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Integrons Containing the VIM-2 Metallo--Lactamase Gene among Imipenem-
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     Infectious Disease Department, The 1st Affiliated Hospital, Medical School, Zhejiang University, The Key Laboratory of the Infectious Diseases of Public Health Ministry, Hangzhou, Zhejiang, China

    Respiratory Department, The 1st Affiliated Hospital, Medical School, Zhejiang University, Hangzhou, Zhejiang, China

    Department of Pathology and Microbiology, University of Bristol, Bristol, United Kingdom

    ABSTRACT

    A total of 140 nonrepetitive strains of imipenem-resistant Pseudomonas aeruginosa were isolated from five different Chinese hospitals. Fourteen isolates were confirmed to contain the VIM-2 metallo--lactamase gene. Twelve isolates harbored two kinds of class 1 integron, containing both VIM-2- and aminoglycoside-resistant genes.

    TEXT

    The emergence of metallo--lactamase (MBL)-producing pathogens is an increasing therapeutic problem. Four types of MBLs, IMP, VIM, SPM-1, and GIM-1, have been reported in Pseudomonas aeruginosa, which is a leading cause of nosocomial infections (3, 11, 29). A new type of MBL, SIM-1, was identified in Acinetobacter baumannii isolated in Korea (15). Strains producing VIM-type MBLs were originally reported in European countries (11, 17). VIM-type enzymes also appear in the Far East, such as VIM-2 in Japan and Korea and VIM-3 in Taiwan (13, 31, 34). In recent years, P. aeruginosa-possessing MBLs have constituted nearly 20% of nosocomial isolates in some countries, whereas in other countries, the number is still comparatively small (5, 12). In P. aeruginosa, blaVIM alleles were found to be carried on mobile elements known as gene cassettes but are inserted into class 1 integrons (16, 20, 21, 23, 24). Integron-located resistance genes provide them with an increased potential for expression and dissemination. In this report, we study the characteristics of the blaVIM-2 gene cassette-harboring class 1 integrons identified in P. aeruginosa clinical strains isolated from hospitals in different geographical areas of China.

    A total of 140 nonrepetitive, imipenem-resistant Pseudomonas aeruginosa (IRPA) isolates, including 27 strains from a big teaching hospital in Beijing, 32 strains from Shanghai, 22 strains from Chengdu, 32 strains from Guangzhou, and 27 strains from Hangzhou, were selected by a disk diffusion method from hospitals in five different regions throughout China from July 2004 to December 2005 (Fig. 1). The disk diffusion method was carried out according to standards of the Clinical Laboratory Standards Institute (3a).

    The MICs of piperacillin-tazobactam and cefoperazone/sulbactam were determined by Etest (AB Biodisk, Solna, Sweden), and the susceptibilities to other antimicrobial agents were tested by an agar dilution method including aztreonam (Sigma, Deisenhofen, Germany), ceftazidime (Sigma, Deisenhofen, Germany), cefepime (Sino-American Shanghai Squibb Pharmaceuticals, Ltd., Shanghai, China), ciprofloxacin (Bayer, Leverkusen, Germany), amikacin (Sigma, Deisenhofen, Germany), netilmicin (Sigma, Deisenhofen, Germany), imipenem (Merck KGaA, Darmstadt, Germany), and meropenem (Sumitomo Pharmaceuticals Co., Ltd., Osaka, Japan) according to reference 3a. P. aeruginosa ATCC 27853 was used as a control. The susceptibilities of the 140 IRPA isolates are shown in Table 1. The meropenem resistance rates for IRPA isolates from Beijing and Shanghai were obviously higher than those for IRPA isolates from other cities, while the resistance rates for IRPA isolates from Guangzhou against aminoglycosides such as amikacin and netimicin were highest among strains from the other four cities. MBL-producing isolates were screened by the imipenem-EDTA double-disk synergy test. The method was modified for the use of a disk containing 750 μg of EDTA plus 2 mg of sodium mercaptoacetic acid (14). Among the 140 IRPA isolates collected, the double-disk synergy test gave positive results for 12 strains. The MBL producer was not the major mechanism of IRPA in our study.

    The blaIMP-1, blaIMP-2, blaVIM-2, blaGIM, blaSPM, and blaSIM genes were detected by PCR using the primers as previously described (3, 11, 21, 22, 25). Fourteen strains were confirmed by PCR to be VIM-2 positive, while no other metallo--lactamases were detected. Among the five different regions, Beijing, Shanghai, Hangzhou, Guangzhou, and Chengdu, the ratios of MBL-positive strains in IRPA isolates were 11.1% (3/27), 18.8% (6/32), 14.8% (4/27), 3.1% (1/32), and 0.0% (0/22), respectively. Ten percent of the 140 imipenem-resistant isolates produced metallo--lactamases. The MICs of 10 antimicrobial agents for the 14 strains carrying the blaVIM-2 gene are shown in Table 2.

    PCR was carried out to characterize the variable region of the class 1 integron by using the 5' conserved segment (5'-CS)P1 (5'-GGCATCCAAGCAGCAAG-3') and the 3' conserved segment (3'-CS)P2 (5'-AAAGCAGACTTGACCTGA-3') primers. The PCR products of the integron carrying blaVIM-2 were detected in 12 of the 14 P. aeruginosa strains. The other two strains were class 1 integrase positive and class 2 and 3 integrase gene negative by PCR using the primers as previously described (18). The sizes of the class 1 integrons carrying the blaVIM-2 gene varied among the isolates and were approximately 2.5 and 3 kb. Analysis of the nucleotide sequences for integrons containing blaVIM-2 revealed that the integrons in seven P. aeruginosa isolates from Beijing, Hangzhou, and Guangzhou (B2, B3, B28, H19, H26, H54, and G4) were In72. They contained aacA4 [aac(6')-Ib] followed immediately downstream by blaVIM-2 (Fig. 2) (19). The integrons in the five P. aeruginosa isolates from Shanghai (S5, S9, S10, S11, and S12) were different from In72 due to the fact that aacA4 was the first cassette, followed by blaVIM-2 and aadB (Fig. 2). The blaVIM-2 gene failed to be detected in the integrons of the other two strains (S15 and H22). In these two strains, the blaVIM-2 gene might be located in untypical integrons, which need to be further studied.

    Conjugation experiments were performed as described previously (32), with streptomycin- and rifampin-resistant Escherichia coli C600 as the recipient (2). Tryptic soy agar plates supplemented with rifampin (512 μg/ml) and meropenem (2 μg/ml) were used to select for transconjugants. Plasmids in 14 P. aeruginosa strains producing metallo--lactamases were extracted and electroporated into Escherichia coli DH5 by following a method previously described, and transformants were selected on meropenem-containing (2 μg/ml) Mueller-Hinton agar plates (30). However, repeated attempts to transfer the MBL gene by conjugation and electrotransformation failed.

    Genomic DNA was digested overnight with XbaI as recommended by Tenover et al. (27) and was subjected to pulsed-field gel electrophoresis (PFGE) as previously described (32). Results from the PFGE analysis are shown in Fig. 3. The XbaI-digested fragments of the total DNA for 14 strains harboring the blaVIM-2 gene showed different patterns among various regions (Fig. 3). The six strains from Shanghai belonged to one PFGE type, while the three strains from Beijing belonged to two PFGE types, similar to those from Hangzhou.

    Repeated attempts to detect the presence of plasmids capable of hybridizing with the blaVIM-2 probe failed. However, the fragments of the XbaI-digested genomic DNA of clinical isolates showed positive hybridization signals by Southern hybridization. DNA fragments digested with XbaI showed two hybridization results. The VIM-2 probe hybridized with a 50-kbp XbaI fragment for strains G4, S5, S9, S10, S11, S12, S15, B3, B28, H19, and H54, and there was no hybridization reaction for H22, H26 and B2 (Fig. 3).

    The prevalence of MBL-producing strains among imipenem-resistant P. aeruginosa isolates from hospitals in China was estimated at 10.0%, less than what was found for Japan. It is striking that metallo--lactamase found in China is not the IMP-1 type, which is disseminated around Japan (10), but the VIM type, which is often reported in East Asia and European countries (1, 7, 29). In this study, P. aeruginosa strains harboring the blaVIM-2 gene were disseminated in four hospitals from different regions of China, confirming that the blaVIM-2 gene is widely spread across China. Earlier studies suggested that blaVIM-2 is located on an approximately 45-kb plasmid (20) or on XbaI-digested genomic DNA fragments (13). PFGE of XbaI-digested genomic DNA showed that organisms from different regions are grouped into various PFGE types. These findings suggest that the transmission of the blaVIM-2 gene among clinical strains with different genetic backgrounds may be associated with mobile genetic elements, such as transposons and transferable plasmids, instead of a clonal expansion of a VIM-2-carrying strain throughout China.

    Acquired MBL genes in P. aeruginosa are often carried on plasmids and are usually nontransferable by conjugation, at least to E. coli (8, 9, 11). In the present study, the MBL genes in our isolates could not be transferred to E. coli by conjugation. Southern hybridization showed that 11 P. aeruginosa isolates carried the blaVIM-2 gene on an XbaI-digested genomic DNA fragment of approximately 50 kb.

    Our results demonstrate that 7 of the 14 blaVIM-2 gene-positive P. aeruginosa isolates from different regions and with different PFGE types harbored integrons similar to In72, containing two resistant cassettes (aacA4 and blaVIM-2). This suggests a horizontal spread of the integron itself. The five blaVIM-2 gene-positive strains from Shanghai harbored the other kind of integron, containing three resistant cassettes (aacA4, blaVIM-2, and aadB). This indicates that the blaVIM-2 gene cassette has been disseminated among various integrons. Interestingly, the structures of the integrons from these strains were the same as those of integrons from strains commonly found in the intensive-care units of hospitals in Shaoxing, Zhejiang Province, as previously reported (33). Through clinical investigation, it was concluded that the clonal expansion in the Shanghai hospital could be due to the transfer of a patient from Shaoxing to Shanghai.

    Although integrons themselves are not mobile, several class 1 integrons have been found in Tn21 and Tn21-related transposons (4, 6, 26, 28), which enables the integrons to be transposed. These findings raise the possibility that the class 1 integrons described in this study are also parts of transposons. This increases the threat of the blaVIM-2 gene being disseminated among diverse genera of bacteria.

    ACKNOWLEDGMENTS

    This work was supported by a research grant from the National Natural Science Foundation of China (no. NSFC30270074) and by a grant from the Program for New Century Excellent Talents in University in China (no. NCET-04-0552).

    FOOTNOTES

    Corresponding author. Mailing address: Respiratory Department, First Affiliated Hospital, Medical School, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang, China. Phone: 86-57187236876. Fax: 86-57187236756. E-mail: wwwjjj312@tom.com.

    Published ahead of print on 27 September 2006.

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