistant colony, designated DB96M, was retained and was homogeneously resistant on retesting. Another variant, DB96R, was obtained after repeated subculture and showed no growth at imipenem concentrations above 3 μg/ml. MICs were determined by NCCLS broth microdilution (7). Organisms DB96 and DB96M had identical resistance levels (±1 dilution) (Table ​(Table1)1) and had high-level resistance to all β-lactams including carbapenems; DB96R was less resistant only to carbapenems. All three variants retained the pI 9.0 carbapenemase, as detected by isoelectric focusing. Imipenemase specific activities were 0.69, 0.75, and 0.87 μmol of imipenem/min/mg of protein for DB96, DB96M, and DB96R, respectively, as determined by the method of Livermore and Williams (6); these values were not significantly different.    TABLE 1  MICs for K. pneumoniae isolates, as determined by NCCLS broth dilution    Using primers based on published sequences for P. aeruginosa 101/1477 (5), two amplicons were generated from DB96. First, primers blaIMP-1 1up (5′-GTGGGTCGATGTTTGATGTTAT-3′; positions 1400 to 1421) and blaIMP-1 6dn (5′-TGCGCGTTGTGGAATACTTTGC-3′; positions 2298 to 2319), which flank the open reading frame by ca. 60 bp upstream and 70 bp downstream, respectively, were used to generate an amplicon of approximately 1 kb. Secondly, primers blaIMP-1 2up (5′-CTTGATGAAGGCGTTTATGTT-3′; positions 1572 to 1592) and blaIMP-1 5dn (5′-TAACCGCCTGCTCTAATGTAAG-3′; positions 2160 to 2181), which were ca. 90 bp and 70 bp internal to the start and stop codons, respectively, were used to generate an amplicon of ca. 600 bp internal to the 1-kb amplicon. Both amplicons were then sequenced using primers blaIMP-1 3up (5′-ACGGTAAGGTTCAAGCCACAA-3′; positions 1864 to 1884) and blaIMP-1 4dn (5′-TTTCAGGCAACCAAACCACTA-3′; positions 1969 to 1989), which were central to the open reading frame and original primers. The aligned sequence was submitted to BLAST 2.0 and found to be identical to blaIMP-1 from S. marcescens (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"S71932","term_id":"560551"}}S71932) (8), P. aeruginosa ({"type":"entrez-nucleotide","attrs":{"text":"AJ223604","term_id":"4210822"}}AJ223604) (5), and K. pneumoniae ({"type":"entrez-nucleotide","attrs":{"text":"D29636","term_id":"473725"}}D29636). This is the first confirmation of a classical blaIMP-1 outside Japan. The patient had no history of recent travel to Japan. Undetected importation by other patients or travellers is possible; alternatively, blaIMP-1 may have escaped to plasmids independently in Singapore.  Outer membrane proteins (OMPs) were extracted (6) and electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4). Organisms DB96 and DB96M showed greatly diminished expression of a major 39-kDa OMP compared with DB96R and with carbapenem-susceptible Klebsiella controls (Fig. ​(Fig.1).1). DB96, DB96M, and DB96R all lacked the minor 41-kDa OMP present in the controls. It seems likely, from its mass, that the 39-kDa OMP corresponds to a major porin (3) and that high-level resistance to carbapenems demands impermeability as well as an IMP β-lactamase. This conclusion is supported by the low imipenem MICs (2 μg/ml) for IMP-1-positive E. coli transconjugants of strain DB96 (Koh et al., letter, 1999). Because IMP-1 alone does not confer high-level carbapenem resistance in Enterobacteriaceae, it might spread without attracting attention, and microbiologists should be aware that gram-negative bacteria with borderline susceptibility to carbapenems could be IMP producers. Suspicious isolates should have carbapenem MICs checked and be examined for carbapenemase activity.    FIG. 1  Outer membrane profiles of K. pneumoniae isolates in SDS-PAGE. Lanes 1 and 7, molecular weight markers (in kilodaltons); lane 2, carbapenem-susceptible control isolate 207; lane 3, carbapenem-susceptible control isolate 504; lane 4, DB96R; lane 5, DB96; ...

Carbapenem-Resistant Klebsiella pneumoniae in Singapore Producing IMP-1 β-Lactamase and Lacking an Outer Membrane Protein