Molecular characterization of metallo-b-lactamase-producing Acinetobacter baumannii and Acinetobacter genomospecies 3 from Korea: identification of two new integrons carrying the blaVIM-2 gene cassettes

Jong Hwa Yum1, Keonsoo Yi1, Hyukmin Lee1, Dongeun Yong1, Kyungwon Lee1,*, June Myung Kim1, Gian Maria Rossolini2 and Yunsop Chong1

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.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Carbapenem-resistant Acinetobacter spp. used to be rare, but are increasingly isolated in Korea. Among 28 isolates of imipenem-resistant Acinetobacter spp. found in a Korean hospital in 1998 and 1999, 14 produced metallo-b-lactamases. The blaVIM-2 gene was detected, by PCR, in 11 and two isolates of Acinetobacter baumannii and Acinetobacter genomospecies 3, respectively, and blaIMP-1 in one isolate of A. baumannii. The MICs of imipenem for the isolates were 8–32 mg/L. PFGE analysis of SmaI-digested genomic DNA gave identical patterns in eight of 11 blaVIM-2-positive A. baumannii isolates from respiratory specimens of ICU patients. The blaVIM-2 gene cassettes in the isolates are identical to those from Pseudomonas aeruginosa isolates in Europe, but are inserted into new class I integrons In105 and In106. The attC site of the last cassette of the array in In106 is interrupted by the insertion of a putative class II intron. This is the first report of VIM-2 b-lactamase-producing A. baumannii and Acinetobacter genomospecies 3. Production of the VIM-2 enzyme presents an emerging threat of carbapenem resistance among Acinetobacter spp. in Korea.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Carbapenems are very useful for the treatment of infections caused by Gram-negative bacilli that are resistant to other b-lactam antibiotics. However, carbapenem-resistant Gram-negative bacilli are being isolated with increasing frequency from clinical sources. Pseudomonas aeruginosa, Acinetobacter baumannii and a number of species of Enterobacteriaceae producing IMP-type metallo-b-lactamases have been found in several hospitals in Japan since the early 1990s, and identical or related enzymes have subsequently been detected elsewhere.1 More recently, the emergence of new metallo-b-lactamases VIM-1 and VIM-2 in P. aeruginosa isolates from Europe was reported,2 and Pseudomonas spp. carrying blaVIM-2 have been reported to be in circulation in Korea.3 Both blaVIM and blaIMP are found on mobile gene cassettes inserted in the variable region of integrons.1,2 Therefore, horizontal spread of these resistance determinants, in addition to clonal spread of individual strains, can be anticipated. In this paper, we report the detection of VIM-2 and IMP-1 metallo-b-lactamase-producing Acinetobacter spp. in Korea, and the characteristics of novel blaVIM-2-carrying integrons.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains and in vitro susceptibility testing

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 1’Étoile, 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 EDTA–disc 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 manufacturer’s instructions using precast gels with pH gradient 3–10, 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{alpha}.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.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Acinetobacter spp. are often resistant to most b-lactams, but are generally susceptible to carbapenems. Of the 28 imipenem-resistant Acinetobacter spp. isolated in the hospital study, 14 were positive for metallo-b-lactamases, as determined by both modified Hodge and EDTA–disc synergy tests. Enzyme assays of crude cell extracts confirmed the presence of carbapenemase activity, which was susceptible to inhibition by EDTA, but not clavulanate (data not shown). This finding indicates that metallo-b-lactamase production is an important mechanism of carbapenem resistance in Acinetobacter spp. in Korea.

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 8–32 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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Table 1.  Antimicrobial susceptibilities of metallo-b-lactamase-producing A. baumannii and Acinetobacter genomospecies 3 isolates. MICs of the same drugs for eight randomly selected imipenem-resistant isolates that did not produce metallo-b-lactamase are shown for comparison
 
All 11 blaVIM-2-positive A. baumannii isolates were from respiratory specimens, while two blaVIM-2- and one blaIMP-1-positive Acinetobacter genomospecies 3 were from urine specimens. PFGE showed that among the 11 A. baumannii isolates, eight gave identical fragmentation patterns and one had a closely related pattern and had been isolated from ICU patients. The two blaVIM-2-positive Acinetobacter genomospecies 3 isolates were unrelated (data not shown).

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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Figure 1. Comparison of the structures of the variable regions of In105 and In106 with those of other blaVIM-2 cassette-containing class I integrons: In56 (AF191564), In72 (AF302086), In58 (AF263519) and In59 (AF263520). Hatched rectangles, 5'- and 3'-conserved segments (5'-CS and 3'-CS); black arrows, cassette-borne resistance genes; empty rectangles, attC sites of the cassettes; grey circles, recombination core sites and inverse core sites. The insertion and orientation of the putative class II intron in the attC site of the last cassette of In106 is indicated.

 
In conclusion, this is the first report of the VIM-2 metallo-b-lactamase in Acinetobacter spp. The blaVIM-2 gene cassette was found to be part of the two new integrons In105 and In106. Furthermore, the attC site of the last cassette of the array in In106 is interrupted by the insertion of a putative class II intron. Production of the VIM-2 b-lactamase presents an emerging threat of carbapenem resistance among Acinetobacter spp. in Korea.


    Acknowledgements
 
This study was supported in part by the BK21 Project for Medical Sciences, Yonsei University in 2001. This study was presented in part at the 7th Western Pacific Congress of Chemotherapy & Infectious Diseases, Hong Kong, 11–14 December 2000.


    Footnotes
 
* Correspondence address. Department of Clinical Pathology, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. Tel: +82-2-361-5866; Fax: +82-2-313-0956; E-mail: leekcp{at}yumc.yonsei.ac.kr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Riccio, M. L., Franceschini, N., Boschi, L., Caravelli, B., Cornaglia, G., Fontana, R. et al. (2000). Characterization of the metallo-b-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of blaIMP allelic variants carried by gene cassettes of different phylogeny. Antimicrobial Agents and Chemotherapy 44, 1229–35.[Abstract/Free Full Text]

2 . Poirel, L., Naas, T., Nicolas, D., Collet, L., Bellais, S., Cavallo, J.-D. et al. (2000). Characterization of VIM-2, a carbapenem-hydrolyzing metallo-b-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrobial Agents and Chemotherapy 44, 891–7.[Abstract/Free Full Text]

3 . Lee, K., Chong, Y., Shin, H. B., Kim, Y. A., Yong, D. & Yum, J. H. (2001). Modified Hodge and EDTA–disk synergy tests to screen metallo-b-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clinical Microbiology and Infection 7, 88–91.[ISI][Medline]

4 . Schreckenberger, P. C. & von Graevenitz, A. (1999). Acinetobacter, Alcaligenes, Moraxella, Methylobacterium, and other nonfermentative gram-negative rods. In Manual of Clinical Microbiology, 7th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 539–60. American Society for Microbiology, Washington, DC.

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. 590–4. 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.

8 . Tenover, F. C., Arbert, R. D., Goering, R. V., Michelsen, P. A., Murray, B. E., Persing, D. H. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Free Full Text]

9 . Cousineau, B., Lawrence, S., Smith, D. & Belfort, M. (2000). Retrotransposition of a bacterial group II intron. Nature 404, 1018–21.[ISI][Medline]

10 . Fluit, A. C. & Schmitz, F. J. (1999). Class 1 integrons, gene cassettes, mobility, and epidemiology. European Journal of Clinical Microbiology and Infectious Diseases 18, 761–70.[ISI][Medline]