1Department of Clinical Pathology, Research Institute of Bacterial Resistance, and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul, Korea; 2Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, I-53100 Siena, Italy
Received 7 September 2001; returned 9 November 2001; revised 11 December 2001; accepted 28 February 2002.
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Abstract |
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Introduction |
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Materials and methods |
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Twenty-eight non-repetitive isolates of imipenem-resistant Acinetobacter spp. were obtained from patients in a tertiary care hospital in Seoul, Korea, in 1998 and 1999. The species were identified by the conventional method4 and the ATB 32 GN System (bioMérieux SA, Marcy 1Étoile, France). For these isolates, resistance to imipenem was detected by disc diffusion test.5 MICs of antimicrobial agents were determined by an agar dilution method.5
ß-Lactamase assays
The modified Hodge and EDTAdisc synergy tests3 were used to screen for metallo-b-lactamase production by imipenem-resistant isolates. b-Lactamase activities in crude cell sonicates were determined using 100 µM b-lactams in 50 mM phosphate buffer (pH 7.0) at 30°C using a UV1601 spectrophotometer (Shimadzu, Tokyo, Japan). Enzyme activities were also determined after incubation of cell extracts with 10 mM EDTA or 50 µM clavulanate for 30 min at 30°C.2 Analytical isoelectric focusing (IEF) was carried out following the manufacturers instructions using precast gels with pH gradient 310, and a ThermoFlow Electrophoresis Temperature Control System (Novex Experimental Technologies, San Diego, CA, USA). Detection of b-lactamase bands, EDTA inhibition and bioassays were carried out as described previously with minor modification.1
Molecular analysis techniques
The presence of blaVIM-2 and blaIMP-1 was detected by PCR. Previously described blaVIM-2 primers2 and blaIMP-1 primers IMP1-F (5'-CATGGTTTGGTGGTTCTTGT-3') and IMP1-R (5'-ATAATTTGGCGGACTTTGGC-3') were used. PCR mixes contained l µL of heat-extracted template DNA, 1 µL (20 pmol) of each primer and PCR Premix containing 1 U of Taq DNA polymerase (Bioneer, Cheongwon, Korea) in a final volume of 20 µL. The thermocycle protocol used was: an initial denaturation step at 94°C for 5 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, elongation at 72°C for 45 s, plus a final extension step at 72°C for 7 min.
Gene cassette arrays inserted into class I integrons were amplified using the primers of Riccio et al.1 The amplication protocol was that of Levesque & Roy6 with minor modification. Briefly, the reaction was carried out in 100 µL, using 10 µL of heat-extracted template DNA, 20 pmol of each primer and 3 U of LA Taq DNA polymerase (Takara, Shiga, Japan).
PCR-generated amplicons were separated by agarose gel electrophoresis and recovered using a DNA extraction kit (Qiagen, Hilden, Germany). When necessary, the amplicon was inserted into the pGEM-T-easy vector (Promega, Madison, WI, USA) and cloned in Escherichia coli DH5.7
Southern hybridization was carried out using a digoxigenin (DIG)-labelled blaVIM-2-containing PCR amplicon. Labelling and detection of the probe were carried out using a DIG DNA labelling and detection kit (Roche Diagnostics, Mannheim, Germany).
Sequencing was carried out by the dideoxynucleotide-chain termination method with an automatic DNA sequencer (ABI 3700; Perkin-Elmer, Foster City, CA, USA) and custom-designed primers. Both strands of two independently cloned PCR amplicons originating from each isolate were sequenced.
PFGE
Genomic DNAs of the Acinetobacter isolates were digested with SmaI, as suggested by the manufacturer. The fragments were separated using a CHEF-DR II System (Bio-Rad, Hercules, CA, USA). The band patterns were interpreted according to the recommendations of Tenover et al.8
Nucleotide sequence accession numbers
The nucleotide sequences of the blaVIM-2-carrying integrons have been assigned the GenBank accession nos AF324464 for that from A. baumannii YMC 98/7/363, and AF369871 for that from Acinetobacter genomospecies 3 YMC 99/11/160.
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Results and discussion |
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The blaIMP-1 and blaVIM-2 determinants were detected by PCR in one and 13 isolates, respectively. IMP-1-producing Acinetobacter spp. have been reported in Japan, but this is the first report of a VIM-2 enzyme in Acinetobacter spp. Bands of b-lactamase activity, approximate pIs 5.3 and >8.5, were detected by analytical IEF in cell extracts of blaVIM-2- and blaIMP-1-positive isolates, respectively. These enzymes were susceptible to EDTA inhibition and active against carbapenems in bioassays. Additional b-lactamases with different pIs and resistant to EDTA inhibition were also present in these isolates (data not shown).
The MICs of imipenem were 832 mg/L for both blaVIM-2-positive and metallo-b-lactamase-negative isolates (Table 1). The MIC range of imipenem for blaVIM-2-positive Acinetobacter spp. was lower than that reported for blaVIM-2-positive P. aeruginosa, 8>128 mg/L, at our hospital. It is significant that 50% of VIM-2-producing Acinetobacter spp. were found to be inhibited by 8 mg/L imipenem (intermediate category). All of the isolates were either susceptible or displayed intermediate resistance to meropenem when results were interpreted according to the criteria of the National Committee for Clinical Laboratory Standards.5 Relatively low-level carbapenem resistance is not uncommon among metallo-b-lactamase-producing bacteria and underscores the need for reliable screening procedures to detect such isolates.
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The VIM-2 gene has been found on a class I integron.2 PCR products of c. 3 kb from 12 A. baumannii isolates and of c. 5 kb from one Acinetobacter genomospecies 3 isolate were obtained using integron-directed primers. These products hybridized to a blaVIM-2 probe in Southern hybridization experiments (data not shown). EaeI digestion of the 3 kb amplicons from the 12 isolates yielded identical six fragment band patterns, indicating that these isolates have identical or very similar integron structures.
The 3 and 5 kb amplicons obtained from isolates of A. baumannii (YMC 98/7/363) and Acinetobacter genomospecies 3 (YMC 99/11/160), respectively, were cloned into pGEM- T-easy. The inserts recovered were sequenced, revealing two new gene cassette arrays inserted between the 5'- and 3'-conserved segments of class I integrons. The 3 kb integron, named In105, carries the blaVIM-2, aacA7 and aadA1 cassettes (Figure 1). The 5 kb integron, named In106, carries the blaVIM-2, aacA4 and aadA1 cassettes, and a putative class II intron inserted into the attC site of the last cassette of the array (Figure 1). This finding indicates that this type of mobile genetic element can provide a target for retro-homing of class II introns.9 The two blaVIM-2 cassettes were identical to those of P. aeruginosa isolates from Europe.2 The sequences of the 5'-conserved segments of the two integrons were identical, except for a putative G to A transition at position 10 in the smaller integron (GenBank accession No. AF324464). The integrons contain two putative promoters, a strong-type Pc and a non-functional P2.10
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Acknowledgements |
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Footnotes |
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References |
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2
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3 . Lee, K., Chong, Y., Shin, H. B., Kim, Y. A., Yong, D. & Yum, J. H. (2001). Modified Hodge and EDTAdisk synergy tests to screen metallo-b-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clinical Microbiology and Infection 7, 8891.[ISI][Medline]
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5 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing. Eleventh Informational Supplement. M100-S11. NCCLS, Wayne, PA.
6 . Levesque, C. & Roy, P. H. (1993). PCR analysis of integrons. In Diagnostic Molecular Microbiology: Principles and Applications, (Persing, D. H., Smith, T. F., Tenover, F. C. & White, T. J., Eds), pp. 5904. American Society for Microbiology, Washington, DC.
7 . Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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