Phenotypic and molecular identification of carbapenemase-producing Enterobacteriaceae - challenges in diagnosis and treatment

Open access


Introduction: Infections due to carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CPCRE) are an emerging global public health threat. The purpose of this study was to investigate phenotypic and genotypic features of CP-CRE strains isolated from hospitalized patients. Material and methods: Between 1st of January - 1st of July 2017, in the Department of Microbiology, “Dr. Constantin Opriş” County Emergency Hospital Baia Mare, Romania, 1110 strains of Enterobacteriaceae were isolated from bronchial secretions, urine, wounds and blood cultures. Bacterial identification and antimicrobial susceptibility tests were performed by conventional methods, Vitek 2 Compact and M.I.C.E. strips. We analysed all Enterobacteriaceae strains non-susceptible to carbapenems according to CLSI 2017 criteria. The modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM) and the combination disks test (KPC, MBL, OXA-48 Confirm kit, Rosco Diagnostica) were used for phenotypic confirmation, whereas a multiplex PCR assay for genes blaKPC, blaNDM and blaOXA-48 was used for genetic confirmation. Results: 19 non-duplicate strains isolated from 16 patients were phenotypically identified as CP-CRE: Klebsiella pneumoniae (n=14), Escherichia coli (n=2), Providencia stuartii (n=2) and Serratia marcescens (n=1). Most strains were isolated from bronchial secretions (n=9). The carbapenem-hydrolizing enzymes were identified by the combination disks test as: KPC (n=9), OXA-48-like (n=5) and MBL (n=5). Molecular confirmation was performed in 18 phenotypically positive isolates with 100% concordant results with mCIM and combination disks test. Discrepant results were noticed with the MHT in case of 4 NDM-producers confirmed by PCR. All CP-CRE strains were resistant to all tested cephems. Three out of 9 K. pneumoniae strains tested against colistin were found resistant. Conclusions: The most common carbapenemase detected was KPC. Therapeutic options were limited in all positive cases. Rapid and reliable detection of CP-CRE is critical for preventing the spread of these pathogens

1. Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends in Mol Med 2012;18(5):263-72. DOI: 10.1016/j.molmed.2012.03.003

2. Lutgring JD, Limbago BM. The problem of carbapenemase- producing-carbapenem-resistant Enterobacteriaceae detection. J Clin Microbiol 2016;54:529-534. DOI: 10.1128/JCM.02771-15

3. Popescu GA, Codiţă I, Szekely E, Şerban R, Ruja G, Tălăpan D. Ghid privind Enterobacteriaceae producătoare de carbapenemaze: diagnosticul, prevenirea transmiterii interumane şi tratamentul infecţiilor produse. Centrul Naţional de Supraveghere şi Control a Bolilor Transmisibile 2015. Available from:

4. Yamanda K, Kashiwa M, Arai K, Nagano N, Saito R. Evaluation of the modified carbapenem inactivation method and sodium mercaptoacetate-combination method for the detection of metallo-β-lactamase production by carbapenemase-producing Enterobacteriaceae. J Microbiol Meth 2017;132:112-115.

5. Tangdén T, Giske CG. Global dissemination of extensively drug-resistant carbapenemase-producing Enterobacteriaceae: clinical perspectives on detection, treatment and infection control. J Intern Med 2015;277:501-512. DOI: 10.1111/joim.12342

6. European Centre for Disease Prevention and Control. Rapid risk assessment: carbapenem-resistant Enterobacteriaceae - 8th April 2016. Stockholm: ECDC; 2016. Available from:

7. World Health Organization. Global priority list of antibiotic- resist¬ant bacteria to guide research, discovery, and development of new antibiotics. Geneva: WHO; 2017. Available from:

8. Butler-Wu SM, Abbott AN. Is this the carbapenemase test we’ve been waiting for? A multi-center evaluation of the Modified Carbapenem Inactivation Method (mCIM). J Clin Microbiol 2017;55:2321-2333. DOI: 10.1128/JCM.00538-17

9. Székely E, Damjanova I, Jánvári L, Vas KE, Molnár S, Bilca DV, et al. First description of blaNDM-1, blaOXA-48, blaOXA-181 producing Enterobacteriaceae strains in Romania. Int J of Med Microbiol 2013;303(8):697-700. DOI: 10.1016/j.ijmm.2013.10.001

10. Dortet L, Flonta M, Boudehen YM, Creton E, Bernabeub S, Vogel A, et al. Dissemination of carbapenemase- producing Enterobacteriaceae and Pseudomonas aeruginosa in Romania. Antimicrob Agents Chemother 2015;59(11):7100-7103. DOI: 10.1128/AAC.01512-15

11. Lixandru BE, Cotar AI, Straut M, Usein CR, Cristea D, Ciontea S, et al. Carbapenemase-producing Klebsiella pneumoniae in Romania: A Six- Month Survey. PLoS ONE 2015;10(11): e0143214. DOI: 10.1371/journal.pone.0143214

12. Rafila A, Talapan D, Dorobăţ OM, Popescu GA, Piţigoi D, Florea D, et al. Emergence of carbapenemase-producing Enterobacteriaceae, a public health threat: a Romanian infectious disease hospital based study. Revista Română de Medicină de Laborator 2015;23(3):295-301. DOI: 10.1515/rrlm-2015-0024

13. Popescu C, Popescu GA, Dorobăț O, Rafila A, Tănase D, Mikula C, et al. OXA-48-carbapenemase- producing Klebsiella pneumoniae infections - the first cases diagnosed in Romanian National Institute of Infectious Diseases. Revista Română de Medicină de Laborator 2017;25(1):55-61. DOI: 10.1515/rrlm-2017-0006

14. Performance Standards for antimicrobial susceptibility testing, 27th informational supplement. CLSI document M100-S27. Clinical and Laboratory Standards Institute, 2017.

15. Duin vD, Doi Y. The global epidemiology of carbapenemase-producing Enterobacteriaceae. Virulence 2017;8(4):460-469. DOI: 10.1080/21505594.2016.1222343

16. Centers for Disease Control and Prevention. Vital signs: carbapenem-resistant Enterobacteriaceae. MMWR Morbid. Mortal. Wkly. Rep. 2013;62(09):165-170.

17. European Centre for Disease Prevention and Control. Surveillance of antimicrobial resistance in Europe 2016. Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net), November 2017. Available from:

18. Albiger B, Glasner C, Struelens MJ, Grundmann H, Monnet DL, the European Survey of Carbapenemase- Producing Enterobacteriaceae (EuSCAPE) working group. Carbapenemase-producing Enterobacteriaceae in Europe: assessment by national experts from 38 countries, May 2015. Euro Surveill 2015;20(45):piiD30062;PMID:26675038. DOI: 10.2807/15607917.ES.2015.20.45.30062

19. Wang X, Chen G, Wu X, Wang L, Cai J, Chan EW, et al. Increased prevalence of carbapenem resistant Enterobacteriaceae in hospital setting due to cross-species transmission of the blaNDM-1 element and clonal spread of progenitor resistant strains. Front. Microbiol. 2015;6:595. DOI: 10.3389/fmicb.2015.00595

20. Bano JR, Cisneros JM, Cobos-Trigueros N, Fresco G, Navarro-San Francisco C, Gudiol C, et al. Diagnosis and antimicrobial treatment of invasive infections due to multidrug-resistant Enterobacteriaceae. Guidelines of the Spanish Society of Infectious Diseases and Clinical Microbiology. Enferm Infecc Microbiol Clin 2015;33(5):337.e1-337.e21. DOI: 10.1016/j.eimc2014.11.009

21. Pasteran F, Gonzalez LJ, Albornoz E, Bahr G, Vila AJ, Corso A. Triton Hodge Test: Improved protocol for modified Hodge test for enhanced detection of NDM and other carbapenemase producers. J Clin Microbiol 2016;54(3):640-649. DOI: 10.1128/JCM.01298-15

22. Sun K, Xu X,Yan Y, Zhang L. Evaluation of six phenotypic methods for the detection of carbapenemases in Gram-negative bacteria with characterized resistance mechanisms. Ann Lab Med 2017;37:305-312. DOI: 10.3343/alm.2017.37.4.305

23. Nordmann P, Poirel L. Strategies for identification of carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother 2013;68:487-489. DOI: 10.1093/jac/dks426

24. Daikos GL, Tsaousi S, Tzouvelekis LS, Anyfantis I, Psichogiou M, Argyropoulou A, et al. Carbapenemase- producing Klebsiella pneumoniae bloodstream infections: lowering mortality by antibiotic combination schemes and the role of carbapenems. Antimicrob Agents Chemother 2014;58(4):2322-2328. DOI: 10.1128/AAC.02166-13

25. Ko JH, Baek JY, Peck KR, Cho SY, Ha YE, Kim SH, et al. Discrepant susceptibility to gentamicin despite amikacin resistance in Klebsiella pneumoniae by VITEK 2 represents false susceptibility associated with the armA 16S rRNA methylase gene. J Med Microbiol 2017;66(10):1448-1450. DOI: 10.1099/jmm.0.000583

26. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. 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 2015;16:161-8; PMID:26603172. DOI: 10.1016/S1473-3099(15)00424-7

27. Chiu SK, Chan MC, Huang LY, Lin YT, Lin JC, Lu PL, et al. Tigecycline resistance among carbapenem- resistant Klebsiella pneumoniae: Clinical characteristics and expression levels of efflux pump genes. PloS ONE 2017;12(4):e0175140. DOI: 10.1371/journal.pone.0175140

Revista Romana de Medicina de Laborator

Romanian Journal of Laboratory Medicine

Journal Information

IMPACT FACTOR 2018: 0,800
5-year IMPACT FACTOR: 0,655

CiteScore 2017: 0.31

SCImago Journal Rank (SJR) 2018: 0.194
Source Normalized Impact per Paper (SNIP) 2018: 0.306


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 282 185 6
PDF Downloads 208 149 5