Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Antimicrobial Agents and Chemotherapy
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Mechanisms of Resistance

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

Spyros Pournaras, Athanassios Tsakris, Maria Maniati, Leonidas S. Tzouvelekis, Antonios N. Maniatis
Spyros Pournaras
Department of Medical Microbiology, University of Thessaly, Larissa
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: spournar@otenet.gr
Athanassios Tsakris
Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Thessaloniki
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Maria Maniati
Department of Medical Microbiology, University of Thessaly, Larissa
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Leonidas S. Tzouvelekis
Department of Microbiology, Medical School, University of Athens, Athens, Greece
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Antonios N. Maniatis
Department of Medical Microbiology, University of Thessaly, Larissa
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AAC.46.12.4026-4028.2002
  • Article
  • Info & Metrics
  • PDF
Loading

ABSTRACT

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 blaVIM-4, a novel variant of blaVIM-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.

There is an increasing number of studies reporting on the emergence of Pseudomonas aeruginosa strains that produce acquired class B metallo-β-lactamases (MBLs), which invariably hydrolyze carbapenems. So far, two distinct MBL types, IMP (11) and VIM (2), have been described. Although these β-lactamase types share less than 40% amino acid sequence identity, they exhibit comparable kinetic properties, inactivating virtually all β-lactams except monobactams (7). Additionally, both blaIMP- and blaVIM-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 blaVIM-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 β-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 DH5α cells with transformant selection on Mueller-Hinton agar containing ampicillin (50 μg/ml).

blaVIM-type genes were detected by PCR with a pair of consensus primers under amplification conditions as described previously (18). Primers specific for blaVIM-1 and blaVIM-2 were also used (19). PCR assays combining primers specific for conserved 5′-CS and 3′-CS sequences (6) with the blaVIM-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 blaVIM-1 (reverse) primers (19) was done by using an ABI Prism 377 DNA sequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, Calif.).

β-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). β-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 β-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 β-lactam-resistant Escherichia coli clone.

Total DNA from P1936 was positive in the PCR assay using the consensus blaVIM 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 blaVIM-2-type genes, P. aeruginosa P1936 was positive for blaVIM-1, producing in the respective PCR assay an amplicon of the expected size (920 bp). PCRs using the 5′-CS and blaVIM-1 (reverse) primers as well as the 3′-CS and blaVIM-1 (forward) primers yielded products of similar sizes (approximately 950 bp), indicating that blaVIM-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 blaVIM-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 blaVIM-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 β-lactamases [3]) is not among those considered significant for β-lactam hydrolysis by VIM and IMP MBLs (3). This novel blaVIM-1 variant was designated blaVIM-4.

Photometric assays showed that crude β-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 blaVIM-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 β-lactamase activity at a pI of 8.8, which probably represented the chromosomal cephalosporinase. Other major β-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, blaVIM-4 was, most likely, the sole gene in the variable region of the detected class 1 integron while the blaVIM-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 blaVIM-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

    • Received 11 April 2002.
    • Returned for modification 16 May 2002.
    • Accepted 11 September 2002.
  • American Society for Microbiology

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-β-lactamase. Clin. Infect. Dis. 31:1119-1125.
    OpenUrlCrossRefPubMedWeb of Science
  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.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Galleni, M., J. Lamotte-Brasseur, G. M. Rossolini, J. Spencer, O. Dideberg, J.-M. Frere, and The Metallo-β-Lactamase Working Group. 2001. Standard numbering scheme for class B β-lactamases. Antimicrob. Agents Chemother. 45:660-663.
    OpenUrlFREE 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 blaVIM, a new integron-borne metallo-β-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 43:1584-1590.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Lee, K., J. B. Lim, J. H. Yum, D. Yong, Y. Chong, J. M. Kim, and D. M. Livermore. 2002. blaVIM-2 cassette-containing novel integrons in metallo-β-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital. Antimicrob. Agents Chemother. 46:1053-1058.
    OpenUrlAbstract/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.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Livermore, D. M., and N. Woodford. 2000. Carbapenemases: a problem in waiting? Curr. Opin. Microbiol. 3:489-495.
    OpenUrlCrossRefPubMedWeb of Science
  8. ↵
    Mavroidi, A., A. Tsakris, E. Tzelepi, S. Pournaras, V. Loukova, and L. S. Tzouvelekis. 2000. Carbapenem-hydrolysing VIM-2 metallo-β-lactamase in Pseudomonas aeruginosa from Greece. J. Antimicrob. Chemother. 46:1041-1043.
    OpenUrlCrossRefPubMedWeb of Science
  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.
    OpenUrlAbstract/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.
    OpenUrlPubMedWeb of Science
  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-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44:891-897.
    OpenUrlAbstract/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 blaVIM-2 carbapenem-hydrolyzing beta-lactamase gene and of two novel aminoglycoside resistance gene cassettes. Antimicrob. Agents Chemother. 45:546-552.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Prats, G., E. Miro, B. Mirelis, L. Poirel, S. Bellais, and P. Nordmann. 2002. First isolation of a carbapenem-hydrolyzing β-lactamase in Pseudomonas aeruginosa in Spain. Antimicrob. Agents Chemother. 46:932-933.
    OpenUrlFREE Full Text
  16. ↵
    Riccio, M. L., L. Pallecchi, R. Fontana, and G. M. Rossolini. 2001. In70 of plasmid pAX22, a blaVIM-1-containing integron carrying a new aminoglycoside phosphotransferase gene cassette. Antimicrob. Agents Chemother. 45:1249-1253.
    OpenUrlAbstract/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 blaVIM metallo-β-lactamase determinants, Italy. Emerg. Infect. Dis. 3:312-313.
    OpenUrl
  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.
    OpenUrlAbstract/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-β-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.
    OpenUrlAbstract/FREE Full Text
View Abstract
PreviousNext
Back to top
Download PDF
Citation Tools
Novel Variant (blaVIM-4) of the Metallo-β-Lactamase Gene blaVIM-1 in a Clinical Strain of Pseudomonas aeruginosa
Spyros Pournaras, Athanassios Tsakris, Maria Maniati, Leonidas S. Tzouvelekis, Antonios N. Maniatis
Antimicrobial Agents and Chemotherapy Dec 2002, 46 (12) 4026-4028; DOI: 10.1128/AAC.46.12.4026-4028.2002

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Antimicrobial Agents and Chemotherapy article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Novel Variant (blaVIM-4) of the Metallo-β-Lactamase Gene blaVIM-1 in a Clinical Strain of Pseudomonas aeruginosa
(Your Name) has forwarded a page to you from Antimicrobial Agents and Chemotherapy
(Your Name) thought you would be interested in this article in Antimicrobial Agents and Chemotherapy.
Share
Novel Variant (blaVIM-4) of the Metallo-β-Lactamase Gene blaVIM-1 in a Clinical Strain of Pseudomonas aeruginosa
Spyros Pournaras, Athanassios Tsakris, Maria Maniati, Leonidas S. Tzouvelekis, Antonios N. Maniatis
Antimicrobial Agents and Chemotherapy Dec 2002, 46 (12) 4026-4028; DOI: 10.1128/AAC.46.12.4026-4028.2002
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • Nucleotide sequence accession number.
    • FOOTNOTES
    • REFERENCES
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About AAC
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AACJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

Copyright © 2019 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0066-4804; Online ISSN: 1098-6596