JAC Advance Access published online on March 6, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm020
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Characterization of In3Mor, a new integron carrying VIM-1 metallo-ß-lactamase and sat1 gene, from Morganella morganii
1 Department of Microbiology, Medical School, University of Athens, Athens, Greece 2 Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece 3 Department of Microbiology, General Hospital of Serres, Serres, Greece
* Corresponding author. Tel: +30-210-746-2010; Fax: +30-210-746-2249; E-mail: atsakris{at}med.uoa.gr
Received 19 November 2006; returned 20 December 2006; revised 27 December 2006; accepted 11 January 2007
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Abstract |
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Objectives: A carbapenem-resistant Morganella morganii clinical isolate that was phenotypically metallo-ß-lactamase (MBL)-positive was recovered from a Greek patient. The aim of the study was to analyse the structure of the integron containing the MBL gene.
Methods: MICs were determined by the broth microdilution method. PCR assays and nucleotide sequencing were used for identification of bla gene types and mapping of the integron carrying the MBL gene. The location of the MBL allele was investigated by mating experiments and plasmid analysis as well as by Southern blotting of the plasmid extract and gene-specific hybridization with a blaVIM-1 probe.
Results: The strain contained In3Mor, a novel class 1 integron carrying a carbapenemase gene (blaVIM-1) associated with a trimethoprim (dfrA1), a streptothricin (sat1) and two aminoglycoside resistance genes (aacA7 and aadA1). Conjugation experiments failed to detect a transferable MBL determinant and plasmid DNA was not visualized. The chromosomal location of blaVIM-1 was confirmed after hybridization of the chromosomal band with the blaVIM-1 probe.
Conclusions: Production of a VIM-type MBL in a M. morganii clinical isolate is documented in this study for the first time. Also, the dfrA1-sat1-aadA1 array which is typically described in the variable region of class 2 integrons consistent with that on Tn7 transposons, is originally detected herein in a class 1 integron.
Key Words: M. morganii , resistance , carbapenems , MBLs
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Introduction |
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During the last decade, acquired metallo-ß-lactamases (MBLs) have emerged among Pseudomonas aeruginosa isolates and more recently among other Gram-negative pathogens such as Acinetobacter baumannii and several enterobacterial species.1 Two groups of MBL genes, the blaIMP- and blaVIM-type genes, have been mainly reported in Gram-negative species (http:\\www.lahey.org\studies). IMP-type enzymes have been detected in Asian countries, and also in some European and American regions while VIM-type enzymes have been disseminated mostly in Mediterranean countries of Europe and several Far East countries and also in some American regions.1 The MBLs readily hydrolyse most ß-lactams including carbapenems and are predominantly encoded by genes carried in class 1 integrons. These genetic elements have variable structures among isolates and the order of the gene cassettes in their variable region seems to reflect antibiotic pressure in different hospital environments.
Morganella morganii is an opportunistic pathogen with significant relevance in hospital infections, particularly among critically ill patients. Carbapenems have a potent activity against this enterobacterial species and in some cases are the drugs of choice for treating infections due to AmpC-hyperproducing multidrug-resistant M. morganii isolates. MBLs have not been described in this pathogen, apart from a blaIMP-1-producing M. morganii isolate identified among 434 M. morganii clinical isolates in a multicentre study conducted in Japan.2 In this study, we report the detection of a M. morganii clinical isolate in Greece carrying a blaVIM-1 allele. The gene was located in a new class 1 integron structure containing a variable structure that is typically described in class 2 integrons.
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Materials and methods |
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A 78-year-old woman having a cardiovascular surgery was hospitalized in two Greek hospitals from May to September 2005. During this time the patient received antibiotic treatment, including ceftazidime, ticarcillin/clavulanate, piperacillin/tazobactam, fluoroquinolones and meropenem. After 20 days of meropenem and ciprofloxacin therapy, urine cultures yielded a M. morganii strain (A209) resistant to most ß-lactams including imipenem and meropenem. Species identification was confirmed by using the API 20E system (bioMérieux, Marcy l'Étoile, France) and conventional biochemical assays. Antimicrobial susceptibility testing was performed by the broth microdilution method recommended by the CLSI.3 Phenotypic detection of MBL production was performed using the Etest MBL assay (AB Biodisk, Solna, Sweden).
PCR detection of various bla gene types, including blaIMP, blaVIM, blaTEM and blaSHV, was performed using consensus primers and amplification conditions as described previously.4–6 For integron mapping, PCR assays combining primers specific for conserved sequences 5'-CS and 3'-CS with primers specific for blaVIM, aacA, dfrA, aadA, qacE
1 and sul genes were performed. PCR products were purified using ExoSAP-IT reagent (USB Corporation, Cleveland, OH, USA) and used as templates for sequencing on both strands with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA).
The potential for conjugational transfer of carbapenem resistance was performed by a plate mating method using Escherichia coli 20R764 (lac+ rifr) as the recipient. Selection of transconjugants was made on MacConkey agar plates containing rifampicin (100 mg/L) and imipenem (0.5–2 mg/L) or ceftazidime (4 mg/L). Plasmid extraction was performed with two different lysis methods using E. coli 39R861 as control. The chromosomal location of the blaVIM-1 allele was detected by Southern blotting after electrophoresis of the plasmid extract and gene-specific hybridization using a digoxigenin-labelled blaVIM-1 probe.5
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Results and discussion |
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M. morganii A209 was resistant to carbapenems (MICs of imipenem and meropenem were 32 mg/L), penicillin/inhibitor combinations, expanded-spectrum cephalosporins, aztreonam, colistin, trimethoprim, tetracycline, tobramycin, netilmicin and ciprofloxacin, and also exhibited reduced susceptibility to gentamicin (MIC of 8 mg/L) and amikacin (MIC of 8 mg/L). The EDTA inhibited carbapenemase activity in Etest MBL suggesting production of an MBL.
Initial screening using pairs of consensus primers was positive for a blaVIM-type gene but was negative for the remaining bla genes. To further investigate the nature of the detected blaVIM gene, primers designed to amplify the entire sequence of the blaVIM-1 gene6 produced an amplicon of the expected size (920 bp). Nucleotide sequencing confirmed that the entire gene cassette was identical to the blaVIM-1 gene originally described in P. aeruginosa, including its cassette boundaries (GenBank accession number Y18050 [GenBank] ). However, conjugation experiments failed to detect that the MBL determinant could be transferred from A209 to 20R764 and plasmid DNA was not visualized by either of the extraction methods in agarose gel electrophoresis. These results suggested the chromosomal location of blaVIM-1, which was confirmed after hybridization of the chromosomal band with the blaVIM-1 probe.
The structure of the blaVIM-1-containing integron was characterized. Assembly of the nucleotide sequences of overlapping PCR products revealed a novel class 1 integron of 
5950 bp named In3Mor (Figure 1). A typical 5'-CS containing an intI1 gene with a strong P1 promoter followed directly by an inactive P2 promoter (without a GGG insertion prior to the –10 hexamer) and an attI1 site was identified. The variable region of 3600 bp contained five gene cassettes, including (5'–3') the blaVIM-1 cassette, an aacA7 cassette conferring resistance to aminoglycosides, a dfrA1 cassette conferring resistance to trimethoprim, a sat1 cassette conferring resistance to streptothricin and an aadA1 cassette conferring resistance to streptomycin. The blaVIM-1 gene cassette with its 59-base element was identical to those reported in other Gram-negative bacilli in Greece.7 The last cassette was followed by a qacE1 allele, typical of the 3'-CS of sul1-associated class 1 integrons.
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Production of a VIM-type MBL in a M. morganii isolate is documented here for the first time. The structure of the gene cassette region of In3Mor was also different from previously described MBL-encoding integrons and this is the first description of a class 1 integron containing an MBL gene associated with a sat gene. The unique structure of In3Mor might suggest a different origin of this integron in comparison with other MBL-containing integron structures. It is of interest that the sat1 gene, which encodes a streptothricin acetyltransferase, is typically detected in the variable region of class 2 integrons.8,9 However, the determinant has also been scarcely sequenced in class 1 integrons of Gram-negatives.10 It is noteworthy that the dfrA1-sat1-aadA1 array that was detected herein is a novel arrangement in the class 1 integrons, while it is typically described in the variable region of class 2 integrons consistent with that on Tn7 transposons.8,9 It could be hypothesized that a simple excision of a sat1 cassette from Tn7 was followed by its integration into a class 1 integron that already had aadA1.
Compared with the previously described blaVIM-1-containing integrons in Greece,7 the gene cassette region of In3Mor differed by the presence of a sat1 gene cassette but also by eight point mutations: one point mutation in the aacA7 cassette, two point mutations in the 59-base element of the dfrA1 cassette and five point mutations upstream of the aadA1 cassette (at the intergenic region between sat1 and aadA1). It is probable that the new blaVIM-1-containing integron has arisen within our hospitals where VIM-1-producing Gram-negative pathogens have become common, indicating a widespread environmental reservoir of the respective gene. Epidemiological surveillance, restriction of carbapenem usage and adjustment of the infection control measures should be considered to monitor the spread of these determinants among our Gram-negative species.
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The nucleotide sequence of In3Mor has been submitted to the EMBL/GenBank nucleotide sequence database under accession number DQ522239 [GenBank] .
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None to declare.
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