IMP-1 and a Novel Metallo-β-Lactamase, VIM-6, in Fluorescent Pseudomonads Isolated in Singapore

Abstract

Four carbapenem-resistant Pseudomonas spp. were isolated from patients in Singapore. One Pseudomonas putida isolate contained a bla_IMP-1 identical to that first described in Japan. The sequence of a variant bla_IMP-1 in Pseudomonas fluorescens contained four silent mutations compared with the original sequence. The remaining P. putida isolates contained bla_VIM-6, a novel VIM gene variant.

The Ambler class B metallo-β-lactamases are able to hydrolyze all β-lactam antimicrobials except aztreonam. These enzymes are intrinsic to certain rare pathogens like Stenotrophomonas maltophilia (1) and Chryseobacterium spp. (13). However, acquired class B enzymes of the IMP and VIM types pose a greater threat because they can be transmitted between different species, including common nosocomial pathogens like Pseudomonas aeruginosa.

We have previously described IMP-1 in a single clinical isolate of Klebsiella pneumoniae in Singapore (T. H. Koh, L. H. Sng, G. S. Babini, N. Woodford, D. M. Livermore, and L. M. Hall, Letter, Antimicrob. Agents Chemother. 45:1939-1940, 2001). Since then, we have not detected any more carbapenemase-producing Klebsiella isolates. We therefore undertook a study to see if metallo-β-lactamase genes could be found in other gram-negative bacilli in Singapore. Pseudomonas putida DB33824/00, DU25151/00, and DU25165/00 and Pseudomonas fluorescens DU6027/00 were collected and identified retrospectively during a survey of carbapenem-resistant Pseudomonas aeruginosa strains isolated from patients in a large tertiary-care hospital in 2000.

The MICs of six antipseudomonal β-lactams were determined by the E-test (AB Biodisk, Solna, Sweden) method (Table 1).

TABLE 1.

Characteristics of carbapenem-resistant Pseudomonas spp.

Strain no.	Species	Source	β-Lactamase pI	MIC (μg/ml)a						Presence of:
				IPM	MEM	ATM	CAZ	FEP	TZP	blaIMP-1	blaVIM-6
DU6027/00	P. fluorescens	Urine	7.8, 8.8	>32	>32	32	>256	>256	>256	+	−
DB33824/00	P. putida	Blood	6.2, 8.6	>32	>32	64	>256	>256	>256	+	−
DU25151/00	P. putida	Urine	4.8, 5.6, 6.2, 6.9	>32	>32	128	>256	>256	>256	−	+
DU25165/00	P. putida	Urine	4.8, 5.8, 6.3, 7.0	>32	>32	128	>256	>256	>256	−	+

^aIPM, imipenem; MEM, meropenem; ATM, aztreonam, CAZ, ceftazidime, FEP, cefepime; TZP, piperacillin-tazobactam.

Isoelectric focusing was performed with crude cell extracts. Strains DB33824/00 and DU6027/00 each had a β-lactamase with an isoelectric point (pI) of approximately 9.0, in keeping with an IMP-type enzyme. This band was not present in extracts from DU25151/00 and DU25165/00, which instead had β-lactamases with pIs of approximately 5.0, in keeping with a VIM-type enzyme.

The IMP and VIM genes were amplified from the appropriate strains as described by Yan et al. (15) and sequenced.

On the basis of the translated amino acid sequence, 13 IMP-type metallo-β-lactamases and 5 VIM-type metallo-β-lactamases have been described so far. As far as we are aware, classic IMP-1, the first acquired metallo-β-lactamase, has been described only in Japan, the United Kingdom, and Singapore. P. putida DB33824/00 contained a bla_IMP-1 identical to that first described in Japan (9) and previously described in K. pneumoniae from Singapore. On the other hand, the variant bla_IMP-1 in P. fluorescens DU6027/00 contained four silent mutations at nucleotide positions 189 (C to T), 273 (C to T), 496 (T to C), and 702 (G to A) compared with the nucleotides in the most closely matching bla_IMP-1 sequence in the GenBank database (GenBank accession number S71932). Surprisingly, the closest DNA sequence match was with bla_IMP-3 from Shigella flexneri from Japan (accession number AB010417), which shared the same bases at nucleotide positions 189, 273, 496, and 702 but which differed by three bases at other positions (4). Because IMP-3 has a lower level of hydrolyzing activity for penicillins and carbapenems than IMP-1, it has been suggested that bla_IMP-3 may in fact be an ancestor of bla_IMP-1. If so, the sequence of variant bla_IMP-1 in Singapore may represent an intermediate in the evolution of the original bla_IMP-1 from bla_IMP-3. The gene for IMP-1 from Acinetobacter junii in the United Kingdom also contained seven silent mutations compared with the original bla_IMP-1 sequence (14). This suggests that bla_IMP-1 in these countries may have evolved separately from local environmental sources rather than through international spread. Despite the apparent similarities in their DNA sequences, the sequence of IMP-3 differs from that of IMP-1 by two amino acids.

The complete VIM genes from P. putida DU25165/00 and DU25151/00 were sequenced, and their sequences were found to differ from that of VIM-2 at nucleotide positions 179 (A to G) and 443 (A to G) and from that of VIM-3 at nucleotide positions 178 (A to C) and 179 (A to G). By following the BBL numbering scheme (3), this novel enzyme, now named VIM-6, differs from VIM-3 at amino acid 59 (K to R) and from VIM-2 at amino acids 59 (Q to R) and 165 (N to S).

The genomic DNAs of the isolates were digested with the restriction endonuclease SpeI (New England Biolabs, Beverly, Mass.), and pulsed-field gel electrophoresis (PFGE) was performed as described previously (7). P. putida DU25151/00 and DU25165/00 had identical PFGE patterns (data not shown) which differed from that of P. putida DB33824/00 by more than seven bands. They were isolated from two patients who had been in adjacent beds in the same ward. The second patient was admitted a day after the discharge of the first patient, and strain DU25165/00 was isolated from her on the day of admission, suggesting a common environmental source rather than cross infection.

PCR for the detection of class I integron sequences was performed with the primers described by Lombardi et al. (8). PCR products of approximately 1,500 and 1,300 bp were obtained for P. fluorescens DU6027/00 but not for the other strains. The gel extract of the 1,500-bp product was positive for bla_IMP by PCR, whereas that of the 1,300-bp product was negative, showing that bla_IMP-1 resides on the larger integron.

Plasmids were extracted from all four strains by using Wizard Plus SV minipreps (Promega, Madison, Wis.). DNA probes were synthesized by using the PCR DIG Probe Synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany) and primers specific for bla_IMP-1 and bla_VIM-2, as described by Yan et al. (15). DNA extracts from P. putida DB33824/00 and DU25151/00 served as templates.

Southern blot analysis of plasmid DNA was carried out by the methods and with the reagents described in the DIG Application Manual for Filter Hybridization (Roche). The bla_VIM-6 probe hybridized to plasmid extracts from P. putida DU25165/00 and DU25151/00, and the bla_IMP-1 probe hybridized to the plasmid extract from P. fluorescens DU6027/00 (data not shown).

We were unable to obtain transconjugants in broth mating experiments (12) using P. putida DU25165/00 and P. fluorescens DU6027/00 as donors and One Shot TOP10 Electrocompetent Escherichia coli (Invitrogen, Singapore, Singapore) as the recipient.

Since VIM-1 and VIM-2 were first described in Italy (5) and France (10), respectively, it was initially thought that VIM-type enzymes were to be found mainly in southern Europe. VIM-type enzymes have since been found in Greece (11) and in Turkey and Portugal (2), including the new variants VIM-4 and VIM-5 (GenBank accession number AY144612). The other focus for VIM-type enzymes appears to be the Far East, notably, VIM-2 in Korea (6) and VIM-3 in Taiwan (15). VIM-2, VIM-3, and VIM-6 appear to form a cluster (with at least 99.2% amino acid identity within the cluster) whose amino acid sequences differ from those of the other VIM enzymes by about 10% (Fig. 1).

FIG. 1.

Comparison of the amino acid sequences of VIM β-lactamases. Amino acids which distinguish the VIM-2 cluster are highlighted. (asterisks, identical residues; colons, conserved substitutions; dots, semiconserved substitutions.

P. putida and P. fluorescens belong to the fluorescent group of pseudomonads. These bacteria are of low virulence and are usually not clinically significant. All four isolates in this study were not thought to be causing true infections, and none of the patients from whom these isolates were recovered received antimicrobial therapy specifically targeting these organisms. One patient was receiving imipenem at the time of specimen collection, whereas the other three patients had not received any antimicrobial chemotherapy.

Although P. putida and P. fluorescens are uncommonly isolated from clinical specimens, they are likely to be widespread in the hospital environment and may represent a reservoir of resistance determinants.

Nucleotide sequence accession numbers.

The sequences of the following strains were submitted to GenBank: P. putida DU25165/00 bla_VIM-6 (accession number AY165025), P. putida DB33824/00 bla_IMP-1 (accession number AY251052), and P. fluorescens DU6027/00 bla_IMP-1 (accession number AY250709).