Korean J Clin Microbiol KJCM Korean Journal of Clinical Microbiology 1229-0025 The Korean Society of Clinical Microbiology 10.5145/KJCM.2012.15.4.131 Original Article Diversity of Integrons Carrying blaVIM-2 Cassette in Pseudomonas spp. and Acinetobacter spp. Yum Jonghwa 1 Shin Hee Bong 2 Yong Dongeun 3 Chong Yunsop 3 Department of Clinical Laboratory Science, College of Nursing and Healthcare Sciences, Dong-eui University, Busan, Korea. Department of Laboratory Medicine, Soonchunhyang University Hospital, Bucheon, Korea. Department of Laboratory Medicine and Research Institute of Bacterial Resistance, College of Medicine, Yonsei University, Seoul, Korea. Correspondence: Jonghwa Yum, Department of Clinical Laboratory Science, College of Nursing and Healthcare Sciences, Dong-eui University, 995, Eomgwang-ro, Busanjin-gu, Busan 614-714, Korea. (Tel) 82-51-890-2682, (Fax) 82-51-890-1469, auxotype@deu.ac.kr 12 2012 21 12 2012 15 4 131 138 06 08 2012 06 10 2012 08 10 2012 Copyright © 2012 The Korean Society of Clinical Microbiology 2012 Background

Metallo-β-lactamase-mediated carbapenem resistance has been increasingly reported in Pseudomonas, Acinetobacter and other Gram-negative bacilli (GNB) in many countries. A few studies showed highly variable structure of MBL-gene cassette-carrying integrons. The aim of this study was to determine the structure of blaVIM-2-carrying integrons in Pseudomonas and Acinetobacter.

 Methods

blaVIM-2-carrying GNB were isolated at a Korean hospitals during the years 1995-1999 and 2005. The size of blaVIM-2-carrying integrons was estimated by the PCR products. Representative integrons were sequenced by the dideoxy-chain termination method. The MICs of antimicrobial agents were tested by the CLSI agar dilution methods.

 Results

During the years 1995-1999 and 2005, the approximate size of the blaVIM-2-carrying class 1 integrons was 3-7 kb in 35 Pseudomonas isolates and 3-5 kb in 24 Acinetobacter isolates. The integrons carried one-five resistance gene cassettes in addition to the blaVIM-2 cassette. Other resistance gene cassettes found were blaOXA-1, aacA1, aac(6')-I, and aac(6')-II. Interestingly, sequences homologous to part of a putative class II intron were inserted into the recombination site of the last cassette in four of nine integrons. The class 1 integron from P. aeruginosa isolates had fused orf/IntI1 in a downstream leftward inverted repeat (IRi).

 Conclusion

According to period, the size and structure of blaVIM-2-carrying integrons are quite variable, but an identical one is also present in a different genus, indicating high mobility of the blaVIM-2 cassette and horizontal transfer of the whole integron. We suggest that the class 1 integron containing the blaVIM-2 gene is spreading horizontally among Gram-negative bacilli and is undergoing continuous development in Korea.

 Gram-negative bacilli Integron Metallo-β-lactamase Papp-Wallace KM Endimiani A Taracila MA Bonomo RA Carbapenems: past, present, and future Antimicrob Agents Chemother 2011 55 4943 4960 Lee K Yong D Choi YS Yum JH Kim JM Woodford N Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 beta-lactamases co-mediated by porin loss Int J Antimicrob Agents 2007 29 201 206 Lee K Yum JH Yong D Lee HM Kim HD Docquier JD Novel acquired metallo-beta-lactamase gene, blaSIM-1, in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea Antimicrob Agents Chemother 2005 49 4485 4491 Pallecchi L Riccio ML Docquier JD Fontana R Rossolini GM Molecular heterogeneity of blaVIM-2-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-beta-lactamase FEMS Microbiol Lett 2001 195 145 150 Hall RM Collis CM Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination Mol Microbiol 1995 15 593 600 Riccio ML Franceschini N Boschi L Caravelli B Cornaglia G Fontana R Characterization of the metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of blaIMP allelic variants carried by gene cassettes of different phylogeny Antimicrob Agents Chemother 2000 44 1229 1235 Poirel L Naas T Nicolas D Collet L Bellais S Cavallo JD Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France Antimicrob Agents Chemother 2000 44 891 897 Lee K Trend of bacterial resistance for the past 50 years in Korea and future perspectives-Gram-negative bacteria Infect Chemother 2011 43 458 467 Clinical and Laboratory Standards Institute Document M100-S15 Performance standards for antimicrobial susceptibility testing; fifteenth informational supplement 2005 Wayne, PA Clinical and Laboratory Standards Institute Nobuta K Tolmasky ME Crosa LM Crosa JH Sequencing and expression of the 6'-N-acetyltransferase gene of transposon Tn1331 from Klebsiella pneumoniae J Bacteriol 1988 170 3769 3773 Walsh TR Toleman MA Poirel L Nordmann P Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev 2005 18 306 325 Gupta V Metallo beta lactamases in Pseudomonas aeruginosa and Acinetobacter species Expert Opin Investig Drugs 2008 17 131 143 Poirel L Rodríguez-Martínez JM Al Naiemi N Debets-Ossenkopp YJ Nordmann P Characterization of DIM-1, an integron-encoded metallo-beta-lactamase from a Pseudomonas stutzeri clinical isolate in the Netherlands Antimicrob Agents Chemother 2010 54 2420 2424 Yong D Toleman MA Giske CG Cho HS Sundman K Lee K Characterization of a new metallo-beta-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India Antimicrob Agents Chemother 2009 53 5046 5054 Sekiguchi J Morita K Kitao T Watanabe N Okazaki M Miyoshi-Akiyama T KHM-1, a novel plasmid-mediated metallo-beta-lactamase from a Citrobacter freundii clinical isolate Antimicrob Agents Chemother 2008 52 4194 4197 Yum JH Yi K Lee H Yong D Lee K Kim JM Molecular characterization of metallo-beta-lactamase-producing Acinetobacter baumannii and Acinetobacter genomospecies 3 from Korea: identification of two new integrons carrying the blaVIM-2 gene cassettes J Antimicrob Chemother 2002 49 837 840 Yum JH Yong D Lee K Kim HS Chong Y A new integron carrying VIM-2 metallo-beta-lactamase gene cassette in a Serratia marcescens isolate Diagn Microbiol Infect Dis 2002 42 217 219 Jeong SH Lee K Chong Y Yum JH Lee SH Choi HJ Characterization of a new integron containing VIM-2, a metallo-beta-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae J Antimicrob Chemother 2003 51 397 400 Lee K Lim JB Yum JH Yong D Chong Y Kim JM blaVIM-2 cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital Antimicrob Agents Chemother 2002 46 1053 1058 Levesque C Roy PH Persing DH Smith TF PCR Analysis of Integrons Diagnostic Molecular Microbiology: Principles and Applications 1993 Washington, DC American Society for Microbiology 590 594 Mingeot-Leclercq MP Glupczynski Y Tulkens PM Aminoglycosides: activity and resistance Antimicrob Agents Chemother 1999 43 727 737 Walsh TR Toleman MA Hryniewicz W Bennett PM Jones RN Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program J Antimicrob Chemother 2003 52 116 119 Poirel L Lambert T Türkoglü S Ronco E Gaillard J Nordmann P Characterization of Class 1 integrons from Pseudomonas aeruginosa that contain the blaVIM-2 carbapenem-hydrolyzing beta-lactamase gene and of two novel aminoglycoside resistance gene cassettes Antimicrob Agents Chemother 2001 45 546 552 Lee H Yong D Yum JH Roh KH Lee K Yamane K Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea Diagn Microbiol Infect Dis 2006 56 305 312

Comparison of the structures of variable regions of class 1 integrons determined in this study. Hatched rectangles indicate the 5'- and 3'-conserved segments (5'-CS and 3'-CS), black arrows the cassette-borne resistance genes, empty rectangles the attC sites of the cassettes, small rectangle the IRi sites of the integron, and gray circles the recombination core sites and inverse core sites. The insertion and orientation of part of putative class II intron in the recombination site of the last cassette of In106 and integron in P. putida YMC 99/10/431, P. aeruginosa YMC 95/1/704, and P. aeruginosa YMC 05/5/1103 are indicated.

Deduced amino acid changes of aac(6')-I detected in this study, compared to aac(6')-I deduced amino acid reported by Nobuta et al. [10], P. aeruginosa YMC 95/1/2105 and P. putida YMC 99/10/431 had a mutation T269C and deduced amino acid change of L90S. P. aeruginosa YMC 95/1/2105 also had G298A and T512A mutations, resulting in amino acid changes of G100S and V171D. Isolates with mutations in this study: Study No. 3, 6 in Table 2. P. aeruginosa YMC 95/1/704 and P. putida YMC 99/10/431; No. 2. Acinetobacter genomospecies 3 YMC 99/11/U160; No. 4. P. aeruginosa YMC 95/1/2105.

Charteristics of P. aeruginosa, P. putida and Acinetoabcter isolates with blaVIM-2 carrying integrons

Abbreviations: STR, streptomycin; SPT, spectinomycin; KAN, kanamycin; AMK, amikacin; GEN, gentamicin; ISP, isepamicin; NET, netilmicin; TOB, tobramycin; S, susceptible; I, intermediate; R, resistant; NT, not tested because probably due to presence of the gene in other genetic elements.

MIC of antimicrobial agents for blaVIM-2-positive gram-negative bacilli with different position of blaVIM-2 cassette in class 1 integrons

Abbreviations: PIP, piperacillin; CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam; IPM, imipenem; MEM, meropenem.

Amino acid substitutions in aac(6')-I type aminoglycoside modifying enzymes

Abbreiations: Leu, leucine; Gly, glycine; Val, valine; Ser, serine; Asp, asparagine.