Jpn. J. Infect. Dis., 57, 130-132, 2004

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Laboratory and Epidemiology Communication

Identification of Pseudomonas aeruginosa Clinical Strains Harboring the blaVIM-2 Metallo-b-Lactamase Gene in Akita Prefecture, Japan

Jun Yatsuyanagi1*, Shioko Saito1, Yuko Ito2, Kazuko Ohta3, Jun Kato4, Seizaburo Harata1, Noriyuki Suzuki1 and Ken-ichi Amano1,5

1Akita Prefectural Institute of Public Health, Akita 010-0874, 2Akita Kumiai General Hospital, Akita 011-0911, 3Odate Municipal Hospital, Oodate 027-0885, 4Yuri Kumiai General Hospital, Akita 015-8511 4 and 5Central Research Laboratory, Akita University School of Medicine, Akita 010-8543

Communicated by Haruo Watanabe

Accepted June 1, 2004

*Corresponding author: Mailing address: Akita Prefectural Institute of Public Health, 6-6 Sensyu kubota-machi, Akita 010-0874, Japan. Tel: +81-18-832-5005, Fax: +81-18-832-5938, E-mail: jyatsu@spica.freemail.ne.jp

Gram-negative bacilli producing metallo-b-lactamases (MBLs) show resistance to carbapenems, such as imipenem, which are often used to treat infections caused by Gram-negative bacteria that are resistant to other b-lactam antibiotics. Therefore, the emergence of MBL-producing bacteria is becoming a severe therapeutic problem. Two carbapenem-hydrolyzing MBLs, IMP and VIM, have been reported (1). An IMP-type MBL, IMP-1, was identified in Pseudomonas aeruginosa in Japan (2) and variants of the IMP-type MBL have subsequently been identified in Asian countries (3,4), and in European countries (5). Strains producing the VIM-type MBLs, on the other hand, were originally identified in European countries (6,7). Thereafter, VIM-3, an MBL closely related to VIM-2, was identified in Taiwan (8), indicating that the strains producing VIM-type MBLs have disseminated in Europe and Asia, but there have been few reports describing VIM-type MBL-producing bacteria in Japan. We have previously reported that four P. aeruginosa clinical strains isolated in one hospital in Akita Prefecture, Japan, harbored a class 1 integron containing ORF1, a gene of unknown function, blaVIM-2, and aacA4 gene cassettes, suggesting that the class 1 integron spread horizontally among these four strains (9). In this report, we describe the antimicrobial resistance and pulsed-field gel electrophoresis (PFGE) patterns of blaVIM-2 gene-positive P. aeruginosa clinical strains isolated in Akita.

Gram-negative clinical isolates were screened for MBL production by a disk-diffusion test using commercially available disks containing sodium mercaptoacetate, as described previously (9). The presence of blaIMP or blaVIM genes was confirmed by PCR in the screening-positive isolates using consensus primer pairs for blaIMP (IMP S: 5'-AAA GAT ACT GAA AAG TTA GT-3' and IMP AS: 5'-TCY CCA AYT TCA CTR TGA CT-3', amplicon size: 446 bp), and for blaVIM, (VIM S: 5'-CCG ATG GTG TTT GGT CGC AT-3', and VIM AS: 5'-GAA TGC GCA GCA CCA GGA-T-3', amplicon size: 391 bp), respectively, as described previously (9). The VIM gene-type was determined by direct sequencing as described previously (9), or by PCR using blaVIM type-specific primer pairs for blaVIM-1 (VIM-1A: 5'-TAG TAG TTT ATT GGT CTA CA-3' and VIM-1B: 5'-TGT GCT TTG ACA ACG TTC GC-3', amplicon size: 762 bp), blaVIM-2 (VIM-2A: 5'-ATG TTC AAA CTT TTG AGT AAG-3' and VIM-2B: 5'-CTC AAC GAC TGA GCG ATT TG-3', amplicon size: 798 bp), and blaVIM-3 (VIM-3S: 5'-TTG GTC GCA TAT CGC AAC GA-3' and VIM-3AS: 5'-AGA GTG CGT GGG AAT CTC GC-3', amplicon size: 304 bp), respectively. The minimum inhibitory concentrations (MICs) of the isolates were determined by the broth microdilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (10,11) using a commercially available plate, Dryplate 'Eiken' DP-25 (Eiken Kagaku Co., Tokyo, Japan). PFGE was performed using SpeI as described previously (9).

From September 2001 to October 2003, 42 isolates were shown to be positive for MBL production using the disk diffusion-screening test in the clinical laboratories of several hospitals in Akita. Among the 42 isolates, PCR using the consensus primer pairs revealed eight strains positive for the blaVIM gene and 24 strains positive for the blaIMP gene (data not shown). The remaining ten strains were negative for both blaIMP and blaVIM, and the mechanism involved in the resistance of the ten strains has not been further examined. The blaVIM gene detected in the eight strains was blaVIM-2 (data not shown). Isolation date, source, and the antibiotic susceptibility patterns of the eight blaVIM-2 gene-harboring strains are summarized in Table 1. These eight strains were isolated in one hospital. Strains Mb-20 and Mb-40 were isolated from the same patient. All these strains were sensitive to piperacillin, ciprofloxacin, and tosufloxacin. Strains except Mb-33 and Mb-35 were resistant to imipenem and meropenem according to the NCCLS resistance breakpoint of >/=16 mg/ml. Strains Mb-2, Mb-6, Mb-7, and Mb-9 which were previously shown to harbor the aacA4 gene (9) were resistant to gentamicin. Only strain Mb-9 was resistant to ceftazidime. The SpeI PFGE patterns of the eight strains are shown in Fig. 1. Strains Mb-2, Mb-6, Mb-7, and Mb-9 showed similar PFGE patterns with only a few band differences, indicating that they belong to a closely related clone, as described previously (9). Strains Mb-20 and Mb-40 which were isolated from identical patient showed quite similar PFGE patterns with a minor one band difference, and the pattern was markedly different from those of strains Mb-2, Mb-6, Mb-7, and Mb-9. The PFGE patterns of strains Mb-33 and Mb-35 were different from those of other strains, indicating that four independent clones of blaVIM-2 gene-harboring P. aeruginosa, Mb-2 to Mb-9, Mb-20 and Mb-40, Mb-33, and Mb-35, have emerged in one hospital in Akita.

VIM-type MBL-producing strains were first reported only in European countries (6,7). Recently, Yan et al. isolated P. aeruginosa strain-producing the VIM-3 type MBL, a novel variant of the VIM-2 type, in Taiwan (8) and pointed out that the VIM-2-related MBLs are the most prevalent MBLs in Taiwan. VIM-2-producing P. aeruginosa, Paeudomonas putida (12), Serratia marcescens (13), and Acinetobacter baumannii (14) have also been reported in Korea. We have previously shown that P. aeruginosa strains that harbor the blaVIM-2 gene were also present in Akita (9), and VIM-2-producing P. aeruginosa strains have been increasingly isolated in hospitals in Japan, even though there have been few reports. Our present results provide molecular epidemiological evidence that blaVIM-2 gene-harboring P. aeruginosa strains belonging to independent clones have emerged in one hospital in Akita. Strains Mb-33 and Mb-35 were imipenem- and meropenem-sensitive, even though they were positive for the blaVIM-2 gene. The reason for this discrepancy is unclear, but low VIM-2 enzyme expression or altered membrane permeability of these strains against imipenem and meropenem is a possible candidate for this mechanism, which should be further elucidated.

Bacterial strains harboring the blaVIM-2 gene are a severe therapeutic problem, but their emergence in Japan still has not been well demonstrated. Our present results along with previous ones pose the possibility that these strains have already emerged in Japan as in the other Asian countries, and that this possibility should be further elucidated.

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