Current Microbiology

, Volume 75, Issue 8, pp 977–987 | Cite as

Tracking Multidrug-Resistant Klebsiella pneumoniae from an Italian Hospital: Molecular Epidemiology and Surveillance by PFGE, RAPD and PCR-Based Resistance Genes Prevalence

  • Giancarlo Ripabelli
  • Manuela Tamburro
  • Giuliana Guerrizio
  • Incoronata Fanelli
  • Romeo Flocco
  • Massimiliano Scutellà
  • Michela L. Sammarco


Antimicrobial-resistant Klebsiella pneumoniae represent a global public health concern. K. pneumoniae strains isolated during 2010 and 2014–2016 within a single hospital of Molise Region, Central Italy, were analyzed testing antimicrobial susceptibility, clonality by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD)-PCR, and prevalence of carbapenem resistance genes by PCR. Forty isolates (23 wild-type in 2010 and 17 non-wild-type in 2014–2016) were collected from hospitalized patients (65.2 ± 18.1 years old, 75% male, 80% from intensive care unit—ICU). K. pneumoniae showed multidrug-resistant profiles and 15 resistotypes were identified (discriminatory power D = 0.88). The 69.6 and 17.4% of isolates in 2010 resulted intermediate and resistant to imipenem, respectively, and 91.3% was sensitive to meropenem, while 88.2% of isolates of 2014–2016 were resistant to both antibiotics. PFGE identified 16 clusters versus 23 by RAPD, 26 pulsotypes versus 33 RAPD patterns (D ≥ 0.97). PFGE separated strains according to isolation period and identified an outbreak occurred in the ICU during December 2014 and January 2015. No strains harbored blaGES, blaIMP, blaNDM−1, and blaOXA−48 genes, as well as AmpC plasmid-mediated beta-lactamases genes. Only K. pneumoniae isolated during 2014–2016 were blaKPC positive. Prevalence of blaVIM was 87 and 76.5% during 2010 and 2014–2016, respectively. No strains colistin-resistant harbored mcr-1 plasmid-mediated resistance gene. The study findings underline an increased circulation of multidrug-resistant K. pneumoniae within the hospital, and the acquisition of carbapenem resistance mechanism. The implementation of surveillance and molecular characterization of isolates are needed to identify outbreaks, reduce the spread of resistance, and guide empirical therapy.



The authors acknowledge Dr. Jim McLauchlin, Public Health England (London, UK) for the helpful comments.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Agodi A, Barchitta M, Quattrocchi A, Maugeri A, Aldisio E, Marchese AE, Mattaliano AR, Tsakris A (2015) Antibiotic trends of Klebsiella pneumoniae and Acinetobacter baumannii resistance indicators in an intensive care unit of Southern Italy, 2008–2013. Antimicrob Resist Infect Control 4:43CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Barbarini D, Russello G, Brovarone F, Capatti C, Colla R, Perilli M, Moro ML, Carretto E (2015) Evaluation of carbapenem-resistant enterobacteriaceae in an Italian setting: report from the trench. Infect Genet Evol 30:8‒14CrossRefGoogle Scholar
  3. 3.
    Barrett TJ, Gerner-Smidt P, Swaminathan B (2006) Interpretation of pulsed-field gel electrophoresis patterns in foodborne disease investigations and surveillance. Foodborne Pathog Dis 3:20–31CrossRefPubMedGoogle Scholar
  4. 4.
    Bartolini A, Basso M, Franchin E, Menegotto N, Ferrari A, De Canale E, Andreis S, Scaggiante R, Stefani S, Palù G, Parisi SG (2017) Prevalence, molecular epidemiology and intra-hospital acquisition of Klebsiella pneumoniae strains producing carbapenemases in an Italian teaching hospital from January 2015 to September 2016. Int J Infect Dis 59:103‒109CrossRefGoogle Scholar
  5. 5.
    Bonura C, Giuffrè M, Aleo A, Fasciana T, Di Bernardo F, Stampone T, Giammanco A, MDR-GN Working Group, Palma DM, Mammina C (2015) An update of the evolving epidemic of blaKPC carrying Klebsiella pneumoniae in Sicily, Italy, 2014: emergence of multiple non-st258 clones. PLoS ONE 10:e0132936CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cannatelli A, Giani T, Antonelli A, Principe L, Luzzaro F, Rossolini GM (2016) First detection of the mcr-1 colistin resistance gene in Escherichia coli in Italy. Antimicrob Agents Chemother 60:3257‒3258CrossRefGoogle Scholar
  7. 7.
    Cantón R, Akóva M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, Livermore DM, Miriagou V, Naas T, Rossolini GM, Samuelsen Ø, Seifert H, Woodford N, Nordmann P; European Network on Carbapenemases (2012) Rapid evolution and spread of carbapenemases among enterobacteriaceae in Europe. Clin Microbiol Infect 18:413‒431CrossRefGoogle Scholar
  8. 8.
    Carnevali C, Morganti M, Scaltriti E, Bolzoni L, Pongolini S, Casadei G (2016) Occurrence of mcr-1 in colistin-resistant Salmonella enterica isolates recovered from humans and animals in Italy, 2012 to 2015. Antimicrob Agents Chemother 60(12):7532–7534PubMedPubMedCentralGoogle Scholar
  9. 9.
    Corbella M, Mariani B, Ferrari C, Comandatore F, Scaltriti E, Marone P, Cambieri P (2017) Three cases of mcr-1-positive colistin-resistant Escherichia coli bloodstream infections in Italy, August 2016 to January 2017. Euro Surveill 22:pii: 30517CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Dallenne C, Da Costa A, Decré D, Favier C, Arlet G (2010) Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in enterobacteriaceae. J Antimicrob Chemother 65:490‒495CrossRefGoogle Scholar
  11. 11.
    Del Franco M, Paone L, Novati R, Giacomazzi CG, Bagattini M, Galotto C et al (2015) Molecular epidemiology of carbapenem resistant Enterobacteriaceae in Valle d’Aosta region, Italy, shows the emergence of KPC-2 producing Klebsiella pneumoniae clonal complex 101 (ST101 and ST1789). BMC Microbiol 15:260CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Di Pilato V, Arena F, Tascini C, Cannatelli A, Henrici De Angelis L, Fortunato S, Giani T, Menichetti F, Rossolini GM (2016) mcr-1.2, a new mcr variant carried on a transferable plasmid from a colistin-resistant KPC carbapenemase-producing Klebsiella pneumoniae strain of sequence type 512. Antimicrob Agents Chemother 60:5612‒5615CrossRefGoogle Scholar
  13. 13.
    Doi Y, Paterson DL (2015) Carbapenemase-producing enterobacteriaceae. Semin Respir Crit Care Med 36:74‒84Google Scholar
  14. 14.
    European Centre for Disease Prevention and Control (ECDC). Plasmid-mediated colistin resistance in Enterobacteriaceae. Stockolm: ECDC; (2016) Available from:
  15. 15.
    Falcone M, Russo A, Iacovelli A, Restuccia G, Ceccarelli G, Giordano A, Farcomeni A, Morelli A, Venditti M (2016) Predictors of outcome in ICU patients with septic shock caused by K. pneumoniae carbapenemase-producing K. pneumoniae. Clin Microbiol Infect 22:444‒450CrossRefGoogle Scholar
  16. 16.
    Giamarellou H (2016) Epidemiology of infections caused by polymyxin-resistant pathogens. Int J Antimicrob Agents 48:614‒621CrossRefGoogle Scholar
  17. 17.
    Giani T, Pini B, Arena F, Conte V, Bracco S, Migliavacca R, Survey AMCLI-CRE, Participants, Pantosti A, Pagani L, Luzzaro F, Rossolini GM (2013) Epidemic diffusion of KPC carbapenemase-producing Klebsiella pneumoniae in Italy: results of the first countrywide survey. Euro Surveill 18:20489PubMedGoogle Scholar
  18. 18.
    Girmenia C, Serrao A, Canichella M (2016) Epidemiology of carbapenem resistant Klebsiella pneumoniae infections in Mediterranean countries. Mediterr J Hematol Infect Dis 8:e2016032CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Girometti N, Lewis RE, Giannella M, Ambretti S, Bartoletti M, Tedeschi S, Tumietto F, Cristini F, Trapani F, Gaibani P, Viale P (2014) Klebsiella pneumoniae bloodstream infection: epidemiology and impact of inappropriate empirical therapy. Medicine 93:298‒309CrossRefGoogle Scholar
  20. 20.
    Grundmann H, Glasner C, Albiger B, Aanensen DM, Tomlinson CT, Andrasević AT et al (2017) Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing enterobacteriaceae (EuSCAPE): a prospective, multinational study. Lancet Infect Dis 17:153‒163CrossRefGoogle Scholar
  21. 21.
    Han H, Zhou H, Li H, Gao Y, Lu Z, Hu K, Xu B (2013) Optimization of pulse-field gel electrophoresis for subtyping of Klebsiella pneumoniae. Int J Environ Res Public Health 10:2720‒2731CrossRefGoogle Scholar
  22. 22.
    Hunter PR, Gaston MA (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol 26:2465–2466PubMedPubMedCentralGoogle Scholar
  23. 23.
    Kramer A, Schwebke I, Kampf G (2006) How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infect Dis 6:130CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Lee CR, Lee JH, Park KS, Kim YB, Jeong BC, Lee SH (2016) Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options and detection methods. Front Microbiol 7:895PubMedPubMedCentralGoogle Scholar
  25. 25.
    Li B, Zhao Y, Liu C, Chen Z, Zhou D (2014) Molecular pathogenesis of Klebsiella pneumoniae. Future Microbiol 9:1071‒1081Google Scholar
  26. 26.
    Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J et al (2015) Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 16:161‒168Google Scholar
  27. 27.
    MacVane SH (2017) Antimicrobial resistance in the intensive care unit: a focus on Gram-negative bacterial infections. J Intensive Care Med 32:25‒37CrossRefGoogle Scholar
  28. 28.
    Magliano E, Grazioli V, Deflorio L, Leuci AI, Mattina R, Romano P, Cocuzza CE (2012) Gender and age-dependent etiology of community-acquired urinary tract infections. Sci World J 2012:349597CrossRefGoogle Scholar
  29. 29.
    Mathlouthi N, Al-Bayssari C, Bakour S, Rolain JM, Chouchani C (2017) Prevalence and emergence of carbapenemases-producing Gram-negative bacteria in Mediterranean basin. Crit Rev Microbiol 43:43‒61CrossRefGoogle Scholar
  30. 30.
    Melot B, Colot J, Guerrier G (2015) Bacteremic community-acquired infections due to Klebsiella pneumoniae: clinical and microbiological presentation in New Caledonia, 2008–2013. Int J Infect Dis 41:29‒31CrossRefGoogle Scholar
  31. 31.
    Moradigaravand D, Martin V, Peacock SJ, Parkhill J (2017) Evolution and Epidemiology of multidrug-resistant Klebsiella pneumoniae in the United Kingdom and Ireland. MBio 8:e01976–16PubMedPubMedCentralGoogle Scholar
  32. 32.
    Morrill HJ, Pogue JM, Kaye KS, LaPlante KL (2015) Treatment options for carbapenem-resistant enterobacteriaceae infections. Open Forum Infect Dis 2:ofv050CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Nordmann P, Poirel L (2014) The difficult-to-control spread of carbapenemase producers among enterobacteriaceae worldwide. Clin Microbiol Infect 20:821‒830CrossRefGoogle Scholar
  34. 34.
    Nordmann P, Poirel L (2016) Plasmid-mediated colistin resistance: an additional antibiotic resistance menace. Clin Microbiol Infect 22:398‒400CrossRefGoogle Scholar
  35. 35.
    Olaitan AO, Li J (2016) Emergence of polymyxin resistance in Gram-negative bacteria. Int J Antimicrob Agents 48:581‒582CrossRefGoogle Scholar
  36. 36.
    Onori R, Gaiarsa S, Comandatore F, Pongolini S, Brisse S, Colombo A, Cassani G, Marone P, Grossi P, Minoja G, Bandi C, Sassera D, Toniolo A (2015) Tracking nosocomial Klebsiella pneumoniae infections and outbreaks by whole-genome analysis: small-scale Italian scenario within a single hospital. J Clin Microbiol 53:2861‒2868CrossRefGoogle Scholar
  37. 37.
    Oteo J, Ortega A, Bartolomé R, Bou G, Conejo C, Fernández-Martínez M et al (2015) Prospective multicenter study of carbapenemase-producing enterobacteriaceae from 83 hospitals in Spain reveals high in vitro susceptibility to colistin and meropenem. Antimicrob Agents Chemother 59:3406‒3412CrossRefGoogle Scholar
  38. 38.
    Pantel A, Richaud-Morel B, Cazaban M, Bouziges N, Sotto A, Lavigne JP (2016) Environmental persistence of OXA-48-producing Klebsiella pneumoniae in a French intensive care unit. Am J Infect Control 44:366‒368CrossRefGoogle Scholar
  39. 39.
    Poulou A, Voulgari E, Vrioni G, Xidopoulos G, Pliagkos A, Chatzipantazi V, Markou F, Tsakris A (2012) Imported Klebsiella pneumoniae carbapenemase-producing K. pneumoniae clones in a Greek hospital: impact of infection control measures for restraining their dissemination. J Clin Microbiol 50:2618‒2623CrossRefGoogle Scholar
  40. 40.
    Ricciardi W, Giubbini G, Laurenti P (2016) Surveillance and control of antibiotic resistance in the Mediterranean Region. Mediterr J Hematol Infect Dis 8:e2016036CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Ripabelli G, Sammarco ML, Flocco R, Scutellà M, Recchia L, Grasso GM, Tamburro M (2017) Klebsiella pneumoniae isolated from intensive care unit patients with respiratory tract infections: characterization by pulsed-field gel electrophoresis, antimicrobial resistance and pcrs for carbapenemase genes detection. J Respir Med Lung Dis 2:1008Google Scholar
  42. 42.
    Russotto V, Cortegiani A, Graziano G, Saporito L, Raineri SM, Mammina C, Giarratano A (2015) Bloodstream infections in intensive care unit patients: distribution and antibiotic resistance of bacteria. Infect Drug Resist 8:287‒296Google Scholar
  43. 43.
    Sammarco ML, Ripabelli G, Tamburro M (2014) Molecular epidemiology of infectious diseases: analytical methods and results interpretation. Ann Ig 26:10‒45Google Scholar
  44. 44.
    Shahcheraghi F, Aslani MM, Mahmoudi H, Karimitabar Z, Solgi H, Bahador A, Alikhani MY (2017) Molecular study of carbapenemase genes in clinical isolates of enterobacteriaceae resistant to carbapenems and determining their clonal relationship using pulsed-field gel electrophoresis. J Med Microbiol 66:570‒576Google Scholar
  45. 45.
    Silva CD, Silva Jr M (2015) Strategies for appropriate antibiotic use in intensive care unit. Einstein 13:448‒453Google Scholar
  46. 46.
    Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233‒2239Google Scholar
  47. 47.
    The European Committee on Antimicrobial Susceptibility Testing (2016) Breakpoint tables for interpretation of MICs and zone diameters. Version 6.0, 2016. Accessed Apr 2017
  48. 48.
    Thurlow CJ, Prabaker K, Lin MY, Lolans K, Weinstein RA, Hayden MK; Centers for Disease Control and Prevention Epicenters Program (2013) Anatomic sites of patient colonization and environmental contamination with Klebsiella pneumoniae carbapenemase-producing enterobacteriaceae at long-term acute care hospitals. Infect Control Hosp Epidemiol 34:56‒61CrossRefGoogle Scholar
  49. 49.
    Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL (2012) Carbapenemases in Klebsiella pneumoniae and other enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev 25:682‒707CrossRefGoogle Scholar
  50. 50.
    Zhou H, Liu W, Qin T, Liu C, Ren H (2017) Defining and evaluating a core genome multilocus sequence typing scheme for whole-genome sequence-based typing of Klebsiella pneumoniae. Front Microbiol 8:371PubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Giancarlo Ripabelli
    • 1
  • Manuela Tamburro
    • 1
  • Giuliana Guerrizio
    • 1
  • Incoronata Fanelli
    • 1
  • Romeo Flocco
    • 2
  • Massimiliano Scutellà
    • 3
  • Michela L. Sammarco
    • 1
  1. 1.Department of Medicine and Health Sciences “Vincenzo Tiberio”University of MoliseCampobassoItaly
  2. 2.Anesthesia and Resuscitation Unit of “Antonio Cardarelli” HospitalAzienda Sanitaria Regionale MoliseCampobassoItaly
  3. 3.Laboratory Medicine Unit of “Antonio Cardarelli” HospitalAzienda Sanitaria Regionale MoliseCampobassoItaly

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