Rapid Characterization of β-Lactamases by Multiplex PCR

  • Neil Woodford
Part of the Methods in Molecular Biology book series (MIMB, volume 642)


The rising prevalence of the members of the Enterobacteriaceae, Pseudomonas spp. and Acinetobacter baumannii that produce extended-spectrum β-lactamases (ESBLs) and carbapenem-hydrolysing β-lactamases (carbapenemases) represents one of the largest “new” resistance problems faced by clinicians and microbiologists during the last 10 years. These diverse enzymes have emerged globally and represent serious health challenges, compromising therapeutic choice and complicating patient management. The rapid detection of strains that produce these β-lactamases in clinical bacteriology laboratories allows appropriate therapy to be implemented promptly, which reduces patient mortality. This chapter describes three multiplex PCR assays, which may be used to detect genes that encode five families of CTX-M-type ESBLs (groups 1, 2, 8, 9, and 25), five families of metallo-carbapenemases (IMP, VIM, SPM, GIM, and SIM enzymes), and four families of OXA-carbapenemases (OXA-23-like, OXA-40-like, OXA-51-like, and OXA-58-like enzymes). The CTX-M ESBLs are the most prevalent of these enzyme groups, particularly, though not exclusively, in isolates of Escherichia coli and in Klebsiella spp.; metallo-carbapenemases are often found in Pseudomonas spp. and other “non-fermenters,” but are also emerging problems in members of the Enterobacteriaceae in some countries and locales; with a few exceptions, the OXA-carbapenemases detected by the assay described are limited to isolates of Acinetobacter spp. These assays are suitable for deployment in national reference laboratories, but should also be considered for use in regional centres and in tertiary referral hospitals.

Key words

Antibiotic resistance β-Lactam Carbapenem Multiplex PCR Molecular diagnostics 


  1. 1.
    Kresse H, Belsey MJ, Rovini H (2007) The antibacterial drugs market. Nat Rev Drug Discov 6:19–20CrossRefPubMedGoogle Scholar
  2. 2.
    Abraham EP, Chain E (1988) An enzyme from bacteria able to destroy penicillin. 1940. Rev Infect Dis 10:677–678PubMedGoogle Scholar
  3. 3.
    Bradford PA (2001) Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 14:933–951CrossRefPubMedGoogle Scholar
  4. 4.
    Bonnet R (2004) Growing group of extended-spectrum β-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 48:1–14CrossRefPubMedGoogle Scholar
  5. 5.
    Walther-Rasmussen J, Hoiby N (2004) Cefotaximases (CTX-M-ases), an expanding family of extended-spectrum β-lactamases. Can J Microbiol 50:137–165CrossRefPubMedGoogle Scholar
  6. 6.
    Canton R, Coque TM (2006) The CTX-M β-lactamase pandemic. Curr Opin Microbiol 9:466–475CrossRefPubMedGoogle Scholar
  7. 7.
    Livermore DM, Canton R, Gniadkowski M, Nordmann P, Rossolini GM, Arlet G, Ayala J, Coque TM, Kern-Zdanowicz I, Luzzaro F, Poirel L, Woodford N (2007) CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother 59:165–174CrossRefPubMedGoogle Scholar
  8. 8.
    Rossolini GM, D’Andrea MM, Mugnaioli C (2008) The spread of CTX-M-type extended-spectrum β-lactamases. Clin Microbiol Infect 14:33–41CrossRefPubMedGoogle Scholar
  9. 9.
    Humeniuk C, Arlet G, Gautier V, Grimont P, Labia R, Philippon A (2002) β-lactamases of Kluyvera ascorbata, probable progenitors of some plasmid-encoded CTX-M types. Antimicrob Agents Chemother 46:3045–3049CrossRefPubMedGoogle Scholar
  10. 10.
    Poirel L, Kampfer P, Nordmann P (2002) Chromosome-encoded Ambler class A β-lactamase of Kluyvera georgiana, a probable progenitor of a subgroup of CTX-M extended-spectrum β-lactamases. Antimicrob Agents Chemother 46:4038–4040CrossRefPubMedGoogle Scholar
  11. 11.
    Rodriguez MM, Power P, Radice M, Vay C, Famiglietti A, Galleni M, Ayala JA, Gutkind G (2004) Chromosome-encoded CTX-M-3 from Kluyvera ascorbata: a possible origin of plasmid-borne CTX-M-1-derived cefotaximases. Antimicrob Agents Chemother 48:4895–4897CrossRefPubMedGoogle Scholar
  12. 12.
    Olson AB, Silverman M, Boyd DA, McGeer A, Willey BM, Pong-Porter V, Daneman N, Mulvey MR (2005) Identification of a progenitor of the CTX-M-9 group of extended-spectrum β-lactamases from Kluyvera georgiana isolated in Guyana. Antimicrob Agents Chemother 49:2112–2115CrossRefPubMedGoogle Scholar
  13. 13.
    Potz NAC, Hope R, Warner M, Johnson AP, Livermore DM, on behalf of the London & South East ESBL Project Group (2006) Prevalence and mechanisms of cephalosporin resistance in Enterobacteriaceae in London and South-East England. J Antimicrob Chemother 58:320–326CrossRefPubMedGoogle Scholar
  14. 14.
    Perez-Perez FJ, Hanson ND (2002) Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153–2162CrossRefPubMedGoogle Scholar
  15. 15.
    Woodford N, Dallow JWT, Hill RLR, Palepou MF, Pike R, Ward ME, Warner M, Livermore DM (2007) Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory. Int J Antimicrob Agents 29:456–459CrossRefPubMedGoogle Scholar
  16. 16.
    Lartigue MF, Poirel L, Poyart C, Reglier-Poupet H, Nordmann P (2007) Ertapenem resistance of Escherichia coli. Emerg Infect Dis 13:315–317CrossRefPubMedGoogle Scholar
  17. 17.
    Bradford PA, Bratu S, Urban C, Visalli M, Mariano N, Landman D, Rahal JJ, Brooks S, Cebular S, Quale J (2004) Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 β-lactamases in New York City. Clin Infect Dis 39:55–60CrossRefPubMedGoogle Scholar
  18. 18.
    Woodford N, Tierno PM Jr, Young K, Tysall L, Palepou MF, Ward E, Painter RE, Suber DF, Shungu D, Silver LL, Inglima K, Kornblum J, Livermore DM (2004) Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A β-lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother 48:4793–4799CrossRefPubMedGoogle Scholar
  19. 19.
    Leavitt A, Navon-Venezia S, Chmelnitsky I, Schwaber MJ, Carmeli Y (2007) Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli hospital. Antimicrob Agents Chemother 51:3026–3029CrossRefPubMedGoogle Scholar
  20. 20.
    Villegas MV, Lolans K, Correa A, Jose Suarez C, Lopez JA, Vallejo M, Quinn JP, the Colombian Nosocomial Resistance Study Group (2006) First detection of the plasmid-mediated class A carbapenemase KPC-2 in clinical isolates of Klebsiella pneumoniae from South America. Antimicrob Agents Chemother 50:2880–2882CrossRefPubMedGoogle Scholar
  21. 21.
    Wei ZQ, Du XX, Yu YS, Shen P, Chen YG, Li LJ (2007) Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. Antimicrob Agents Chemother 51:763–765CrossRefPubMedGoogle Scholar
  22. 22.
    Cuzon G, Naas T, Demachy MC, Nordmann P (2008) Plasmid-mediated carbapenem-hydrolyzing β-lactamase KPC-2 in Klebsiella pneumoniae isolate from Greece. Antimicrob Agents Chemother 52:796–797CrossRefPubMedGoogle Scholar
  23. 23.
    Toleman MA, Simm AM, Murphy TA, Gales AC, Biedenbach DJ, Jones RN, Walsh TR (2002) Molecular characterization of SPM-1, a novel metallo-β-lactamase isolated in Latin America: report from the SENTRY antimicrobial surveillance programme. J Antimicrob Chemother 50:673–679CrossRefPubMedGoogle Scholar
  24. 24.
    Gales AC, Menezes LC, Silbert S, Sader HS (2003) Dissemination in distinct Brazilian regions of an epidemic carbapenem-resistant Pseudomonas aeruginosa producing SPM metallo-β-lactamase. J Antimicrob Chemother 52:699–702CrossRefPubMedGoogle Scholar
  25. 25.
    Castanheira M, Toleman MA, Jones RN, Schmidt FJ, Walsh TR (2004) Molecular characterization of a β-lactamase gene, blaGIM-1, encoding a new subclass of metallo-β-lactamase. Antimicrob Agents Chemother 48:4654–4661CrossRefPubMedGoogle Scholar
  26. 26.
    Lee K, Yum JH, Yong D, Lee HM, Kim HD, Docquier JD, Rossolini GM, Chong Y (2005) Novel acquired metallo-β-lactamase gene, bla SIM-1, in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea. Antimicrob Agents Chemother 49:4485–4491CrossRefPubMedGoogle Scholar
  27. 27.
    Gulmez D, Woodford N, Palepou MF, Mushtaq S, Metan G, Yakupogullari Y, Kocagoz S, Uzun O, Hascelik G, Livermore DM (2008) Carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from Turkey with OXA-48-like carbapenemases and outer membrane protein loss. Int J Antimicrob Agents 31:523–526CrossRefPubMedGoogle Scholar
  28. 28.
    Poirel L, Heritier C, Nordmann P (2004) Chromosome-encoded Ambler class D β-lactamase of Shewanella oneidensis as a progenitor of carbapenem-hydrolyzing oxacillinase. Antimicrob Agents Chemother 48:348–351CrossRefPubMedGoogle Scholar
  29. 29.
    Heritier C, Poirel L, Nordmann P (2004) Genetic and biochemical characterization of a chromosome-encoded carbapenem-hydrolyzing Ambler class D β-lactamase from Shewanella algae. Antimicrob Agents Chemother 48:1670–1675CrossRefPubMedGoogle Scholar
  30. 30.
    Brown S, Amyes S (2006) OXA β-lactamases in Acinetobacter: the story so far. J Antimicrob Chemother 57:1–3CrossRefPubMedGoogle Scholar
  31. 31.
    Poirel L, Figueiredo S, Cattoir V, Carattoli A, Nordmann P (2008) Acinetobacter radioresistens as a silent source of carbapenem resistance for Acinetobacter spp. Antimicrob Agents Chemother 52:1252–1256CrossRefPubMedGoogle Scholar
  32. 32.
    Turton JF, Woodford N, Glover J, Yarde S, Kaufmann ME, Pitt TL (2006) Identification of Acinetobacter baumannii by detection of the bla OXA-51-like carbapenemase gene intrinsic to this species. J Clin Microbiol 44:2974–2976CrossRefPubMedGoogle Scholar
  33. 33.
    Turton JF, Ward ME, Woodford N, Kaufmann ME, Pike R, Livermore DM, Pitt TL (2006) The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol Lett 258:72–77CrossRefPubMedGoogle Scholar
  34. 34.
    Woodford N, Fagan EJ, Ellington MJ (2006) Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases. J Antimicrob Chemother 57:154–155CrossRefPubMedGoogle Scholar
  35. 35.
    Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, Amyes SG, Livermore DM (2006) Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 27:351–353CrossRefPubMedGoogle Scholar
  36. 36.
    Ellington MJ, Kistler J, Livermore DM, Woodford N (2007) Multiplex PCR for rapid detection of genes encoding acquired metallo-β-lactamases. J Antimicrob Chemother 59:321–322CrossRefPubMedGoogle Scholar
  37. 37.
    Johnson AP, Woodford N (1998) Plasmid analysis. Methods Mol Med 15:51–62Google Scholar
  38. 38.
    Birkett CI, Ludlam HA, Woodford N, Brown DFJ, Brown NM, Roberts MTM, Milner N, Curran MD (2007) Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum β-lactamases. J Med Microbiol 56:52–55CrossRefPubMedGoogle Scholar
  39. 39.
    Naas T, Oxacelay C, Nordmann P (2007) Identification of CTX-M-type extended-spectrum-β-lactamase genes using real-time PCR and pyrosequencing. Antimicrob Agents Chemother 51:223–230CrossRefPubMedGoogle Scholar
  40. 40.
    Nyberg SD, Osterblad M, Hakanen AJ, Huovinen P, Jalava J, Resistance TF (2007) Detection and molecular genetics of extended-spectrum β-lactamases among cefuroxime-resistant Escherichia coli and Klebsiella spp. isolates from Finland, 2002–2004. Scand J Infect Dis 39:417–424CrossRefPubMedGoogle Scholar
  41. 41.
    Ensor VM, Livermore DM, Hawkey PM (2007) A novel reverse-line hybridization assay for identifying genotypes of CTX-M-type extended-spectrum β-lactamases. J Antimicrob Chemother 59:387–395CrossRefPubMedGoogle Scholar
  42. 42.
    Xu L, Evans J, Ling T, Nye K, Hawkey P (2007) Rapid genotyping of CTX-M extended-spectrum β-lactamases by denaturing high-performance liquid chromatography. Antimicrob Agents Chemother 51:1446–1454CrossRefPubMedGoogle Scholar
  43. 43.
    Batchelor M, Hopkins KL, Liebana E, Slickers P, Ehricht R, Mafura M, Aarestrup F, Mevius D, Clifton-Hadley FA, Woodward MJ, Davies RH, Threlfall EJ, Anjum MF (2008) Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria. Int J Antimicrob Agents 31:440–451CrossRefPubMedGoogle Scholar
  44. 44.
    Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, Pignatari ACC, Tufik S (2007) Rapid detection and identification of metallo-β-lactamase encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 45:544–577CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Neil Woodford
    • 1
  1. 1.Antibiotic Resistance Monitoring and Reference Laboratory, Centre for InfectionsHealth Protection AgencyLondonUK

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