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Antimicrobial Agents and Chemotherapy, June 2001, p. 1939-1940, Vol. 45, No. 6
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.6.1939-1940.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

LETTERS TO THE EDITOR

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

	LETTER

IMP metallo--lactamases hydrolyze virtually all -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 bla_IMP-1 has not been confirmed outside Japan, but related enzymesIMP-2 and -4have 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 bla_IMP. 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 1) and had high-level resistance to all -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.

View this table: [in this window] [in a new window]   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 bla_IMP-1 1up (5'-GTGGGTCGATGTTTGATGTTAT-3'; positions 1400 to 1421) and bla_IMP-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 bla_IMP-1 2up (5'-CTTGATGAAGGCGTTTATGTT-3'; positions 1572 to 1592) and bla_IMP-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 bla_IMP-1 3up (5'-ACGGTAAGGTTCAAGCCACAA-3'; positions 1864 to 1884) and bla_IMP-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 bla_IMP-1 from S. marcescens (GenBank accession number S71932) (8), P. aeruginosa (AJ223604) (5), and K. pneumoniae (D29636). This is the first confirmation of a classical bla_IMP-1 outside Japan. The patient had no history of recent travel to Japan. Undetected importation by other patients or travellers is possible; alternatively, bla_IMP-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. 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 -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.

View larger version (49K): [in this window] [in a new window]   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; lane 6, DB96M. (The photograph has been cropped, so not all molecular weight markers can be seen.)

	ACKNOWLEDGMENTS

This work was supported by an MRCPath project grant from the British Society for Antimicrobial Chemotherapy.

We are grateful to Brigid Duke, Jeff Maskell, Mei Yuan, and David Griffiths for advice and assistance.

	FOOTNOTES

^* Phone: 65-321-4505 Fax: 65-222-6826 E-mail: gptthk{at}sgh.com.sg

	REFERENCES

1.	Chu, Y.-W., M. Afzal-Shah, E. T. S. Houang, M.-F. Palepou, D. J. Lyon, N. Woodford, and D. M. Livermore. 2001. IMP-4, a novel metallo--lactamase from nosocomial Acinetobacter spp. collected in Hong Kong between 1994 and 1998. Antimicrob. Agents Chemother. 45:710-714[Abstract/Free Full Text].
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6.	Livermore, D. M., and J. D. Williams. 1996. -Lactams: mode of action and mechanisms of bacterial resistance, p. 502-578. In V. Lorian (ed.), Antibiotics in laboratory medicine, 4th ed. Williams & Wilkins, Baltimore, Md.
7.	National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa.
8.	Osano, E., Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato. 1994. Molecular characterization of an enterobacterial metallo -lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance. Antimicrob. Agents Chemother. 38:71-78[Abstract/Free Full Text].
9.	Riccio, M. L., N. Franceschini, L. Boschi, B. Caravelli, G. Cornaglia, R. Fontana, G. Amicosante, and G. M. Rossolini. 2000. Characterization of the metallo--lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of blaIMP alleleic variants carried by gene cassettes of different phylogeny. Antimicrob. Agents Chemother. 44:1229-1235[Abstract/Free Full Text].

					Tse H. Koh* Li-Hwei Sng Singapore General Hospital Singapore 169608, Singapore
					Gioia S. Babini Neil Woodford David M. Livermore Central Public Health Laboratory London, United Kingdom
					Lucinda M. C. Hall St. Bartholomew's and the Royal London   School of Medicine and Dentistry London, United Kingdom

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