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Antimicrobial Agents and Chemotherapy, February 2004, p. 626-628, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.626-628.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Class 1 Integron Containing Metallo-ß-Lactamase Gene blaVIM-2 in Pseudomonas aeruginosa Clinical Strains Isolated in Japan

Jun Yatsuyanagi,1* Shioko Saito,1 Seizaburo Harata,1 Noriyuki Suzuki,1 Yuko Ito,2 Ken-ichi Amano,3 and Katsuhiko Enomoto4

Akita Prefectural Institute of Public Health, 6-6 Sensyu kubota-machi, Akita 010-0874,1 Akita Kumiai General Hospital, 273-1 Iijima aza nishibukuro, Akita 011-0911,2 Central Research Laboratory,3 Department of Pathology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan4

Received 8 July 2003/ Returned for modification 2 October 2003/ Accepted 22 October 2003


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ABSTRACT
 
Four blaVIM-2 gene-harboring Pseudomonas aeruginosa strains were identified. These strains possessed a class 1 integron harboring ORF1, blaVIM-2, and aacA4 gene cassettes. The transposon-mediated horizontal spread of the blaVIM-2 gene among these strains was suggested, which increases the threat that the blaVIM-2 gene will disseminate among diverse genera of bacteria.


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INTRODUCTION
 
The emergence of metallo-ß-lactamase (MBL)-producing bacilli that are resistant to carbapenems is becoming a severe therapeutic problem (9). Two types of MBLs, IMP and VIM, have been reported (13). IMP-1 was identified in Pseudomonas aeruginosa in Japan in 1991 (15). Strains producing IMP-type MBLs have also been reported in Hong Kong (3), Taiwan (17), and Italy (12). Strains producing VIM-type MBLs were originally reported in European countries. VIM-1 was identified in P. aeruginosa in Italy in 1999 (7), and VIM-2 was identified in France (10). Thereafter, VIM-3 was identified in Taiwan (16). However, there have been few reports describing VIM-type-MBL-producing bacteria in Japan. The genes of both IMP- and VIM-type MBLs (blaIMP and blaVIM, respectively) are often encoded on mobile gene cassettes inserted into class 1 integrons (1, 7, 10). The class 1 integrons are genetic elements capable of integrating gene cassettes by a site-specific recombination mechanism (4). Gene cassettes are mobile units composed of a gene, most often an antibiotic resistance gene, and a recombination site, the 59-base element (4). Integrons are sometimes found as a part of transposons (4), which is probably the reason that they are found in many different genetic locations. In this work, we report on the characterization of the blaVIM-2 gene cassette-harboring class 1 integron identified in the P. aeruginosa clinical strains isolated in one hospital in Akita Prefecture, Japan.

Clinical isolates were screened for MBL production by a disk diffusion test (2) modified for use with disks containing 3 mg of sodium mercaptoacetate. An increase of more than 5 mm in the diameter of the inhibition zone around the ceftazidime disk (30 µg) in the presence of the sodium mercaptoacetate disk indicated a screening test positive for MBL production. The blaIMP and blaVIM genes were detected by PCR by using the consensus primer pairs IMP S and IMP AS for the blaIMP genes and VIM S and VIM AS for the blaVIM genes (Table 1). The blaVIM gene was typed by direct sequencing by using the VIMseq S and VIMseq AS primers (Table 1). Four blaVIM-2 gene-positive P. aeruginosa strains, Mß-2, Mß-6, Mß-7, and Mß-9, were employed in this study. Two fragments, INT5CS-VIM AS and VIM S-INT3CS, were amplified from strain Mß-7 by PCR with two primer sets, INT5/CSBH and VIM AS and VIM S and INT3/CSJY2ER, and sequenced by using the primers listed in Table 1, as described previously (18). The annealing sites of these primers are shown schematically in Fig. 1. Pulsed-field gel electrophoresis (PFGE) was performed as described by Speijer et al. (13) by using SpeI. The chromosomal DNA fragments were analyzed by Southern blot hybridization as described previously (18) by using the blaVIM-2 DNA probe, which was prepared by PCR by using the VIMseq S and VIMseq AS primers (Table 1). INT5CS-VIM AS and VIM S-INT3CS fragments amplified from strains Mß-2, Mß-6, and Mß-9 were analyzed by Southern blot hybridization for the presence of the ORF1, blaVIM-2, and aacA4 genes. The ORF1 DNA probe and the aacA4 DNA probe were prepared by PCR by using the ORF1 S and ORF1 AS primers and the AACA4 S and AACA4 AS primers (Table 1), respectively.


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  		TABLE 1. Primers used for detection of MBL genes and genes comprising the blaVIM-2 gene-containing integron and for sequencing of the blaVIM-2 gene containing integron



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  		FIG. 1. Schematic structure (not to scale) of the approximately 3-kb integron of P. aeruginosa strain Mß-7, containing the blaVIM-2 gene cassette. The gene cassettes are boxed. Arrows indicate transcriptional orientation. The attI1 recombination site is represented by a white circle, and the 59-base elements are indicated by black circles. Primers used for sequencing are shown under the integron structure.

From September 2001 to October 2002, 16 isolates tested positive for MBL production by the disk diffusion screening test. Five strains, Mß-3, Mß-4, Mß-5, Mß-8, and Mß-12, were positive for the blaIMP gene, and four P. aeruginosa isolates, Mß-2, Mß-6, Mß-7, and Mß-9, were positive for the blaVIM-2 gene. Sequence analysis of the integron from strain Mß-7 revealed a class 1 integron structure. As shown in Fig. 1, this class 1 integron contained three gene cassettes. The first cassette contained a 399-bp open reading frame of unknown function, ORF1. The second cassette contained the blaVIM-2 gene, and the third cassette contained the aacA4 gene, which encodes aminoglycoside acetyltransferase. Southern blot hybridization analysis of the INT5CS-VIM AS and VIM S-INT3CS fragments amplified from strains Mß-2, -6, and -9 revealed that all of these strains also contained the integron harboring the ORF1, blaVIM-2, and aacA4 genes (data not shown). SpeI PFGE patterns for the four isolates differed only within three bands, indicating that these isolates are closely related (Fig. 2A). The Southern blot analysis of the SpeI-digested chromosomal PFGE fragments revealed that the blaVIM-2 gene is located on an approximately 60-kb fragment in Mß-2, a 280-kb fragment in Mß-7, and a 240-kb fragment in Mß-9 but on no fragment in Mß-6 (Fig. 2B).



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  		FIG. 2. Southern hybridization analysis of the SpeI-digested chromosomal DNA fragments from Mß-2, -6, -7, and -9. PFGE patterns (A) and results of Southern hybridization using a blaVIM-2 DNA probe (B) are shown. Lanes: M, lambda molecular weight ladder; 2, Mß-2; 6, Mß-6; 7, Mß-7; 9, Mß-9.

We have shown in this study that P. aeruginosa strains harboring the blaVIM-2 gene have been disseminated in one hospital in Akita Prefecture, Japan, confirming that blaVIM-2 gene-harboring strains are now widespread in eastern Asian countries. Earlier studies reported that blaVIM-1 is located on the chromosome of P. aeruginosa strain VR-143/97 (7) but that blaVIM-2 is located on an approximately 45-kb plasmid (10) or on the fragments of XbaI-digested genomic DNA (8). In this study, the blaVIM-2 gene was found in various genetic locations, which suggests the horizontal spread of the blaVIM-2 gene among these four P. aeruginosa strains. The precise mechanism by which the blaVIM-2 gene achieved a horizontal spread among these four strains is unclear. Our results demonstrate that the four blaVIM-2 gene-positive P. aeruginosa isolates harbored integrons of the same size and containing three genes, ORF1, blaVIM-2, and aacA4, which suggests the horizontal spread of the integron itself, rather than of the blaVIM-2 gene-containing gene cassette among different integrons. Although integrons themselves are not mobile, several class 1 integrons have been found in Tn21 and Tn21-related transposons (4, 5, 14), which enables the integrons to be transposed. These findings raise the possibility that the class 1 integron described in this study is also part of a transposon. The structures of several blaVIM-2 gene-containing integrons (6, 8, 10, 11), including the integron identified in strain Mß-7, are unique, indicating that the blaVIM-2 gene cassette has disseminated among various integrons worldwide. Moreover, our present results suggest the possibility that the blaVIM-2 gene cassette-harboring integron is associated with a transposon, which increases the threat that the blaVIM-2 gene will disseminate among diverse genera of bacteria.


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Nucleotide sequence accession numbers.
 
Sequences for the blaVIM-2 genes from isolates Mß-2, Mß-6, Mß-7, and Mß-9 were submitted to GenBank under accession no. AY242981 to AY242984. The sequence for the integron from strain Mß-7 was submitted under accession no. AY294333.


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FOOTNOTES
 
* Corresponding author. Mailing address: Akita Prefectural Institute of Public Health, 6-6 Sensyu kubota-machi, Akita 010-0874, Japan. Phone: 81-18-832-5005. Fax: 81-18-832-5938. E-mail: jyatsu{at}spica.freemail.ne.jp. Back


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Antimicrobial Agents and Chemotherapy, February 2004, p. 626-628, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.626-628.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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