Skip to main content
SpringerLink
Log in

Multiplex real-time PCR probe-based for identification of strains producing: OXA48, VIM, KPC and NDM

  • Original Paper
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The spread of multi-resistant enterobacteria, particularly carbapenem-resistant Enterobacteriaceae (CRE), in both community and hospital settings is a global problem. The phenotypic identification of CRE is complex, occasionally inconclusive and time consuming. However, commercially available molecular assays are very expensive, and many do not allow the simultaneous identification of all genetic markers of resistance that have been recognised in CRE (bla KPC, bla OXA-48, bla VIM and bla NDM). The aim of the present study is to describe a new test: a multiplex real time PCR probe-based assay designed for the simultaneous detection of KPC, OXA-48, VIM and NDM in a short time (no longer than 90 min from the extraction of DNA to detection). Our assay correctly identified 63 CRE isolates and all standard reference strains tested, in agreement with and extending the results of phenotypic identification tests; additionally, a KPC–VIM co-expressing Enterobacter aerogenes isolate was identified using the new assay, whereas traditional methods failed to detect it. The assay was also able to correctly detect 28 CRE-producers from 50 positive blood cultures, again detecting, in four specimens, the presence of CRE co-expressing KPC and VIM, which were only partially identified by traditional methods. Finally, when used directly on rectal swabs, the assay enabled the identification of CRE-carrier patients, for whom isolation is mandatory in a hospital setting.

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
Fig. 2

Similar content being viewed by others

References

  • Adler A, Navon-Venezia S, Moran-Gilad J, Marcos E, Schwartz D, Carmeli Y (2012) Laboratory and clinical evaluation of screening agar plates for detection of carbapenem-resistant Enterobacteriaceae from surveillance rectal swabs. J Clin Microbiol 49:2239–2242

    Article  Google Scholar 

  • CDC (2014) Carbapenem-resistant Enterobacteriaceae in healthcare settings. Available at CDC home page http://www.cdc.gov/hai/organisms/cre/. Accessed 10 June 2014

  • Chen L, Mediavilla JR, Endimiani A, Rosenthal ME, Zhao Y, Bonomo RA, Kreiswirth BN (2011) Multiplex real-time PCR assay for detection and classification of Klebsiella pneumoniae carbapenemase gene (bla KPC) variants. J Clin Microbiol 49:579–585

    Article  CAS  Google Scholar 

  • Cunningham SA, Noorie T, Meunier D, Woodford N, Patel R (2013) Rapid and simultaneous detection of genes encoding Klebsiella pneumoniae carbapenemase (blaKPC) and New Delhi metallo-β-lactamase (blaNDM) in Gram-negative bacilli. J Clin Microbiol 51:1269–1271

    Article  CAS  Google Scholar 

  • Dhillon RKP, Clark J (2012) ESBLs: a clear and present danger? Crit Care Res Pract. doi:10.1155/2012/625170

    Google Scholar 

  • Doi Y, Park YS, Rivera JI, Adams-Haduch JM, Hingwe A, Sordillo EM, Lewis JS II, Howard WJ, Johnson LE, Olsky B, Jorgensen JH, Richter SS, Shutt KA, Paterson DL (2013) Community-associated extended-spectrum β-lactamase-producing Escherichia coli infection in the United States. Clin Infect Dis 56:641–648. doi:10.1093/cid/cis942

    Article  Google Scholar 

  • Evans BA, Amyes SBJ (2014) OXA beta-lactamases. Clin Microbiol Rev 27:241–263

    Article  CAS  Google Scholar 

  • Favaro M, Savini V, Favalli C, Fontana C (2013) A multi-target real-time PCR assay for rapid identification of meningitis-associated microorganisms. Mol Biotechnol 53:74–79

    Article  CAS  Google Scholar 

  • Fontana C, Favaro M, Sarmati L, Natoli S, Altieri A, Bossa MC, Minelli S, Leonardis F, Favalli C (2010) Emergence of KPC-producing Klebsiella pneumoniae in Italy. BMC Res Notes 3:40

    Article  Google Scholar 

  • Giani T, D’Andrea MM, Pecile P, Borgianni L, Nicoletti P, Tonelli F, Bartoloni A, Rossolini GM (2009) Emergence in Italy of Klebsiella pneumoniae sequence type 258 producing KPC-3 carbapenemase. J Clin Microbiol 47:3793–3794

    Article  Google Scholar 

  • Giani T, Conte V, Di Pilato V, Aschbacher R, Weber C, Larcher C, Rossolini GM (2012) Escherichia coli from Italy producing OXA-48 carbapenemase encoded by a novel Tn1999 transposon derivative. Antimicrob Agents Chemother 56:2211–2213

    Article  CAS  Google Scholar 

  • Giani T, Pini B, Arena F, Conte V, Bracco S, Migliavacca R, the AMCLI-CRE Survey 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. Eurosurveill 18:22

    Google Scholar 

  • Hanemaaijer NM, Nijhuis RH, Slotboom BJ, Mascini EM, van Zwet AA (2014) New screening method to detect carriage of carbapenemase-producing Enterobacteriaceae in patients within 24 hours. J Hosp Infect 87:47–49

    Article  CAS  Google Scholar 

  • Hindiyeh M, Smollan G, Grossman Z, Ram D, Robinov J, Belausov N, Ben-David D, Tal I, Davidson Y, Shamiss A, Mendelson E, Keller N (2011) Rapid detection of blaKPC carbapenemase genes by internally controlled real-time PCR assay using bactec blood culture bottles. J Clin Microbiol 49:2480–2484

    Article  Google Scholar 

  • Jacoby G, Bush K (2005) Beta-lactam resistance in the 21st century. In: White DG, Alekshun MN, McDermott PF (eds) Frontiers in antimicrobial resistance: a tribute to Stuart B. Levy. ASM Press, Washington, pp 53–65

  • Kaase M, Szabados F, Wassill L, Gatermann SG (2012) Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR. J Clin Microbiol 50:3115–3118

    Article  CAS  Google Scholar 

  • Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF, Sumpradit N, Vlieghe E, Hara GL, Gould IM, Goossens H, Greko C, So AD, Bigdeli M, Tomson G, Woodhouse W, Ombaka E, Peralta AQ, Qamar FN, Mir F, Kariuki S, Bhutta ZA, Coates A, Bergstrom R, Wright GD, Brown ED, Cars O (2013) Antibiotic resistance-the need for global solutions. Lancet Infect Dis 13:1057–1098

    Article  Google Scholar 

  • Magiorakos AP (2011a) Scientific advice: a European perspective on CPE: the necessity to turn the tide. http://www.ecdc.europa.eu/en/activities/diseaseprogrammes/arhai/presentations2011warsaw/arhai-networks-meeting_plenary-session-two-4-anna-pelagia-magiorakos.pdf. Accessed 10 June 2014

  • Magiorakos AP (2011b) Carbapenemase-producing bacteria in Europe- update from ECDC and future directions. http://www.ecdc.europa.eu/en/activities/diseaseprogrammes/arhai/presentations2011warsaw/arhai-networks-meeting_parallel-session-seven-3-anna%20pelagia%20magiorakos.pdf. Accessed 10 June 2014

  • Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, Pignatari AC, Tufik S (2007) Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 45:544–547

    Article  CAS  Google Scholar 

  • Monteiro J, Widen RH, Pignatari ACC, Kubasek C, Silbert S (2012) Rapid detection of carbapenemase genes by multiplex real-time PCR. J Antimicrob Chemother 67:906–909

    Article  CAS  Google Scholar 

  • Nijhuis R, Samuelsen Ø, Savelkoul P, van Zwet A (2013) Evaluation of a new real-time PCR assay (Check-Direct CPE) for rapid detection of KPC, OXA-48, VIM, and NDM carbapenemases using spiked rectal swabs. Diagn Microbiol Infect Dis 77:316–320

    Article  CAS  Google Scholar 

  • Nordmann P, Poirel L (2013) Strategies for identification of carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother 68:487–489

    Article  CAS  Google Scholar 

  • Nordmann P, Gniadkowski M, Giske CG, Poirel L, Woodford N, Miriagou V, European Network on Carbapenemases, (2012) Identification and screening of carbapenemase-producing Enterobacteriacae. Clin Microbiol Infect 18:432–438

    Article  CAS  Google Scholar 

  • Paterson DL, Singh N, Rihs JD, Squier C, Rihs BL, Muder RR (2001) Control of an outbreak of infection due to extended-spectrum β-lactamase-producing Escherichia coli in a liver transplantation unit. Clin Infect Dis 33:1126–1128

    Article  Google Scholar 

  • Perilli M, Mezzatesta ML, Falcone M, Pellegrini C, Amicosante G, Venditti M, Stefani S (2008) Class I integron-borne bla (VIM-1) carbapenemase in a strain of Enterobacter cloacae responsible for a case of fatal pneumonia. Microb Drug Resist 14:45–47

    Article  CAS  Google Scholar 

  • Pitout JDD, Laupland KB (2008) Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public health concern. Lancet Infect Dis 3:159–166

    Article  Google Scholar 

  • Pitout JDD, Nordmann P, Laupland KP, Poirel L (2005) Emergence of Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) in the community. J Antimicrob Chemother 56:52–59

    Article  CAS  Google Scholar 

  • Rosdahl VT, Pedersen KB (1998) Report from the invitational European Union conference on “The Microbial Threat” hosted by the Danish Government in Copenhagen, Denmark, 9–10 September 1998. www.microbial.threat.dk

  • Struelens MJ, Monnet DL, Magiorakos AP, Santos O’Connor F, Giesecke J (2010) European NDM-1 survey participants. New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae: emergence and response in Europe. Euro Surveill 15(46):19716

  • Swayne RL, Ludlama HA, Shet VG, Woodfordb N, Curran MD (2011) Real-time TaqMan PCR for rapid detection of genes encoding five types of non-metallo- (class A and D) carbapenemases in Enterobacteriaceae. Int J Antimicrob Agents 38:35–38

    Article  CAS  Google Scholar 

  • Voulgari E, Poulou A, Koumaki V, Tsakris A (2013) Carbapenemase-producing Enterobacteriaceae: now that the storm is finally here, how will timely detection help us fight back? Future Microbiol 8:27–39

    Article  CAS  Google Scholar 

Download references

Conflict of interest

The authors have no conflict of interest to declare.

Author information

Authors and Affiliations

  1. Department of Experimental Medicine and Surgery, “Tor Vergata” University of Rome, Via Montpellier 1, 00133, Rome, Italy

    Marco Favaro & Carla Fontana

  2. Clinical Microbiology Laboratories, Foundation Polyclinic Tor Vergata, V.le Oxford 81, 00133, Rome, Italy

    Carla Fontana

  3. Dipartimento Interaziendale ad Attività Integrata Medicina di Laboratorio ed Anatomia Patologica, Microbiologia Clinica Provinciale Azienda USL di Modena NOCSAE, Baggiovara, Modena, Italy

    Mario Sarti

Authors
  1. Marco Favaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mario Sarti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carla Fontana
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Carla Fontana.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Favaro, M., Sarti, M. & Fontana, C. Multiplex real-time PCR probe-based for identification of strains producing: OXA48, VIM, KPC and NDM. World J Microbiol Biotechnol 30, 2995–3001 (2014). https://doi.org/10.1007/s11274-014-1727-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11274-014-1727-8

Keywords

Search

Navigation