Hospital outbreak of multiple clones of Pseudomonas aeruginosa carrying the unrelated metallo-ß-lactamase gene variants blaVIM-2 and blaVIM-4

S. Pournaras1,*, M. Maniati1, E. Petinaki1, L. S. Tzouvelekis2, A. Tsakris3, N. J. Legakis2 and A. N. Maniatis1

1 Department of Microbiology, University Hospital of Thessaly, Mezourlo, Larissa 412 22; 2 Department of Microbiology, Medical School and 3 Department of Microbiology, Faculty of Nursing, School of Health Sciences, University of Athens, Athens 115 27, Greece

Received 4 January 2003; returned 10 February 2003; revised 6 March 2003; accepted 6 March 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The possible contribution of metallo-ß-lactamases in the frequent detection of carbapenem-resistant Pseudomonas aeruginosa isolates in a tertiary Greek hospital in Central Greece was investigated.

Materials and methods: All carbapenem-resistant (imipenem- and/or meropenem-resistant) P. aeruginosa isolates recovered from separate patients during a 1 year period in the Clinical Microbiology Laboratory at the University Hospital of Thessaly, Larissa, Greece, were studied for metallo-ß-lactamases. They were tested by Etest MBL, PCR analysis and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA.

Results: A blaVIM gene was detected in 47 of the 53 (88.7%) carbapenem-resistant P. aeruginosa isolates. PFGE grouped the blaVIM-positive isolates in six unrelated genotypes; one type included two subtypes. Nucleotide sequencing of the PCR amplicons of a randomly selected isolate from each one of the seven subtypes, detected the variant sequences blaVIM-2 in four and blaVIM-4 in three cases, respectively. They were carried as single gene cassettes or along with an aminoglycoside resistance gene (aacA29a) in class 1 integrons.

Conclusions: These findings suggest that different strains of P. aeruginosa carrying unrelated metallo-ß-lactamase gene variants predominate in our hospital environment.

Keywords: carbapenemases, polyclonal, outbreak, Greece


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carbapenems have a potent antipseudomonal activity and are often used as a last resort for the treatment of infections due to multiresistant Pseudomonas isolates. However, pseudomonads may develop resistance to carbapenems mainly through diminished permeability, stable derepression of chromosomal AmpC ß-lactamases, or over-expression of the upregulating efflux system. During the last decade, carbapenem-resistance among hospital-acquired pseudomonads has been sporadically attributed to the production of the metallo-ß-lactamases (MBLs) IMP and VIM.1 These enzymes readily hydrolyse most ß-lactams, including carbapenems, and are encoded by genes carried in class 1 integrons. blaVIM genes have been detected more recently among carbapenem-resistant nosocomial pseudomonads. First, the gene blaVIM-1 was reported from Italy.2 A variant gene (blaVIM-2) with 90% nucleotide identity was detected soon after in France3 and Greece (reported as blaVIM-1 while unsequenced, but proved to be blaVIM-2),4,5 and also in very distant regions.6 Two variants of the blaVIM genes have been detected, one of them (blaVIM-3) exhibiting two nucleotide differences to blaVIM-26 and lately the gene blaVIM-4 with a single amino acid difference from blaVIM-1.7 Here we report the characteristics of a large outbreak due to carbapenemase-producing P. aeruginosa isolates in a tertiary Greek hospital with a high prevalence of carbapenem-resistant pseudomonads.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the study period (September 2001–September 2002), all carbapenem- (imipenem- and/or meropenem-) resistant (MICs > 8 mg/L) Pseudomonas aeruginosa non-replicate isolates recovered consecutively from nosocomial infection samples of patients hospitalized in the University Hospital of Thessaly, Larissa, Greece, were included. The isolates were identified at the species level using the Vitek system (bioMérieux, Marcy l’Étoile, France) and the API 20NE system (bioMérieux).

The susceptibility of the isolates to a range of anti-pseudomonal antimicrobials was also determined by the Vitek system according to the recommendations of the manufacturer. The MICs of imipenem and meropenem for the isolates were determined in addition by an agar dilution method using Mueller–Hinton agar (Oxoid, Basingstoke, UK) and a final inoculum of 104 cfu/mL. The possible presence of an MBL among carbapenem-resistant P. aeruginosa was tested by the Etest MBL (AB Biodisk, Solna, Sweden).

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA of P. aeruginosa isolates was carried out with a CHEF-DRIII system (Bio-Rad, Hemel Hempstead, UK), as described elsewhere.4 Banding patterns of the strains were compared visually. The possible carriage of the carbapenemase-encoding genes blaIMP and blaVIM was tested by PCR using primers and amplification conditions that have been described previously.4,8 Primers specific for blaVIM-1 and blaVIM-2 genes were also used.6 PCR assays combining primers specific for conserved 5'-CS and 3'-CS sequences5 with the blaVIM-1- and blaVIM-2-specific primers were also carried out to investigate the possible association of the MBL gene with a class 1 integron. Positive controls for detecting blaIMP and blaVIM genes were, respectively, the strains P. aeruginosa 101/1477 (kindly provided by Dr N. Woodford, PHLS, UK), and P. aeruginosa 174.5

Nucleotide sequencing of both strands of the PCR products was performed on amplicons derived using the primers 5'-CS and blaVIM-1 (reverse), and 5'-CS and blaVIM-2 (reverse). The amplicons were sequenced on an ABI PRISM 377 DNA sequence analyser (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the study period, the rate of resistance of P. aeruginosa to carbapenems was 35% and 53 non-replicate carbapenem-resistant isolates were recovered. Most of the isolates exhibited cross-resistance to all tested ß-lactams including aztreonam, implying that in cases of drug-degrading metallo-ß-lactamases, another resistance mechanism was concomitant (increased efflux, OXA-type enzyme or AmpC hyperproduction). The MICs of imipenem for the isolates ranged from 1 to >256 mg/L and of meropenem from 0.5 to 128 mg/L. Of the 53 pseudomonads, 47 (88.7%) exhibited resistance to both carbapenems whereas four were resistant only to imipenem and two only to meropenem. In 44 of these 47 Pseudomonas isolates, the Etest MBL gave a phenotype (imipenem was potentiated greater than eight-fold by the EDTA) that according to the instructions of the manufacturer was strongly indicative of the presence of a metallo-ß-lactamase. In three isolates resistant to both carbapenems, the ratio of the MICs of imipenem/imipenem-EDTA was 6–8, whereas there was essentially no decrease in the imipenem MICs by EDTA for the remaining six P. aeruginosa isolates that were resistant to imipenem or meropenem.

The PCR for the blaVIM gene was repeatedly positive (PCR products of 261 bp) and for the gene blaIMP, negative, in all 47 isolates that were resistant to both carbapenems, and negative for both genes in the six strains that were resistant to imipenem or meropenem. The blaVIM gene was not detected in any of the seven carbapenem-sensitive isolates that were used as negative controls in the PCR experiments. Six unrelated genotypes, each containing three to 16 isolates, were detected among the 47 blaVIM-positive isolates by PFGE (Table 1 and Figure 1). Only one clone (PFGE type I) was relatively more common, including 16 isolates. One genotype (PFGE type III) contained two subtypes with three-band differences from each other, whereas isolates that belonged to the other genotypes were indistinguishable. Four distinct genotypes were also identified among the six isolates that were resistant to imipenem or meropenem and these genotypes were distinct from those detected among the blaVIM-positive isolates by more than five bands (data not shown).


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Table 1.  Characteristics of the 47 blaVIM-positive P. aeruginosa isolates recovered at the University Hospital of Thessaly during 2001–02
 


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Figure 1. PFGE of XbaI-restricted genomic DNA of carbapenemase-producing P. aeruginosa isolates, representative of each one of the detected genotypes. The origin and the PFGE type of each isolate are shown in Table 1. Lane M, molecular mass markers (kb).

 
From each of the seven PFGE types and subtypes, a randomly selected blaVIM-positive isolate was further evaluated in order to investigate the association of the blaVIM gene with a class 1 integron. PCR with combinations of primers for the amplification of the variable region showed that the blaVIM genes were consistently carried in class 1 integrons. In more detail, the size of the amplicon with primers 5'-CS and blaVIM-1-reverse was about 950 bp in three cases, whereas with primers 5'-CS and blaVIM-2-reverse was approximately 1400 bp in one case and 940 bp in two cases. Given that the size of the amplicon with the primers blaVIM-1-forward or blaVIM-2-forward and 3'-CS was around 920 bp in all seven cases, it could be assumed that a sole (blaVIM) gene cassette in six isolates and two cassettes in another isolate were carried within these class 1 integrons. Sequencing of the amplicons showed that three isolates with a 950 bp amplicon carried the variant blaVIM-1 gene, blaVIM-4.7 Three of the remaining amplicons had nucleotide sequences of the blaVIM-2 alleles that were identical to each other and to that of the original VIM-2 producer, P. aeruginosa COL-1,3 whereas sequencing of the last amplicon showed that the integron also carried an identical blaVIM-2 gene, along with an aminoglycoside resistance gene (aacA29a), located upstream of the carbapenemase-encoding gene. The AAC(6')-29a protein had a 100% amino acid identity with that described previously in In59 of the strain RON-2.9 The characteristics, the PFGE type and the variant blaVIM sequence of the seven isolates representative of each clone are shown in Table 1.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The appearance of acquired MBLs among clinical isolates of Enterobacteriaceae and non-fermenter species such as P. aeruginosa, is considered one of the most worrisome developments in the field of bacterial resistance. Reports from the Far East have shown that pseudomonads carrying blaVIM genes usually belong to common clones.6 Also, studies from several European countries have reported that VIM-1 and VIM-2 MBLs are mostly detected among clonally related P. aeruginosa.4,10 Thus, it has been indicated that horizontal spread of the blaVIM genes was less likely to have contributed to their dissemination. However, blaVIM is encoded by an integron-borne gene cassette and therefore has the potential to disseminate.2,3 In the present study, blaVIM-carrying P. aeruginosa isolates were detected in eight different departments of the hospital. Also, as many as six genotypically different strains were identified, indicating that the prevalence of carbapenemase-encoding genes was mainly due to gene spread and to a lesser extent to clonal dissemination.

It is noteworthy that the MBL found in our region is not only VIM-2 type, which is scattered in several European countries,3,5 but also VIM-4. The identification of P. aeruginosa strains that carried either of the variant genes blaVIM-2 or blaVIM-4, in the same setting, taken together with the finding that the host pseudomonads were genetically distinct, possibly indicates a wide distribution of VIM-type genes in the hospital environment. This is further supported by the fact that several integrons of different phylogeny have been described to carry the blaVIM genes.2,9 Our carbapenemase-producing P. aeruginosa isolates carried blaVIM genes mostly as a sole gene cassette in a class 1 integron as reported previously for In56 of the strain COL-1,3 unlike most integrons containing blaVIM genes that usually carry two or more gene cassettes.2,9 In our study, blaVIM was detected only once together with another gene (aacA29a); the latter gene cassette was previously reported only in In59, where it was followed at its 3' end by gene cassettes blaVIM-2 and aacA29b.9

It is of particular concern that VIM enzymes have been detected in widely separated Eurasian countries. The results of this study suggest that if effective infection control approaches are not stringently applied at this stage, horizontal but also clonal spread of VIM-producing Pseudomonas might dramatically increase in our region.


    Footnotes
 
* Corresponding author. Tel: +30-2-410-682509; Fax: +30-2-410-682508; E-mail: pournaras{at}med.uth.gr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Livermore, D. M. & Woodford, N. (2000). Carbapenemases: a problem in waiting? Current Opinions in Microbiology 3, 489–95.[CrossRef]

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3 . Poirel, L., Naas, T., Nicolas, D., Collet, L., Bellais, S., Cavallo, J.-D. et al. (2000). Characterization of VIM-2, a carbapenem-hydrolyzing metallo-ß-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]

4 . Tsakris, A., Pournaras, S., Woodford, N., Palepou, M.-F. I., Babini, G. S., Douboyas, J. et al. (2000). Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. Journal of Clinical Microbiology 38, 1290–2.[Abstract/Free Full Text]

5 . Mavroidi, A., Tsakris, A., Tzelepi, E., Pournaras, S., Loukova, V. & Tzouvelekis, L. S. (2000). Carbapenem-hydrolysing VIM-2 metallo-ß-lactamase in Pseudomonas aeruginosa from Greece. Journal of Antimicrobial Chemotherapy 46, 1041–3.[Free Full Text]

6 . Yan, J.-J., Hsueh, P.-R., Ko, W.-C., Luh, K.-T., Tsai, S.-H., Wu, H.-M. et al. (2001). Metallo-ß-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme. Antimicrobial Agents and Chemotherapy 45, 2224–8.[Abstract/Free Full Text]

7 . Pournaras, S., Tsakris, A., Maniati, M., Tzouvelekis, L. S. & Maniatis, A. N. (2002). Identification of a novel variant of the metallo-ß-lactamase gene blaVIM-1 (blaVIM-4), in a clinical strain of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 46, 4026–8.[Abstract/Free Full Text]

8 . Senda, K., Arakawa, Y., Ichiyama, S., Nakashima, K., Ito, H., Ohsuka, S. et al. (1996). PCR detection of metallo-ß-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum ß-lactams. Journal of Clinical Microbiology 34, 2909–13.[Abstract]

9 . Poirel, L., Lambert, T., Turkoglu, S., Ronco, E., Gaillard, J.-L. & Nordmann, P. (2001). Characterization of class 1 integrons from Pseudomonas aeruginosa that contain the blaVIM-2 carbapenem-hydrolyzing ß-lactamase gene and of two novel aminoglycoside resistance gene cassettes. Antimicrobial Agents and Chemotherapy 45, 546–52.[Abstract/Free Full Text]

10 . Cornaglia, G., Mazzariol, A., Lauretti, L., Rossolini, G. M. & Fontana, R. (2000). Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo-beta-lactamase. Clinical Infectious Diseases 31, 1119–25.[CrossRef][ISI][Medline]