This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pournaras, S. Articles by Maniatis, A. N. Search for Related Content PubMed PubMed Citation Articles by Pournaras, S. Articles by Maniatis, A. N.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, December 2002, p. 4026-4028, Vol. 46, No. 12
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.12.4026-4028.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Novel Variant (bla_VIM-4) of the Metallo-ß-Lactamase Gene bla_VIM-1 in a Clinical Strain of Pseudomonas aeruginosa

Department of Medical Microbiology, University of Thessaly, Larissa,¹ Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki,² Department of Microbiology, Medical School, University of Athens, Athens, Greece³

Received 11 April 2002/ Returned for modification 16 May 2002/ Accepted 11 September 2002

	ABSTRACT

Top Abstract Text References

 
A Pseudomonas aeruginosa isolate highly resistant to carbapenems was collected from a patient with postsurgical cerebrospinal infection in Greece. The isolate carried a class 1 integron that contained as a sole cassette the gene bla_VIM-4, a novel variant of bla_VIM-1, with one nucleotide difference resulting in a Ser-to-Arg change at amino acid position 175 of the VIM-1 enzyme. This is the first detection of a VIM-1 variant after its appearance in Italy.

	TEXT

Top Abstract Text References

 
There is an increasing number of studies reporting on the emergence of Pseudomonas aeruginosa strains that produce acquired class B metallo-ß-lactamases (MBLs), which invariably hydrolyze carbapenems. So far, two distinct MBL types, IMP (11) and VIM (2), have been described. Although these ß-lactamase types share less than 40% amino acid sequence identity, they exhibit comparable kinetic properties, inactivating virtually all ß-lactams except monobactams (7). Additionally, both bla_IMP- and bla_VIM-type genes are carried as gene cassettes by class 1 integrons. Of the VIM MBLs, VIM-1 was the first to be identified, but up to now it had been described only for P. aeruginosa and Achromobacter xylosoxidans from Italy (1, 4, 16). In contrast, VIM-2 enzymes have been detected in clinical Pseudomonas isolates in several Mediterranean countries and the Far East (5, 8, 12, 14, 15, 19). A variant of VIM-2 differing by two amino acids, VIM-3, was also detected in clinical isolates of P. aeruginosa from Taiwan (19). In this study we report on the identification of bla_VIM-4, a novel variant of the VIM-1 MBL gene, found in a clinical strain of P. aeruginosa.

The carbapenem-resistant P. aeruginosa strain P1936 was studied. P. aeruginosa ATCC 27853 was used as a control strain. P. aeruginosa P1936 was isolated on 23 April 2001 in the University Hospital of Thessaly, Larissa, Greece. The strain was repeatedly recovered as a sole isolate from the ventriculo-peritoneal catheter and the cerebrospinal fluid of a 25-year-old man 20 days after a neurosurgical operation for the management of a posttraumatic hydrocephalus. The patient was readmitted to the hospital 18 days after the operation with high fever, respiratory distress, and clinical signs of meningitis. He also had simultaneous positive blood cultures that yielded Acinetobacter baumannii and Enterococcus faecium but not P. aeruginosa. Prior to the isolation of these microorganisms, the patient had been treated with multiple antibiotic courses that included ceftazidime, imipenem, ciprofloxacin, clindamycin, and metronidazole. Based on the results of the susceptibility tests, the patient was treated with imipenem, aztreonam, amikacin, and vancomycin, and the catheter was removed. The patient's clinical condition gradually improved, and the cultures became negative.

The susceptibility of P. aeruginosa P1936 to several antimicrobial agents including ß-lactams, tobramycin, gentamicin, amikacin, netilmicin, and ciprofloxacin was assessed by disk diffusion (10). The MICs of ticarcillin-clavulanic acid, piperacillin-tazobactam, ceftazidime, aztreonam, imipenem, and meropenem were determined by an agar dilution method (9).

Extraction of plasmid DNA was carried out by using the Qiagen Plasmid kit (Hilden, Germany) according to the manufacturer's instructions. These extracts were used to transform Escherichia coli DH5 cells with transformant selection on Mueller-Hinton agar containing ampicillin (50 µg/ml).

bla_VIM-type genes were detected by PCR with a pair of consensus primers under amplification conditions as described previously (18). Primers specific for bla_VIM-1 and bla_VIM-2 were also used (19). PCR assays combining primers specific for conserved 5'-CS and 3'-CS sequences (6) with the bla_VIM-specific ones were also performed to investigate the possible association of the MBL gene with a class 1 integron.

Nucleotide sequencing of both strands of the PCR products derived using the 5'-CS and bla_VIM-1 (reverse) primers (19) was done by using an ABI Prism 377 DNA sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.).

ß-Lactamase extracts from P. aeruginosa P1936 and the control strains were obtained by ultrasonic treatment of cell suspensions and clarified by centrifugation. Protein concentrations were determined with a protein assay kit (Bio-Rad, Richmond, Calif.). Carbapenemase activity was estimated by spectrophotometry as described previously (4). The maximum hydrolysis rate of imipenem was monitored at 299 nm and expressed as units of activity (1 unit is defined as 1 pmol of imipenem hydrolyzed per min per µg of protein). Isoelectric focusing (IEF) was performed in polyacrylamide gels containing ampholytes (pH range, 3.5 to 9.5). ß-Lactamase activity was visualized with nitrocefin.

P. aeruginosa P1936 was examined in the context of the regular screening for MBL producers among carbapenem-resistant nonfermenters (mostly Pseudomonas and Acinetobacter spp.) that have sporadically appeared in the University Hospital of Thessaly since 1999. The strain was highly resistant to all ß-lactam antibiotics except aztreonam, which retained a moderate activity (MIC, 16 µg/ml). It was also resistant to all four aminoglycosides tested and to ciprofloxacin. Transformation experiments did not yield any ß-lactam-resistant Escherichia coli clone.

Total DNA from P1936 was positive in the PCR assay using the consensus bla_VIM primers. PCR products of similar size (261 bp) were also detected with 12 other carbapenem-resistant P. aeruginosa strains isolated during the time period from September 1999 to August 2001 in this hospital (data not shown). While the latter strains were all found to carry bla_VIM-2-type genes, P. aeruginosa P1936 was positive for bla_VIM-1, producing in the respective PCR assay an amplicon of the expected size (920 bp). PCRs using the 5'-CS and bla_VIM-1 (reverse) primers as well as the 3'-CS and bla_VIM-1 (forward) primers yielded products of similar sizes (approximately 950 bp), indicating that bla_VIM-1 was the sole gene cassette in the variable region of this integron.

The DNA sequence of the PCR amplicon encompassed by the 5'-CS and bla_VIM-1 (reverse) oligonucleotides showed an 870-bp segment that corresponded to a fraction of the VIM-1 integron (from nucleotide 1185 to nucleotide 2054; GenBank accession no. Y18050) originally described by Lauretti et al. (4). The sequence included a part of the 5' conserved segment followed by a bla_VIM-1-type gene that possessed a C instead of an A at nucleotide position 1864 (numbering according to Y18050). This transversion resulted in a Ser-to-Arg change at position 175 (numbering according to reference 4) of the VIM-1 MBL. It is notable that position 175 is also occupied by Arg in VIM-2 and VIM-3 enzymes. Residue 175 (position 228 in the recently proposed standard numbering scheme for class B ß-lactamases [3]) is not among those considered significant for ß-lactam hydrolysis by VIM and IMP MBLs (3). This novel bla_VIM-1 variant was designated bla_VIM-4.

Photometric assays showed that crude ß-lactamase preparations from P. aeruginosa P1936 exhibited an activity against imipenem that was comparable to that observed with extracts containing VIM-2 (data not shown). It was therefore assumed that bla_VIM-4 was expressed and was, at least partly, responsible for the high-level resistance of P. aeruginosa P1936 to carbapenems. The enzyme, however, could not be detected in the IEF experiments. Prolonged incubation of the IEF gels with nitrocefin indicated a weak ß-lactamase activity at a pI of 8.8, which probably represented the chromosomal cephalosporinase. Other major ß-lactamase bands were not observed.

Initially, IMP MBLs were restricted to the Far East, while VIM types were found exclusively in southern Europe (7, 13, 17). However, during the last few years, VIM-type genes carried by a variety of integron structures have been found in various gram-negative rods in the Far East (5, 19). VIM-4- and VIM-1-encoding genes differ by only one nucleotide and should be considered alleles derived from a common ancestor. On the other hand, bla_VIM-4 was, most likely, the sole gene in the variable region of the detected class 1 integron while the bla_VIM-1 integrons found in sporadic isolates in Italy included additional resistance gene cassettes (4, 16), indicating different phylogenies. Given also that there was not any apparent epidemiological association of the case described here with those in Italy, it can be assumed that the bla_VIM-4-encoding integron emerged independently.

The results of the present study, taken together with previous findings (18), indicate that VIM-producing P. aeruginosa strains have already been established in Greek hospitals. Epidemiological surveillance in the major tertiary care institutions, restriction of carbapenem usage, and adjustment of the infection control measures should be considered.

Nucleotide sequence accession number. The GenBank accession no. of the nucleotide sequence reported here is AY135661.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Microbiology, Medical School, University of Thessaly, 412 22 Larissa, Greece. Phone: 30 410 682509. Fax: 30 410 682508. E-mail: spournar{at}otenet.gr.

	REFERENCES

Top Abstract Text References

 

1. Cornaglia, G., A. Mazzariol, L. Lauretti, G. M. Rossolini, and R. Fontana. 2000. Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo-ß-lactamase. Clin. Infect. Dis. 31:1119-1125.[CrossRef][Medline]
2. Franceschini, N., B. Caravelli, J. D. Docquier, M. Galleni, J.-M. Frere, G. Amicosante, and G. M. Rossolini. 2000. Purification and biochemical characterization of the VIM-1 metallo-beta-lactamase. Antimicrob. Agents Chemother. 44:3003-3007.[Abstract/Free Full Text]
3. Galleni, M., J. Lamotte-Brasseur, G. M. Rossolini, J. Spencer, O. Dideberg, J.-M. Frere, and The Metallo-ß-Lactamase Working Group. 2001. Standard numbering scheme for class B ß-lactamases. Antimicrob. Agents Chemother. 45:660-663.[Free Full Text]
4. Lauretti, L., M. L. Riccio, A. Mazzariol, G. Cornaglia, G. Amicosante, R. Fontana, and G. M. Rossolini. 1999. Cloning and characterization of bla_VIM, a new integron-borne metallo-ß-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 43:1584-1590.[Abstract/Free Full Text]
5. Lee, K., J. B. Lim, J. H. Yum, D. Yong, Y. Chong, J. M. Kim, and D. M. Livermore. 2002. bla_VIM-2 cassette-containing novel integrons in metallo-ß-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital. Antimicrob. Agents Chemother. 46:1053-1058.[Abstract/Free Full Text]
6. Levesque, C., L. Piche, C. Larose, and P. H. Roy. 1995. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob. Agents Chemother. 39:185-191.[Abstract]
7. Livermore, D. M., and N. Woodford. 2000. Carbapenemases: a problem in waiting? Curr. Opin. Microbiol. 3:489-495.[CrossRef][Medline]
8. Mavroidi, A., A. Tsakris, E. Tzelepi, S. Pournaras, V. Loukova, and L. S. Tzouvelekis. 2000. Carbapenem-hydrolysing VIM-2 metallo-ß-lactamase in Pseudomonas aeruginosa from Greece. J. Antimicrob. Chemother. 46:1041-1043.[Free Full Text]
9. National Committee for Clinical Laboratory Standards. 1999. Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically. Approved standard M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
10. National Committee for Clinical Laboratory Standards. 2001. Performance standards for antimicrobial disk susceptibility tests. Approved standard M2-A7. National Committee for Clinical Laboratory Standards, Wayne, Pa.
11. Osano, E., Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato. 1994. Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance. Antimicrob. Agents Chemother. 38:71-78.[Abstract/Free Full Text]
12. Pallecchi, L., M. L. Riccio, J. D. Docquier, R. Fontana, and G. M. Rossolini. 2001. Molecular heterogeneity of bla(VIM-2)-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-beta-lactamase. FEMS Microbiol. Lett. 195:145-150.[Medline]
13. Poirel, L., T. Naas, D. Nicolas, L. Collet, S. Bellais, J.-D. Cavallo, and P. Nordmann. 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. Antimicrob. Agents Chemother. 44:891-897.[Abstract/Free Full Text]
14. Poirel, L., T. Lambert, S. Turkoglu, E. Ronco, J. Gaillard, and P. Nordmann. 2001. Characterization of class 1 integrons from Pseudomonas aeruginosa that contain the bla_VIM-2 carbapenem-hydrolyzing beta-lactamase gene and of two novel aminoglycoside resistance gene cassettes. Antimicrob. Agents Chemother. 45:546-552.[Abstract/Free Full Text]
15. Prats, G., E. Miro, B. Mirelis, L. Poirel, S. Bellais, and P. Nordmann. 2002. First isolation of a carbapenem-hydrolyzing ß-lactamase in Pseudomonas aeruginosa in Spain. Antimicrob. Agents Chemother. 46:932-933.[Free Full Text]
16. Riccio, M. L., L. Pallecchi, R. Fontana, and G. M. Rossolini. 2001. In70 of plasmid pAX22, a bla_VIM-1-containing integron carrying a new aminoglycoside phosphotransferase gene cassette. Antimicrob. Agents Chemother. 45:1249-1253.[Abstract/Free Full Text]
17. Rossolini, G. M., M. L. Riccio, G. Cornaglia, L. Pagani, C. Lagatolla, L. Selan, and R. Fontana. 2000. Carbapenem-resistant Pseudomonas aeruginosa with acquired bla_VIM metallo-ß-lactamase determinants, Italy. Emerg. Infect. Dis. 3:312-313.
18. Tsakris, A., S. Pournaras, N. Woodford, M.-F. I. Palepou, G. S. Babini, J. Douboyas, and D. M. Livermore. 2000. Outbreak of infections caused by Pseudomonas aeruginosa producing VIM-1 carbapenemase in Greece. J. Clin. Microbiol. 38:1290-1292.[Abstract/Free Full Text]
19. Yan, J.-J., P.-R. Hsueh, W.-C. Ko, K.-T. Luh, S.-H. Tsai, H.-M. Wu, and J.-J. Wu. 2001. Metallo-ß-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme. Antimicrob. Agents Chemother. 45:2224-2228.[Abstract/Free Full Text]

This article has been cited by other articles:

• Li, H., Toleman, M. A., Bennett, P. M., Jones, R. N., Walsh, T. R. (2008). Complete Sequence of p07-406, a 24,179-Base-Pair Plasmid Harboring the blaVIM-7 Metallo-{beta}-Lactamase Gene in a Pseudomonas aeruginosa Isolate from the United States. Antimicrob. Agents Chemother. 52: 3099-3105 [Abstract] [Full Text]  
• Strateva, T., Ouzounova-Raykova, V., Markova, B., Todorova, A., Marteva-Proevska, Y., Mitov, I. (2007). Problematic clinical isolates of Pseudomonas aeruginosa from the university hospitals in Sofia, Bulgaria: current status of antimicrobial resistance and prevailing resistance mechanisms. J Med Microbiol 56: 956-963 [Abstract] [Full Text]  
• Giske, C. G., Libisch, B., Colinon, C., Scoulica, E., Pagani, L., Fuzi, M., Kronvall, G., Rossolini, G. M. (2006). Establishing Clonal Relationships between VIM-1-Like Metallo-{beta}-Lactamase-Producing Pseudomonas aeruginosa Strains from Four European Countries by Multilocus Sequence Typing. J. Clin. Microbiol. 44: 4309-4315 [Abstract] [Full Text]  
• Horianopoulou, M., Legakis, N. J., Kanellopoulou, M., Lambropoulos, S., Tsakris, A., Falagas, M. E. (2006). Frequency and predictors of colonization of the respiratory tract by VIM-2-producing Pseudomonas aeruginosa in patients of a newly established intensive care unit.. J Med Microbiol 55: 1435-1439 [Abstract] [Full Text]  
• Peleg, A. Y., Bell, J. M., Hofmeyr, A., Wiese, P. (2006). Inter-country transfer of Gram-negative organisms carrying the VIM-4 and OXA-58 carbapenem-hydrolysing enzymes. J Antimicrob Chemother 57: 794-795 [Full Text]  
• Fiett, J., Baraniak, A., Mrowka, A., Fleischer, M., Drulis-Kawa, Z., Naumiuk, L., Samet, A., Hryniewicz, W., Gniadkowski, M. (2006). Molecular Epidemiology of Acquired-Metallo-{beta}-Lactamase-Producing Bacteria in Poland. Antimicrob. Agents Chemother. 50: 880-886 [Abstract] [Full Text]  
• Gacar, G. G., Midilli, K., Kolayli, F., Ergen, K., Gundes, S., Hosoglu, S., Karadenizli, A., Vahaboglu, H. (2005). Genetic and Enzymatic Properties of Metallo-{beta}-Lactamase VIM-5 from a Clinical Isolate of Enterobacter cloacae. Antimicrob. Agents Chemother. 49: 4400-4403 [Abstract] [Full Text]  
• Walsh, T. R., Toleman, M. A., Poirel, L., Nordmann, P. (2005). Metallo-{beta}-Lactamases: the Quiet before the Storm?. Clin. Microbiol. Rev. 18: 306-325 [Abstract] [Full Text]  
• Riccio, M. L., Pallecchi, L., Docquier, J.-D., Cresti, S., Catania, M. R., Pagani, L., Lagatolla, C., Cornaglia, G., Fontana, R., Rossolini, G. M. (2005). Clonal Relatedness and Conserved Integron Structures in Epidemiologically Unrelated Pseudomonas aeruginosa Strains Producing the VIM-1 Metallo-{beta}-Lactamase from Different Italian Hospitals. Antimicrob. Agents Chemother. 49: 104-110 [Abstract] [Full Text]  
• Toleman, M. A., Biedenbach, D., Bennett, D. M. C., Jones, R. N., Walsh, T. R. (2005). Italian metallo-{beta}-lactamases: a national problem? Report from the SENTRY Antimicrobial Surveillance Programme. J Antimicrob Chemother 55: 61-70 [Abstract] [Full Text]  
• Libisch, B., Gacs, M., Csiszar, K., Muzslay, M., Rokusz, L., Fuzi, M. (2004). Isolation of an Integron-Borne blaVIM-4 Type Metallo-{beta}-Lactamase Gene from a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate in Hungary. Antimicrob. Agents Chemother. 48: 3576-3578 [Abstract] [Full Text]  
• Koh, T. H., Wang, G. C. Y., Sng, L.-H. (2004). IMP-1 and a Novel Metallo-{beta}-Lactamase, VIM-6, in Fluorescent Pseudomonads Isolated in Singapore. Antimicrob. Agents Chemother. 48: 2334-2336 [Abstract] [Full Text]  
• Patzer, J., Toleman, M. A., Deshpande, L. M., Kaminska, W., Dzierzanowska, D., Bennett, P. M., Jones, R. N., Walsh, T. R. (2004). Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001). J Antimicrob Chemother 53: 451-456 [Abstract] [Full Text]  
• Luzzaro, F., Docquier, J.-D., Colinon, C., Endimiani, A., Lombardi, G., Amicosante, G., Rossolini, G. M., Toniolo, A. (2004). Emergence in Klebsiella pneumoniae and Enterobacter cloacae Clinical Isolates of the VIM-4 Metallo-{beta}-Lactamase Encoded by a Conjugative Plasmid. Antimicrob. Agents Chemother. 48: 648-650 [Abstract] [Full Text]  
• Toleman, M. A., Rolston, K., Jones, R. N., Walsh, T. R. (2004). blaVIM-7, an Evolutionarily Distinct Metallo-{beta}-Lactamase Gene in a Pseudomonas aeruginosa Isolate from the United States. Antimicrob. Agents Chemother. 48: 329-332 [Abstract] [Full Text]  
• Giske, C. G., Rylander, M., Kronvall, G. (2003). VIM-4 in a Carbapenem-Resistant Strain of Pseudomonas aeruginosa Isolated in Sweden. Antimicrob. Agents Chemother. 47: 3034-3035 [Full Text]  
• Walsh, T. R., Toleman, M. A., Hryniewicz, W., Bennett, P. M., Jones, R. N. (2003). Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program. J Antimicrob Chemother 52: 116-119 [Abstract] [Full Text]  
• Pournaras, S., Maniati, M., Petinaki, E., Tzouvelekis, L. S., Tsakris, A., Legakis, N. J., Maniatis, A. N. (2003). Hospital outbreak of multiple clones of Pseudomonas aeruginosa carrying the unrelated metallo-{beta}-lactamase gene variants blaVIM-2 and blaVIM-4. J Antimicrob Chemother 51: 1409-1414 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pournaras, S. Articles by Maniatis, A. N. Search for Related Content PubMed PubMed Citation Articles by Pournaras, S. Articles by Maniatis, A. N.