In Vitro Activity of Single and Combined Antibiotics against Carbapenem Resistant Enterobacteriaceae Clinical Isolates in Relation to their Resistance Genes


  • Inas El-Defrawy Department of Microbiology, Theodor Bilharz Research Institute, Giza, Egypt
  • Aisha Abu Aitta Department of Microbiology, Theodor Bilharz Research Institute, Giza, Egypt
  • Nevine Fam Department of Microbiology, Theodor Bilharz Research Institute, Giza, Egypt
  • Manar Khaled Department of Microbiology, Theodor Bilharz Research Institute, Giza, Egypt
  • Nadia Madany Department of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University, Giza, Egypt
  • Mervat El Damarawy Intensive Care Unit, Theodor Bilharz Research Institute, Giza, Egypt
  • Doaa Gamal Department of Microbiology, Theodor Bilharz Research Institute, Giza, Egypt
  • Mohammed Amr Alkholy Department of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University, Giza, Egypt



Carbapenem, Treatment, Carbapenem-resistant Enterobacteriaceae, Carbapenemase-producing Enterobacteriaceae, Colistin, Tygecycline, Dual carbapenem, NDM, OXA-48


Background: Mortality due to infection with carbapenem-resistant Enterobacteriaceae (CRE) is reported globally and carbapenemase production is the main mechanism of resistance in these isolates. The detection and treatment of carbapenemase-producing Enterobacteriaceae (CPE) is a major challenge in health care facilities.

Objectives: The aim of the current study was to evaluate the in-vitro effect of different single and combined antibiotic agents against CRE clinical isolates.

Methodology: Fifty CRE isolates were detected using disk diffusion test as a screening test. Species identification and antibiotic susceptibility testing was done using Vitek 2 system. Carbapenemase enzyme production was confirmed by Carba NP test. Multiplex PCR was done to detect carbapenem resistance genes. Antibiotics were tested in the form of single agents (colistin and tigecycline) and combined (tigecycline/ colistin, doripenem/ colistin and dual carbapenem therapy (ertapenem and doripenem) against CRE isolates using E-test method.

Results: Most of the CRE isolates were K. pneumoniae, 68%, followed by E. coli, 22%, S. marcescens, 4%, E. cloacae, 4% and C. freundii, 2%. CPE was confirmed in 46 isolates by multiplex PCR; blaNDM-like was the main carbapenem resistance gene in (84%) of the isolates, followed by blaOXA-48-like (6%) and blaKPC-like (2%). Carba NP test detected 90% of CPE isolates. Single use of colistin and tigecycline showed 100% sensitivity against all tested CRE isolates except in blaNDM-like (83%).  Combination of colistin/tigecycline showed synergetic activity in 18% of CRE that was correlated to their carbapenemase R genes showing a significant increase in blaOXA-48-like and blaKPC-like positive isolates (100%) compared to blaNDM-like (7%). Other combinations showed indifferent effect whereas antagonism was not detected in any of the tested combinations.

Conclusions: blaNDM-like is the main carbapenemase-producing gene detected among our CPE isolates followed by blaOXA-48-like. Colistin and tigecycline are still effective when used as single agents, and may offer effective treatment options when used in combination for CRE infections. Characterization of carbapenemases is crucial in determining treatment options. There is urgent demand for the development of novel therapeutic agents against NDM-producing CPE isolates.


Download data is not yet available.


Metrics Loading ...

Plum Analytics Artifact Widget Block


Gajdács M, Urbán E, Stájer A, Baráth Z. Antibiotic resistance in the context of the sustainable development goals: A brief review. Eur J Investig Health Psychol Educ. 2021;11(1):71-82. PMid:34542450 DOI:

Touati A, Mairi A. Epidemiology of carbapenemase-producing Enterobacterales in the Middle East: A systematic review. Expert Rev Anti Infect Ther. 2020;18(3):241-50. PMid:32043905 DOI:

Wauters G, Vaneechoutte M. Approach to the identification of aerobic Gram-negative bacteria. In: Manual of Clinical Microbiology. Washington, DC: ASM Press; 2011. p. 539-48. DOI:

Bush K, Jacoby G. Updated functional classification of beta-lactamases. Antimicrob Agents Chemother. 2010;54(3):969-76. PMid:19995920 DOI:

Betts JW, Phee LM, Hornsey M, Woodford N, Wareham DW. In vitro and in vivo activities of tigecycline-colistin combination therapies against Carbapenem-resistant Enterobacteriaceae. Antimicrob Agents Chemother. 2014;58(6):3541-6. PMid:24687491 DOI:

Nordmann P, Poirel L. Strategies for identification of carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother. 2013;68(3):487-9. PMid:23104494 DOI:

Queenan AM, Bush K. Carbapenemases: The versatile beta-lactamases. Clin Microbiol Rev. 2007;20(3):440-58. PMid:17630334 DOI:

Kashyap A, Gupta R, Sharma R, Verma VV, Gupta S, Goyal P. New Delhi metallo beta lactamase: Menace and its challenges. J Mol Genet Med. 2017;11(4):299. DOI:

Nordmann P, Gniadkowski M, Giske CG, Poirel L, Woodford N, Miriagou V, on Carbapenemases EN. Identification and screening of carbapenemase-producing Enterobacteriaceae. Clinical Microbiology and Infection. 2012;18:432-8. DOI:

Marchaim D, Navon-Venezia S, Schwaber MJ, Carmeli Y. Isolation of imipenem-resistant Enterobacter species: Emergence of KPC-2 carbapenemase, molecular characterization, epidemiology, and outcomes: Antimicrob Agents Chemother. 2008;52(4):1413-8. PMid:18227191 DOI:

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):2322-8. PMid:24514083 DOI:

Falagas ME, Lourida P, Poulikakos P, Rafailidis PI, Tansarli GS. Antibiotic treatment of infections due to carbapenem-resistant Enterobacteriaceae: Systematic evaluation of the available evidence. Antimicrob Agents Chemother. 2014;58(2):654-63. PMid:24080646 DOI:

Zhou YF, Liu P, Zhang CJ, Liao XP, Sun J, Liu YH. Colistin combined with tigecycline: A promising alternative strategy to combat Escherichia coli harboring bla NDM-5 and mcr-1. Front Microbiol. 2020;10:2957. PMid:31969868 DOI:

Horcajada JP, Torre-Cisneros J, Peña C, Fariñas MC. Future alternatives for the treatment of infections caused by carbapenemase producing Enterobacteriaceae: What is in the pipeline? Enferm Infecc Microbiol Clin. 2014;32(Suppl 4):56-60. PMid:25542053 DOI:

European Committee on Antibiotic Susceptibility Testing. Testing Breakpoint Tables for Interpretation of MICs and Zone Diameters Version 5.0, Valid from 2015-01-01. Sweden: European Committee on Antibiotic Susceptibility Testing; 2017.

Nordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: Here is the storm! Trends Mol Med. 2012;18(5):263-72. PMid:22480775 DOI:

White RL, Burgess DS, Manduru M, Bosso JA. Comparison of three different in vitro methods of detecting synergy: Time-kill, checkerboard, and E-test. Antimicrob Agents Chemother. 1996;40(8):1914-8. PMid:8843303 DOI:

Samonis G, Maraki S, Karageorgopoulos DE, Vouloumanou EK, Falagas ME. Synergy of fosfomycin with carbapenems, colistin, netilmicin, and tigecycline against multidrug-resistant Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa clinical isolates: Eur J Clin Microbiol Infect Dis. 2012;31(5):695-701. PMid:21805292 DOI:

Poirel L, Dortet L, Bernabeu S, Nordmann P. Genetic features of blaNDM-1 positive Enterobacteriaceae. Antimicrob Agents Chemother. 2011;55(11):5403-7. PMid:21859933 DOI:

Dundar D, Duymaz Z, Genc S, Er DK, İrvem A, Kandemir N. In-vitro activities of imipenem–colistin, imipenem–tigecycline, and tigecycline–colistin combinations against carbapenem-resistant Enterobacteriaceae. Journal of Chemotherapy. 2018;30:342-7. DOI:

Shanmugam P, Meenakshisundaram J, Jayaraman P. blaKPC gene detection in clinical isolates of carbapenem resistant Enterobacteriaceae in a tertiary care hospital. J Clin Diagn Res. 2013;7(12):2736-8. DOI:

Huang SR, Liu MF, Lin CF, Shi ZY. Molecular surveillance and clinical outcomes of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae infections. J Microbiol Immunol Infect. 2014;47(3):187-96 PMid:23200553 DOI:

Amer WH, Khalil HS, Abdelwahab MA. Risk factors, phenotypic and genotypic characterization of carpabenem resistant Enterobacteriaceae in Tanta university hospitals, Egypt. Int J Infect Control. 2016;12(2):1-11. DOI:

Nagaraj S, Chandran SP, Shamanna P, Macaden R. Carbapenem resistance among Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital in South India: Indian J Med Microbiol. 2012;30(1):93-5. PMid:22361769 DOI:

Lin MY, Lyles-Banks RD, Lolans K, Hines DW, Spear JB, Petrak R, et al. The importance of long-term acute care hospitals in the regional epidemiology of Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae. Clin Infect Dis. 2013;57(9):1246-52. PMid:23946222 DOI:

Dortet L, Agathine A, Naas T, Cuzon G, Poirel L, Nordmann P. Evaluation of the RAPIDEC® CARBA NP, the rapid CARB screen® and the carba NP test for biochemical detection of carbapenemase-producing Enterobacteriaceae. J Antimicrob Chemother. 2015;70(11):3014-22. PMid:26260131 DOI:

Rudresh SM, Ravi GS, Sunitha L, Hajira SN, Kalaiarasan E, Harish BN. Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among gram-negative bacteria. J Lab Physicians. 2017;9(4):303-7. PMid:28966495 DOI:

Kazi M, Drego L, Nikam C, Ajbani K, Soman R, Shetty A, et al. Molecular characterization of carbapenem-resistant Enterobacteriaceae at a tertiary care laboratory in Mumbai. Eur J Clin Microbiol Infect Dis. 2015;34(3):467-72. PMid:25260787 DOI:

Perovic O, Britz E, Chetty V, Singh-Moodley A. Molecular detection of carbapenemase-producing genes in referral Enterobacteriaceae in South Africa: A short report. S Afr Med J. 2016;106(10):975-7. PMid:27725012 DOI:

Jamal WY, Albert MJ, Rotimi VO. High prevalence of New Delhi metallo-β-lactamase-1 (NDM-1) producers among carbapenem-resistant Enterobacteriaceae in Kuwait. PLoS One. 2016;11(3):e0152638. PMid:27031521 DOI:

Osterblad M, Hakanen AJ, Jalava J. Evaluation of the Carba NP test for carbapenemase detection. Antimicrob Agents Chemother. 2014;58(12):7553-6. PMid:25246404 DOI:

Pasteran F, Tijet N, Melano RG, Corso A. Simplified protocol for Carba NP test for enhanced detection of carbapenemase producers directly from bacterial cultures. J Clin Microbiol. 2015;53(12):3908-11. PMid:26424841 DOI:

Giancarlo T, Pini B, Arena F, Conte V, Bracco S, Migliavacca R, et al. Epidemic diffusion of KPC carbapenemase-producing Klebsiella pneumoniae in Italy: Results of the first countrywide survey, 15 May to 30 June 2011. Euro Surveill. 2013;18(22):20489. PMid:23787077 DOI:

Souli M, Karaiskos I, Masgala A, Galani L, Barmpouti E, Giamarellou H. Double-carbapenem combination as salvage therapy for untreatable infections by KPC-2-producing Klebsiella pneumoniae. Eur J Clin Microbiol Infect Dis. 2017;36(7):1305-15. PMid:28210888 DOI:

Chahine EB, Ferrill MJ, Poulakos MN. Doripenem: A new carbapenem antibiotic. Am J Health Syst Pharm. 2010;67(23):2015-24. PMid:21098373 DOI:

Tzouvelekis LS, Markogiannakis A, Piperaki E, Souli M, Daikos GL. Treating infections caused by carbapenemase-producing Enterobacteriaceae. Clin Microbiol Infect. 2014;20(9):862-72. PMid:24890393 DOI:

Bergen PJ, Li J, Nation RL. Dosing of colistin back to basic PK/PD: Curr Opin Pharmacol. 2011;11(5):464-9. PMid:21835694 DOI:

Yahav D, Lador A, Paul M, Leibovici I. Efficacy and safety of tigecycline: A systematic review and meta-analysis. J Antimicrob Chemother. 2011;66(9):1963-71. PMid:21685488 DOI:

Hornsey M, Ellington MJ, Doumith M, Thomas CP, Gordon NC, Wareham DW, et al. AdeABC-mediated efflux and tigecycline MICs for epidemic clones of Acinetobacter baumannii. J Antimicrob Chemother. 2010;65(8):1589-93. PMid:20554571 DOI:

Pournaras S, Vironi G, Neou E, Dendrinos J, Dimitroulia E, Poulou A, et al. Activity of tigecycline alone and in combination with colistin and meropenem against Klebsiella pneumonia carbapenemase (KPC)-producing Enterobacteriaceae by time-kill assay. Int J Antimicrob Agents. 2011;37(3):244-7. PMid:21236643 DOI:

Zavascki AP, Bulitta JB, Landersdorfer CB. Combination therapy for carbapenem-resistant gram-negative bacteria. Expert Rev Anti Infect Ther. 2013;11(12):1333-53. PMid:24191943 DOI:

De Pascale G, Martucci G, Montini L, Panarello G, Cutuli SL, Di Carlo D, et al. Double carbapenem as a rescue strategy for the treatment of severe carbapenemase-producing Klebsiella pneumoniae infections: A two-center, matched case-control study. Crit Care. 2017;21(1):173. PMid:28679413 DOI:

Qamar MU, Lopes BS, Hassan B, Khurshid M, Shafique M, Nisar MA, et al. The present danger of New Delhi metallo- β-lactamase: A threat to public health. Future Microbiol. 2020;15:1759-78. PMid:33404261 DOI:

Tilahun M, Kassa Y, Gedefie A, Ashagire M. Emerging carbapenem-resistant Enterobacteriaceae infection, its epidemiology and novel treatment options. Infect Drug Resist. 2021;14:4363-74. PMid:34707380 DOI:

Durante-Mangoni E, Andini R, Zampino R. Management of carbapenem-resistant Enterobacteriaceae infections. Clin Microbiol Infect. 2019;25(8):943-50. PMid:31004767 DOI:

Li J, Bi W, Dong G, Zhang Y, Wu Q, Dong T, et al. The new perspective of old antibiotic: In Vitro antibacterial activity of TMP-SMZ against Klebsiella Pneumoniae. J Microbiol Immunol Infect. 2020;53(5):757-65. PMid:30857922 DOI:

Lin Q, Wang Y, Yu J, Li S, Zhang Y, Wang H, et al. Bacterial characteristics of carbapenem-resistant Enterobacteriaceae (CRE) colonized strains and their correlation with subsequent infection. BMC Infect Dis. 2021;21(1):638. PMid:34215214 DOI:

Nabti LZ, Sahli F, Olowo-Okere A, Benslama A, Harrar A, Lupande-Mwenebitu D, et al. Molecular characterization of clinical carbapenem-resistant Enterobacteriaceae isolates from Sétif, Algeria. Microb Drug Resist. 2022;28(3):274-9. PMid:34860598 DOI:

Jean SS, Harnod D, Hsueh PR. Global threat of carbapenem resistant gram-negative bacteria. Front Cell Infect Microbiol. 2022;12:823684. PMid:35372099 DOI:




How to Cite

El-Defrawy I, Aitta AA, Fam N, Khaled M, Madany N, El Damarawy M, Gamal D, Alkholy MA. In Vitro Activity of Single and Combined Antibiotics against Carbapenem Resistant Enterobacteriaceae Clinical Isolates in Relation to their Resistance Genes. Open Access Maced J Med Sci [Internet]. 2022 Jul. 21 [cited 2023 Mar. 28];10(A):1600-7. Available from:

Funding data