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

  • M. J. Gude
  • C. SeralEmail author
  • Y. Sáenz
  • M. González-Domínguez
  • C. Torres
  • F. J. Castillo


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.


Cefoxitin Boronic Acid AmpC Phenotypic Method Klebsiella Pneumoniae Carbapenemase 
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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).


  1. 1.
    Bauernfeind A, Chong Y, Schweighart S (1989) Extended broad spectrum beta-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection 17(5):316–321PubMedCrossRefGoogle Scholar
  2. 2.
    Jacoby GA (2009) AmpC beta-lactamases. Clin Microbiol Rev 22(1):161–182PubMedCrossRefGoogle Scholar
  3. 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):703–706PubMedCrossRefGoogle Scholar
  4. 4.
    Philippon A, Arlet G, Jacoby GA (2002) Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother 46(1):1–11PubMedCrossRefGoogle Scholar
  5. 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):563–569PubMedGoogle Scholar
  6. 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):783–788PubMedCrossRefGoogle Scholar
  7. 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):358–365PubMedCrossRefGoogle Scholar
  8. 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):768–772PubMedCrossRefGoogle Scholar
  9. 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):251–252PubMedCrossRefGoogle Scholar
  10. 10.
    Jaurin B, Grundstrom T, Normark S (1982) Sequence elements determining ampC promoter strength in E. coli. EMBO J 1(7):875–881PubMedGoogle Scholar
  11. 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):2153–2162PubMedCrossRefGoogle Scholar
  12. 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):448–454PubMedCrossRefGoogle Scholar
  13. 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):4163–4167PubMedCrossRefGoogle Scholar
  14. 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):165–170PubMedCrossRefGoogle Scholar
  15. 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):294–299PubMedCrossRefGoogle Scholar
  16. 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):467–471PubMedCrossRefGoogle Scholar
  17. 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):552–556CrossRefGoogle Scholar
  18. 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):1319–1321PubMedCrossRefGoogle Scholar
  19. 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):362–367PubMedCrossRefGoogle Scholar
  20. 20.
    CLSI (2009) Performance for antimicrobial susceptibility testing: 17th informational supplement M100-S19. Clinical and Laboratory Standards Institute, Wayne, PA, USAGoogle Scholar
  21. 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):370–372PubMedCrossRefGoogle Scholar
  22. 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):3110–3113PubMedCrossRefGoogle Scholar
  23. 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):1791–1796PubMedGoogle Scholar
  24. 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):383–389PubMedCrossRefGoogle Scholar
  25. 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):866–867PubMedCrossRefGoogle Scholar
  26. 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):459–465PubMedGoogle Scholar
  27. 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):57–63PubMedCrossRefGoogle Scholar
  28. 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):472–476PubMedCrossRefGoogle Scholar
  29. 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):533–537PubMedCrossRefGoogle Scholar
  30. 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):2549–2553PubMedCrossRefGoogle Scholar
  31. 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):1317–1321PubMedCrossRefGoogle Scholar
  32. 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):273–277PubMedCrossRefGoogle Scholar
  33. 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):2924–2932PubMedCrossRefGoogle Scholar
  34. 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):146–149PubMedCrossRefGoogle Scholar
  35. 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):715–721PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • M. J. Gude
    • 1
  • C. Seral
    • 2
    Email author
  • Y. Sáenz
    • 3
  • M. González-Domínguez
    • 1
  • C. Torres
    • 4
  • F. J. Castillo
    • 2
  1. 1.Departamento de MicrobiologíaHospital Clínico Universitario Lozano BlesaZaragozaSpain
  2. 2.Departamento de MicrobiologíaHospital Clínico Universitario Lozano Blesa and Universidad de ZaragozaZaragozaSpain
  3. 3.Área de Microbiología MolecularCentro de Investigación Biomédica de La Rioja (CIBIR)LogroñoSpain
  4. 4.Área de Bioquímica y Biología Molecular and Área de Microbiología MolecularUniversidad de La Rioja and Centro de Investigación Biomédica de La Rioja (CIBIR)LogroñoSpain

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