Skip to main content
SpringerLink
Log in

Evaluation of four phenotypic methods to detect plasmid-mediated AmpC β-lactamases in clinical isolates

  • Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

Four phenotypic methods (three dimensional test, AmpC test, cloxacillin synergy test and cefotetan/cefotetan-cloxacillin E-test) to detect plasmid-mediated AmpC β-lactamases (pAmpC) were compared in 125 clinical Enterobacteriaceae isolates with AmpC profile: 74 E. coli (bla CMY-2: 70; bla DHA-1: 4), five K. pneumoniae (bla CMY-2: 2; bla DHA-1: 3), six P. mirabilis (bla CMY-2: 6) and 40 negative isolates for pAmpC β-lactamases. All evaluated methods showed a good sensitivity (>95%) but low values of specificity (<60%) in E. coli, explained by an increase of AmpC expression caused by chromosomal ampC promoter/attenuator mutations (−42, −18, −1, +58, predominantly). The cefotetan/cefotetan-cloxacillin or cloxacillin synergy test may be advocated as phenotypic screening test, and the AmpC test as confirmatory test for detection of pAmpC in isolates that lack or minimally express chromosomally encoded AmpC β-lactamases. In the case of E. coli, the phenotypic evaluated tests were not able to differentiate between chromosomal ampC overexpression or acquisition of plasmid-encoded ampC genes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  1. Bauernfeind A, Chong Y, Schweighart S (1989) Extended broad spectrum beta-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection 17(5):316–321

    Article  PubMed  CAS  Google Scholar 

  2. Jacoby GA (2009) AmpC beta-lactamases. Clin Microbiol Rev 22(1):161–182

    Article  PubMed  CAS  Google Scholar 

  3. Rodríguez-Martínez JM, Pascual A, Garcia I, Martinez-Martinez L (2003) Detection of the plasmid-mediated quinolone resistance determinant qnr among clinical isolates of Klebsiella pneumoniae producing AmpC-type beta-lactamase. J Antimicrob Chemother 52(4):703–706

    Article  PubMed  Google Scholar 

  4. Philippon A, Arlet G, Jacoby GA (2002) Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother 46(1):1–11

    Article  PubMed  CAS  Google Scholar 

  5. Bradford PA, Urban C, Mariano N, Projan SJ, Rahal JJ, Bush K (1997) Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein. Antimicrob Agents Chemother 41(3):563–569

    PubMed  CAS  Google Scholar 

  6. Caroff N, Espaze E, Gautreau D, Richet H, Reynaud A (2000) Analysis of the effects of -42 and -32 ampC promoter mutations in clinical isolates of Escherichia coli hyperproducing ampC. J Antimicrob Chemother 45(6):783–788

    Article  PubMed  CAS  Google Scholar 

  7. Mulvey MR, Bryce E, Boyd DA, Ofner-Agostini M, Land AM, Simor AE, Paton S (2005) Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals. Antimicrob Agents Chemother 49(1):358–365

    Article  PubMed  CAS  Google Scholar 

  8. Tracz DM, Boyd DA, Bryden L, Hizon R, Giercke S, Van Caeseele P, Mulvey MR (2005) Increase in ampC promoter strength due to mutations and deletion of the attenuator in a clinical isolate of cefoxitin-resistant Escherichia coli as determined by RT-PCR. J Antimicrob Chemother 55(5):768–772

    Article  PubMed  CAS  Google Scholar 

  9. Fernández-Cuenca F, Pascual A, Martinez-Martinez L (2005) Hyperproduction of AmpC beta-lactamase in a clinical isolate of Escherichia coli associated with a 30 bp deletion in the attenuator region of ampC. J Antimicrob Chemother 56(1):251–252

    Article  PubMed  Google Scholar 

  10. Jaurin B, Grundstrom T, Normark S (1982) Sequence elements determining ampC promoter strength in E. coli. EMBO J 1(7):875–881

    PubMed  CAS  Google Scholar 

  11. Pérez-Pérez FJ, Hanson ND (2002) Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40(6):2153–2162

    Article  PubMed  Google Scholar 

  12. Lee W, Jung B, Hong SG, Song W, Jeong SH, Lee K, Kwak HS (2009) Comparison of 3 phenotypic-detection methods for identifying plasmid-mediated AmpC beta-lactamase-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains. Korean J Lab Med 29(5):448–454

    Article  PubMed  CAS  Google Scholar 

  13. Coudron PE (2005) Inhibitor-based methods for detection of plasmid-mediated AmpC beta-lactamases in Klebsiella spp., Escherichia coli, and Proteus mirabilis. J Clin Microbiol 43(8):4163–4167

    Article  PubMed  CAS  Google Scholar 

  14. Pitout JD, Le PG, Moore KL, Church DL, Gregson DB (2010) Detection of AmpC beta-lactamases in Escherichia coli, Klebsiella spp., Salmonella spp. and Proteus mirabilis in a regional clinical microbiology laboratory. Clin Microbiol Infect 16(2):165–170

    Article  PubMed  CAS  Google Scholar 

  15. Tenover FC, Emery SL, Spiegel CA, Bradford PA, Eells S, Endimiani A, Bonomo RA, McGowan JE Jr (2009) Identification of plasmid-mediated AmpC beta-lactamases in Escherichia coli, Klebsiella spp., and Proteus species can potentially improve reporting of cephalosporin susceptibility testing results. J Clin Microbiol 47(2):294–299

    Article  PubMed  CAS  Google Scholar 

  16. Jeong SH, Song W, Park MJ, Kim JS, Kim HS, Bae IK, Lee KM (2008) Boronic acid disk tests for identification of extended-spectrum beta-lactamase production in clinical isolates of Enterobacteriaceae producing chromosomal AmpC beta-lactamases. Int J Antimicrob Agents 31(5):467–471

    Article  PubMed  CAS  Google Scholar 

  17. Giske CG, Gezelius L, Samuelsen O, Warner M, Sundsfjord A, Woodford N (2010) A sensitive and specific phenotypic assay for detection of metallo-beta-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect 17(4):552–556

    Article  Google Scholar 

  18. Pournaras S, Poulou A, Tsakris A (2010) Inhibitor-based methods for the detection of KPC carbapenemase-producing Enterobacteriaceae in clinical practice by using boronic acid compounds. J Antimicrob Chemother 65(7):1319–1321

    Article  PubMed  CAS  Google Scholar 

  19. Tsakris A, Kristo I, Poulou A, Themeli-Digalaki K, Ikonomidis A, Petropoulou D, Pournaras S, Sofianou D (2009) Evaluation of boronic acid disk tests for differentiating KPC-possessing Klebsiella pneumoniae isolates in the clinical laboratory. J Clin Microbiol 47(2):362–367

    Article  PubMed  CAS  Google Scholar 

  20. CLSI (2009) Performance for antimicrobial susceptibility testing: 17th informational supplement M100-S19. Clinical and Laboratory Standards Institute, Wayne, PA, USA

    Google Scholar 

  21. Mirelis B, Rivera A, Miro E, Mesa RJ, Navarro F, Coll P (2006) A simple phenotypic method for differentiation between acquired and chromosomal AmpC beta-lactamases in Escherichia coli. Enferm Infecc Microbiol Clin 24(6):370–372

    Article  PubMed  Google Scholar 

  22. Black JA, Moland ES, Thomson KS (2005) AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 43(7):3110–3113

    Article  PubMed  CAS  Google Scholar 

  23. Coudron PE, Moland ES, Thomson KS (2000) Occurrence and detection of AmpC beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a veterans medical center. J Clin Microbiol 38(5):1791–1796

    PubMed  CAS  Google Scholar 

  24. Navarro F, Perez-Trallero E, Marimon JM, Aliaga R, Gomariz M, Mirelis B (2001) CMY-2-producing Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Escherichia coli strains isolated in Spain (October 1999-December 2000). J Antimicrob Chemother 48(3):383–389

    Article  PubMed  CAS  Google Scholar 

  25. Miró E, Mirelis B, Navarro F, Matas L, Gimenez M, Rabaza C (2005) Escherichia coli producing an ACC-1 class C beta-lactamase isolated in Barcelona, Spain. Antimicrob Agents Chemother 49(2):866–867

    Article  PubMed  Google Scholar 

  26. Caroff N, Espaze E, Berard I, Richet H, Reynaud A (1999) Mutations in the ampC promoter of Escherichia coli isolates resistant to oxyiminocephalosporins without extended spectrum beta-lactamase production. FEMS Microbiol Lett 173(2):459–465

    PubMed  CAS  Google Scholar 

  27. Forward KR, Willey BM, Low DE, McGeer A, Kapala MA, Kapala MM, Burrows LL (2001) Molecular mechanisms of cefoxitin resistance in Escherichia coli from the Toronto area hospitals. Diagn Microbiol Infect Dis 41(1–2):57–63

    Article  PubMed  CAS  Google Scholar 

  28. Mata C, Miro E, Rivera A, Mirelis B, Coll P, Navarro F (2010) Prevalence of acquired AmpC beta-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes at a Spanish hospital from 1999 to 2007. Clin Microbiol Infect 16(5):472–476

    Article  PubMed  CAS  Google Scholar 

  29. Álvarez M, Tran JH, Chow N, Jacoby GA (2004) Epidemiology of conjugative plasmid-mediated AmpC beta-lactamases in the United States. Antimicrob Agents Chemother 48(2):533–537

    Article  PubMed  Google Scholar 

  30. Bou G, Oliver A, Ojeda M, Monzon C, Martinez-Beltran J (2000) Molecular characterization of FOX-4, a new AmpC-type plasmid-mediated beta-lactamase from an Escherichia coli strain isolated in Spain. Antimicrob Agents Chemother 44(9):2549–2553

    Article  PubMed  CAS  Google Scholar 

  31. Li Y, Li Q, Du Y, Jiang X, Tang J, Wang J, Li G, Jiang Y (2008) Prevalence of plasmid-mediated AmpC beta-lactamases in a Chinese university hospital from 2003 to 2005: first report of CMY-2-Type AmpC beta-lactamase resistance in China. J Clin Microbiol 46(4):1317–1321

    Article  PubMed  CAS  Google Scholar 

  32. Hernández-Alles S, Conejo M, Pascual A, Tomas JM, Benedi VJ, Martinez-Martinez L (2000) Relationship between outer membrane alterations and susceptibility to antimicrobial agents in isogenic strains of Klebsiella pneumoniae. J Antimicrob Chemother 46(2):273–277

    Article  PubMed  Google Scholar 

  33. Peter-Getzlaff S, Polsfuss S, Poledica M, Hombach M, Giger J, Bottger EC, Zbinden R, Bloemberg GV (2011) Detection of AmpC beta-lactamase in Escherichia coli: comparison of three phenotypic confirmation assays and genetic analysis. J Clin Microbiol 49(8):2924–2932

    Article  PubMed  CAS  Google Scholar 

  34. Tan TY, Ng LS, He J, Koh TH, Hsu LY (2009) Evaluation of screening methods to detect plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Antimicrob Agents Chemother 53(1):146–149

    Article  PubMed  CAS  Google Scholar 

  35. Ingram PR, Inglis TJ, Vanzetti TR, Henderson BA, Harnett GB, Murray RJ (2011) Comparison of methods for AmpC {beta}-lactamase detection in Enterobacteriaceae. J Med Microbiol 60(Pt 6):715–721

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by Departamento de Ciencia, Tecnología and Universidad del Gobierno de Aragón, Spain (Project DGA/Grupos consolidados, B24-211130). We are grateful to Mirelis B., Miró E., and Navarro F. for providing reference strains. MJG received a grant from the S.E.I.M.C (Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica).

Author information

Authors and Affiliations

  1. Departamento de Microbiología, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain

    M. J. Gude & M. González-Domínguez

  2. Departamento de Microbiología, Hospital Clínico Universitario Lozano Blesa and Universidad de Zaragoza, Zaragoza, Spain

    C. Seral & F. J. Castillo

  3. Área de Microbiología Molecular, Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain

    Y. Sáenz

  4. Área de Bioquímica y Biología Molecular and Área de Microbiología Molecular, Universidad de La Rioja and Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain

    C. Torres

Authors
  1. M. J. Gude
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C. Seral
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Y. Sáenz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. González-Domínguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. C. Torres
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. F. J. Castillo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. Seral.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gude, M.J., Seral, C., Sáenz, Y. et al. Evaluation of four phenotypic methods to detect plasmid-mediated AmpC β-lactamases in clinical isolates. Eur J Clin Microbiol Infect Dis 31, 2037–2043 (2012). https://doi.org/10.1007/s10096-011-1537-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-011-1537-y

Keywords

Search

Navigation