Carbapenem-hydrolysing VIM-2 metallo- ß-lactamase in Pseudomonas aeruginosa from Greece

A. Mavroidia, A. Tsakrisb, E. Tzelepia, S. Pournarasc, V. Loukovad and L. S. Tzouvelekisd,*

a Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens; b Department of Microbiology, Medical School, Aristotle University of Thessaloniki; c Department of Microbiology, AHEPA University Hospital, Thessaloniki; d Laboratory of Antimicrobial Agents, Department of Microbiology, Medical School, University of Athens, M. Asias 75, Athens 11527, Greece

Sir,

Four integron-borne metallo-ß-lactamases, IMP-1, IMP-2, VIM-1 and VIM-2, conferring resistance to carbapenems, have been described.14 The respective bla genes were in the form of cassettes and located within the variable region of class 1 integrons. The IMP-1 ß-lactamase has been detected in various Gram-negative species in Japan,1 the IMP-2 ß-lactamase in a clinical isolate of Acinetobacter baumannii in Italy2 and the closely related VIM-1 and VIM-2 ß-lactamases in Pseudomonas aeruginosa strains from Italy and France, respectively.3,4 Recently, an outbreak caused by a carbapenem-resistant P. aeruginosa strain was described in a Greek hospital. Based on polymerase chain reaction (PCR) assays, it was assumed that resistance was due to the production of a VIM-like metallo-ß-lactamase.5 In order to characterize the respective bla gene and its carrier, a representative isolate, P. aeruginosa 174, was studied.

Escherichia coli JM109 was used in transformation experiments. The plasmids pMON-382016 and pBCSK(+) (Stratagene) were used for cloning and expression of ß-lactamases. Susceptibility testing was performed by an agar dilution method as described by Tsakris et al.5 Plasmid DNA from P. aeruginosa was obtained by various methods as described by Lauretti et al.3 ß-Lactamase preparations were obtained by ultrasonic treatment of cell suspensions. Isoelectric focusing (IEF) was performed in polyacrylamide gels containing ampholytes (pH range 3.5–9.5). ß-Lactamases were detected with nitrocefin (Oxoid). Enzymatic activity was inhibited in situ by application, for 10 min, of paper strips soaked in 0.1 M EDTA. Wholecell DNA from P. aeruginosa was used as template in PCR assays. The following primers were used: VIM-F (5'-AGTGGTGAGTATCCGACAG-3') and VIM-R (5'-ATGAAAGTGCGTGGAGAC-3') for blaVIM-specific amplification; 5'-CS (5'-GGCATCCAAGCAGCAAG-3') and 3'-CS (5'-AAGCAGACTTGACCTGA-3') for amplification of the variable region of the class 1 integron. An integrase-specific primer, INT-F (5'-CGTTCCATACAGAAGCTG-3'; nucleotides 657–674 of GenBank accession no. Y18050), was also used. PCR products were cloned into XcmI-digested pMON-38201, and appropriate fragments were subcloned into pBCSK(+). Nucleotide sequences were determined using Sequenase 2.0 (USB, Cleveland, OH, USA).

P. aeruginosa 174 was highly resistant to carbapenems and to other ß-lactams except aztreonam (TableGo). Electrophoresis of plasmid DNA preparations showed the presence of at least three plasmids with sizes ranging from 10 to >90 kb. Transformation of E. coli with these preparations did not yield clones resistant to ß-lactams. IEF showed that the strain produced one main ß-lactamase (pI 5.6) that was inhibited in situ by EDTA. The isolate was positive in PCR assays with blaVIM-specific oligonucleotides. By performing PCRs using combinations of the VIM- and integronspecific primers, it was found that blaVIM was associated with a class 1 integron. With INT-F and 3'-CS primers, a product of 1529 bp was obtained. This amplicon was cloned into pMON-38201 resulting in plasmid pMON-VIM. A BamHI–EcoRI fragment (1551 bp) was ligated into the polycloning site of pBCSK(+), yielding plasmid pB-VIM. Transformation of E. coli with pB-VIM yielded clones resistant to ß-lactams that produced an EDTA-inhibited ß-lactamase with a pI of 5.6. The transformants were less resistant to imipenem, meropenem and cefepime than P. aeruginosa 174 (TableGo). The nucleotide sequence of the BamHI–EcoRI insert of pB-VIM was identical to a segment of integron In56 (nucleotides 721–2250 of GenBank accession no. AF191564) that included a part of an intI1 integrase gene and the blaVIM-2 gene cassette.4 The sequence downstream of the 59-be (59-base element) was not determined. The size of the PCR products using various pairs of primers showed that blaVIM-2 was the sole cassette present in the variable region of this class 1 integron as in In56.


View this table:
[in this window]
[in a new window]
 
Table. MICs (mg/L) of ß-lactam antibiotics for a P. aeruginosa clinical strain and an E. coli clone producing VIM-2 ß-lactamase
 
It is shown here that high-level resistance to carbapenems in an outbreak strain of P. aeruginosa from a Greek hospital was associated with production of VIM-2. This enzyme and the similar enzyme VIM-1, also found in P. aeruginosa, constitute a novel cluster of class B ß-lactamases distantly related to the carbapenem-hydrolysing ß-lactamase B-II of Bacillus cereus. The unsuccessful attempts to transfer blaVIM-2 indicated that the respective integron was chromosomal, while the blaVIM-2 integron described by Poirel et al.4 was plasmid borne. However, the possibility that the VIM-2 enzyme found here was also of plasmid origin cannot be ruled out, given the difficulty in obtaining intact plasmid DNA from P. aeruginosa. The similar structure of the integrons and, especially, the identity of the nucleotide sequence of the bla gene cassette to that of blaVIM-2, were unexpected. Patients' records did not indicate any epidemiological association between the VIM-2-producing P. aeruginosa strain from Greece and that isolated in France.4 It appears that the VIM-2-encoding integron had been acquired from P. aeruginosa strains on at least two independent occasions; its distribution may, therefore, be wide. Notably, VIM-producing P. aeruginosa strains have emerged in southern France, Italy and Greece. This may reflect an intensive use of carbapenems in these areas. Recent records from Greek hospitals showed a high incidence of imipenem-resistant P. aeruginosa strains. The present findings suggest that spread of metallo-ß-lactamases may have contributed to this development.

Notes

J Antimicrob Chemother 2000; 46: 1041–1043

1 Corresponding author. Tel: +30-1-7709180; Fax: +30-1-7785638; E-mail: ltzouvel{at}cc.uoa.gr Back

References

1 . 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]

2 . Riccio, M. L., Franceschini, N., Boschi, L., Caravelli, B., Cornaglia, G., Fontana, R. et al. (2000). Characterization of the metallo- ß-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]

3 . Lauretti, L., Riccio, M. L., Mazzariol, A., Cornaglia, G., Amicosante, G., Fontana, R. et al. (1999). Cloning and characterization of blaVIM, a new integron-borne metallo-ß-lactamase gene from Pseudomonas aeruginosa clinical isolate. Antimicrobial Agents and Chemotherapy 43, 1584–90.[Abstract/Free Full Text]

4 . 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]

5 . 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]

6 . Borovkov, A. Y. & Rivkin, M. I. (1997). XcmI-containing vector for direct cloning of PCR products. BioTechniques 22, 812–4.[ISI][Medline]