Spread of an OmpK36-modified ST15 Klebsiella pneumoniae variant during an outbreak involving multiple carbapenem-resistant Enterobacteriaceae species and clones

  • Â. Novais
  • C. Rodrigues
  • R. Branquinho
  • P. Antunes
  • F. Grosso
  • L. Boaventura
  • G. Ribeiro
  • L. Peixe


We aim to characterise multiple ertapenem-resistant (ERT-R, n = 15) Enterobacteriaceae isolates identified as presumptive carbapenemase producers in a Portuguese hospital in a short period of time (March–July 2010). Antibiotic susceptibility patterns, β-lactamases, genetic relatedness [pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST)], plasmid content and major enterobacterial porins were investigated. Ertapenem resistance was associated with deficiencies in major porins and, in some cases, extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase production among outbreak and non-outbreak clones. Most isolates (n = 8) corresponded to two ERT-R Klebsiella pneumoniae ST15 PFGE-types: (i) a sporadic variant (Kp-A-ERT, n = 1) presenting a premature stop codon in ompK36 and (ii) an epidemic variant (Kp-B-ERT, n = 7) exhibiting a new OmpK36 porin variant, which differed additionally in plasmid and antibiotic susceptibility profiles. ST14 (n = 1) and ST45 (n = 1) K. pneumoniae, ST131 (n = 1) and ST354 (n = 1) Escherichia coli, Enterobacter asburiae (n = 1), Enterobacter cloacae (n = 1) and Enterobacter aerogenes (n = 1) ERT-R clones were also sporadically detected. Porin changes in these isolates included non-sense mutations [ompK35, ompK36, ompF; minimum inhibitory concentration (MIC) = 4–32 mg/l], IS-mediated porin disruptions (ompK36, ompC; MIC = 12–>32 mg/l) or alterations in the L3 loop (ompK36; MIC = 4–16 mg/l). We describe, for the first time in Portugal, the simultaneous emergence of multiple ERT-R Enterobacteriaceae species and clones in a short period of time. Moreover, our results support that a CTX-M-15-producing ST15 K. pneumoniae with an OmpK36-modified porin might successfully spread in the nosocomial setting.


  1. 1.
    Pfeifer Y, Cullik A, Witte W (2010) Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens. Int J Med Microbiol 300(6):371–379PubMedCrossRefGoogle Scholar
  2. 2.
    Nordmann P, Naas T, Poirel L (2011) Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 17(10):1791–1798PubMedCrossRefGoogle Scholar
  3. 3.
    Doumith M, Ellington MJ, Livermore DM, Woodford N (2009) Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J Antimicrob Chemother 63(4):659–667PubMedCrossRefGoogle Scholar
  4. 4.
    García-Fernández A, Miriagou V, Papagiannitsis CC, Giordano A, Venditti M, Mancini C, Carattoli A (2010) An ertapenem-resistant extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae clone carries a novel OmpK36 porin variant. Antimicrob Agents Chemother 54(10):4178–4184PubMedCrossRefGoogle Scholar
  5. 5.
    Kaczmarek FM, Dib-Hajj F, Shang W, Gootz TD (2006) High-level carbapenem resistance in a Klebsiella pneumoniae clinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35/36 insertional inactivation, and down-regulation of the phosphate transport porin phoE. Antimicrob Agents Chemother 50(10):3396–3406PubMedCrossRefGoogle Scholar
  6. 6.
    Cuzon G, Naas T, Guibert M, Nordmann P (2010) In vivo selection of imipenem-resistant Klebsiella pneumoniae producing extended-spectrum beta-lactamase CTX-M-15 and plasmid-encoded DHA-1 cephalosporinase. Int J Antimicrob Agents 35(3):265–268PubMedCrossRefGoogle Scholar
  7. 7.
    Chudácková E, Bergerová T, Fajfrlík K, Cervená D, Urbásková P, Empel J, Gniadkowski M, Hrabák J (2010) Carbapenem-nonsusceptible strains of Klebsiella pneumoniae producing SHV-5 and/or DHA-1 beta-lactamases in a Czech hospital. FEMS Microbiol Lett 309(1):62–70PubMedGoogle Scholar
  8. 8.
    Mena A, Plasencia V, García L, Hidalgo O, Ayestarán JI, Alberti S, Borrell N, Pérez JL, Oliver A (2006) Characterization of a large outbreak by CTX-M-1-producing Klebsiella pneumoniae and mechanisms leading to in vivo carbapenem resistance development. J Clin Microbiol 44(8):2831–2837PubMedCrossRefGoogle Scholar
  9. 9.
    Gröbner S, Linke D, Schütz W, Fladerer C, Madlung J, Autenrieth IB, Witte W, Pfeifer Y (2009) Emergence of carbapenem-non-susceptible extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates at the university hospital of Tübingen, Germany. J Med Microbiol 58(Pt 7):912–922PubMedCrossRefGoogle Scholar
  10. 10.
    Lartigue MF, Poirel L, Poyart C, Réglier-Poupet H, Nordmann P (2007) Ertapenem resistance of Escherichia coli. Emerg Infect Dis 13(2):315–317PubMedCrossRefGoogle Scholar
  11. 11.
    Leavitt A, Chmelnitsky I, Colodner R, Ofek I, Carmeli Y, Navon-Venezia S (2009) Ertapenem resistance among extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae isolates. J Clin Microbiol 47(4):969–974PubMedCrossRefGoogle Scholar
  12. 12.
    Endimiani A, Perez F, Bajaksouzian S, Windau AR, Good CE, Choudhary Y, Hujer AM, Bethel CR, Bonomo RA, Jacobs MR (2010) Evaluation of updated interpretative criteria for categorizing Klebsiella pneumoniae with reduced carbapenem susceptibility. J Clin Microbiol 48(12):4417–4425PubMedCrossRefGoogle Scholar
  13. 13.
    Martínez-Martínez L (2008) Extended-spectrum beta-lactamases and the permeability barrier. Clin Microbiol Infect 14(Suppl 1):82–89PubMedCrossRefGoogle Scholar
  14. 14.
    Machado E, Coque TM, Cantón R, Novais A, Sousa JC, Baquero F, Peixe L; Portuguese Resistance Study Group (2007) High diversity of extended-spectrum beta-lactamases among clinical isolates of Enterobacteriaceae from Portugal. J Antimicrob Chemother 60(6):1370–1374PubMedCrossRefGoogle Scholar
  15. 15.
    Poirel L, Barbosa-Vasconcelos A, Simões RR, Da Costa PM, Liu W, Nordmann P (2012) Environmental KPC-producing Escherichia coli isolates in Portugal. Antimicrob Agents Chemother 56(3):1662–1663PubMedCrossRefGoogle Scholar
  16. 16.
    Clinical and Laboratory Standards Institute (CLSI) (2010) Performance standards for antimicrobial susceptibility testing; update. CLSI document M100–S20 June 2010 update. CLSI, Wayne, PAGoogle Scholar
  17. 17.
    Novais A, Baquero F, Machado E, Cantón R, Peixe L, Coque TM (2010) International spread and persistence of TEM-24 is caused by the confluence of highly penetrating Enterobacteriaceae clones and an IncA/C2 plasmid containing Tn1696::Tn1 and IS5075-Tn21. Antimicrob Agents Chemother 54(2):825–834PubMedCrossRefGoogle Scholar
  18. 18.
    Doi Y, Potoski BA, Adams-Haduch JM, Sidjabat HE, Pasculle AW, Paterson DL (2008) Simple disk-based method for detection of Klebsiella pneumoniae carbapenemase-type beta-lactamase by use of a boronic acid compound. J Clin Microbiol 46(12):4083–4086PubMedCrossRefGoogle Scholar
  19. 19.
    Galani I, Rekatsina PD, Hatzaki D, Plachouras D, Souli M, Giamarellou H (2008) Evaluation of different laboratory tests for the detection of metallo-beta-lactamase production in Enterobacteriaceae. J Antimicrob Chemother 61(3):548–553PubMedCrossRefGoogle Scholar
  20. 20.
    Poirel L, Castanheira M, Carrër A, Rodriguez CP, Jones RN, Smayevsky J, Nordmann P (2011) OXA-163, an OXA-48-related class D beta-lactamase with extended activity toward expanded-spectrum cephalosporins. Antimicrob Agents Chemother 55(6):2546–2551PubMedCrossRefGoogle Scholar
  21. 21.
    Ellington MJ, Kistler J, Livermore DM, Woodford N (2007) Multiplex PCR for rapid detection of genes encoding acquired metallo-beta-lactamases. J Antimicrob Chemother 59(2):321–322PubMedCrossRefGoogle Scholar
  22. 22.
    Antunes P, Coque TM, Peixe L (2010) Emergence of an IncIγ plasmid encoding CMY-2 β-lactamase associated with the international ST19 OXA-30-producing β-lactamase Salmonella typhimurium multidrug-resistant clone. J Antimicrob Chemother 65(10):2097–2100. doi:10.1093/jac/dkq293 PubMedCrossRefGoogle Scholar
  23. 23.
    Higgins PG, Lehmann M, Seifert H (2010) Inclusion of OXA-143 primers in a multiplex polymerase chain reaction (PCR) for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 35(3):305PubMedCrossRefGoogle Scholar
  24. 24.
    Poirel L, Héritier C, Tolün V, Nordmann P (2004) Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrob Agents Chemother 48(1):15–22PubMedCrossRefGoogle Scholar
  25. 25.
    Poirel L, Le Thomas I, Naas T, Karim A, Nordmann P (2000) Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob Agents Chemother 44(3):622–632PubMedCrossRefGoogle Scholar
  26. 26.
    D’Andrea MM, Nucleo E, Luzzaro F, Giani T, Migliavacca R, Vailati F, Kroumova V, Pagani L, Rossolini GM (2006) CMY-16, a novel acquired AmpC-type beta-lactamase of the CMY/LAT lineage in multifocal monophyletic isolates of Proteus mirabilis from northern Italy. Antimicrob Agents Chemother 50(2):618–624PubMedCrossRefGoogle Scholar
  27. 27.
    Bert F, Branger C, Lambert-Zechovsky N (2002) Identification of PSE and OXA beta-lactamase genes in Pseudomonas aeruginosa using PCR-restriction fragment length polymorphism. J Antimicrob Chemother 50(1):11–18PubMedCrossRefGoogle Scholar
  28. 28.
    Carlone GM, Thomas ML, Rumschlag HS, Sottnek FO (1986) Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 24(3):330–332PubMedGoogle Scholar
  29. 29.
    Coelho A, González-López JJ, Miró E, Alonso-Tarrés C, Mirelis B, Larrosa MN, Bartolomé RM, Andreu A, Navarro F, Johnson JR, Prats G (2010) Characterisation of the CTX-M-15-encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan area: plasmid diversity and chromosomal integration. Int J Antimicrob Agents 36(1):73–78PubMedCrossRefGoogle Scholar
  30. 30.
    Woodford N, Turton JF, Livermore DM (2011) Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 35(5):736–755PubMedCrossRefGoogle Scholar
  31. 31.
    Orsi GB, García-Fernández A, Giordano A, Venditti C, Bencardino A, Gianfreda R, Falcone M, Carattoli A, Venditti M (2011) Risk factors and clinical significance of ertapenem-resistant Klebsiella pneumoniae in hospitalised patients. J Hosp Infect 78(1):54–58PubMedCrossRefGoogle Scholar
  32. 32.
    Lee MY, Ko KS, Kang CI, Chung DR, Peck KR, Song JH (2011) High prevalence of CTX-M-15-producing Klebsiella pneumoniae isolates in Asian countries: diverse clones and clonal dissemination. Int J Antimicrob Agents 38(2):160–163PubMedCrossRefGoogle Scholar
  33. 33.
    Yan JJ, Wu JJ, Lee CC, Ko WC, Yang FC (2010) Prevalence and characteristics of ertapenem-nonsusceptible Escherichia coli in a Taiwanese university hospital, 1999 to 2007. Eur J Clin Microbiol Infect Dis 29(11):1417–1425PubMedCrossRefGoogle Scholar
  34. 34.
    Mammeri H, Guillon H, Eb F, Nordmann P (2010) Phenotypic and biochemical comparison of the carbapenem-hydrolyzing activities of five plasmid-borne AmpC beta-lactamases. Antimicrob Agents Chemother 54(11):4556–4560PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Â. Novais
    • 1
  • C. Rodrigues
    • 1
  • R. Branquinho
    • 1
  • P. Antunes
    • 1
    • 3
  • F. Grosso
    • 1
  • L. Boaventura
    • 2
  • G. Ribeiro
    • 2
  • L. Peixe
    • 1
  1. 1.REQUIMTE, Laboratório de Microbiologia, Faculdade FarmáciaUniversidade do PortoPortoPortugal
  2. 2.Laboratório MicrobiologiaHospitais da Universidade de CoimbraCoimbraPortugal
  3. 3.Faculdade de Ciências da Nutrição e AlimentaçãoUniversidade do PortoPortoPortugal

Personalised recommendations