Original Article

An In-Vitro Study of Molecular Effects of a Combination Treatment with Antibiotics and Nanofluid Containing Carbon Nano-tubes on Klebsiella pneumoniae


Background: We aimed to prepare a nanofluid, containing f-MWCNTs, and investigate the antibacterial efficacy of f-MWCNTs+ ciprofloxacin (cip) on Klebsiella pneumoniae by evaluating the virulence gene expression.

Methods: This study was carried out from 2019 to 2020, in the Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran. The nanofluid containing antibiotic and f-MWCNTs were prepared by the ultrasonic method. The minimum inhibitory concentrations (MICs) of ciprofloxacin and f-MWCNTs were determined using the broth micro dilution MIC tests. For examining the antibacterial effects, the expression level of virulence genes, under the influence of f-MWCNTs, was evaluated by a real-time PCR.

Results: The effect of 8 µg/ml ciprofloxacin + 400 µg/ml f-MWCNTs, completely inhibited the growth of the resistant isolate of K. pneumoniae, while, in the ATCC 700,603 isolate, 2 µg/ml ciprofloxacin with 100 µg/ml f-MWCNT could inhibit a bacterial growth. In the resistant K. pneumoniae clinical isolate, after f-MWCNT+cip treatment, the expression of fimA, fimD, wza, and wzi genes was significantly downregulated, compared to the ciprofloxacin treatment, and upregulated, compared to the negative control. For the ATCC 700,603 isolate treated with f-MWCNT+cip, the expression of fimA, fimD and wza virulence genes showed upregulation, compared to the negative control and downregulated in comparison with the ciprofloxacin treatment.

Conclusion: Simultaneous treatment of resistant isolate of K. pneumoniae with f-MWCNTs +antibiotic could improve the effectiveness of antibiotic at lower doses, due to the reduced expression of virulence genes in comparison with antibiotic treatment, besides the increased cell wall permeability to antibiotics.

1. Jiang W, Yang W, Zhao X, Wang N, Ren H (2020). Klebsiella pneumoniae presents antimicrobial drug resistance for β lactam through the ESBL/PBP signaling pathway. Exp Ther Med, 19(4): 2449-56.
2. Dosunmu E, Chaudhari AA, Singh Sh, et al (2015). Silver-coated carbon nanotubes downregulate the expression of Pseudomonas aeruginosa virulence genes: a potential mechanism for their antimicrobial effect. Int J Nanomedicine, 10: 5025–34.
3. Griffiths J, Maguire JH, Heggenhougen K, Quah S (2010). Public Health and Infectious Diseases. 1st ed. Elsevier. USA. pp.119-124.
4. Marcoleta AE, Varas MA, Ortiz-Severín J, et al (2018). Evaluating Different Virulence Traits of Klebsiella pneumoniae Using Dictyostelium discoideum and Zebrafish Larvae as Host Models. Front Cell Infect Microbiol, 8:30.
5. Kamlesh JRK, Mannheim W (1974). Differentiation of the Oxytocum group from Klebsiella by deoxyribonucleic acid hybridization. Int J Syst Bacteriol, 24 (4): 402-7.
6. Ranjbar R, Fatahian Kelishadrokhi A, Chehelgerdi M (2019). Molecular characterization, serotypes and phenotypic and genotypic evaluation of antibiotic resistance of the Klebsiella pneumoniae strains isolated from different types of hospital-acquired infections. Infect Drug Resist,12: 603-611.
7. Herridge W, Shibu P, O'shea J, Brook TC, Hoyles L (2020). Bacteriophages of Klebsiella spp., their diversity and potential therapeutic uses. J Med Microbiol, 69: 176–94.
8. Jean Sh, Chang YCh, Lin WCh, et al (2020). Epidemiology, Treatment, and Prevention of Nosocomial Bacterial Pneumonia. J Clin Med, 9:275.
9. Hu D, Liu B, Dijkshoorn L, Wang L, Reeves PR (2013). Diversity in the Major Polysaccharide Antigen of Acinetobacter baumannii Assessed by DNA Sequencing, and Development of a Molecular Serotyping Scheme. PLoS One, 8(7): e70329.
10. Catalán-Nájera JC, Garza-Ramos U, Camacho HB (2017). Hypervirulence and hypermucoviscosity: Two different but complementary Klebsiella spp. phenotypes. Virulence, 8(7): 1111–23.
11. Follador R, Heinz E, Wyres KL (2016). The diversity of Klebsiella pneumoniae surface polysaccharides. Microb Genom, 2(8): e000073.
12. Doorduijn J D,Rooijakkers SHM, Schaik WV (2016). Complement Resistance Mechanisms of Klebsiella pneumoniae. Immunobiology, 221(10): 1102-9.
13. Gerlach G, Clegg S, and Allen BL (1989). Identification and Characterization of the Genes Encoding the Type 3 and Type 1 Fimbrial Adhesins of Klebsiella pneumoniae. J Bacteriol, 171(3): 1262–1270.
14. Yeh YCh, Huang TH, Yang Sh, ChenCh, Fang JY (2020). Nano-Based Drug Delivery or Targeting to Eradicate Bacteria for Infection Mitigation: A Review of Recent Advances. Front Chem, 8:286.
15. Murray PR, Rosenthal KS, Pfaller MA (2016). Medical Microbiology.8th ed. Elsevier. Canada.
16. Mirzaie A, Ranjbar R (2021). Antibiotic resistance, virulence-associated genes analysis and molecular typing of Klebsiella pneumoniae strains recovered from clinical samples. AMB Express, 11:122.
17. Zomorodbakhsh Sh, Abbasian Y, Naghinejad M, Sheikhpour M (2020). The Effects Study of Isoniazid Conjugated Multi-Wall Carbon Nanotubes Nanofluid on Mycobacterium tuberculosis. Int J Nanomedicine, 15: 5901–9.
18. O’Neill J (2016). Tackling drug-resistant infections globally: final report and recommendations. Review on antimicrobial Resistance. https://amr-review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf
19. Tacconelli E CE, Savoldi A (2017). Global priority list (ppl) of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. https://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf
20. Rai M, Ingle AP,Pandit R, et al (2017). Broadening the spectrum of small-molecule antibacterials by metallic nanoparticles to overcome microbial resistance. Int J Pharm, 532(1): 139-148.
21. Sheikhpour M, Arabi M, Kasaeian A, et al (2020). Role of Nanofluids in Drug Delivery and Biomedical Technology: Methods and Applications. Nanotechnol Sci Appl, 13: 47–59.
22. Sheikhpour M, Barani L, Kasaeian A (2017). Biomimetics in drug delivery systems: A critical review. J Control Release, 253: 97-109.
23. Sheikhpour M, Golbabaeie A, Kasaeian A (2017). Carbon Nanotubes: A review of novel strategies for cancer treatment and diagnosis. Mater Sci Eng C Mater Biol Appl, 76:1289-1304.
24. Zardini HZ, Davarpanah M, Shanbedi M, et al (2014). Microbial Toxicity of Ethanolamines-Multi Walled Carbon nanotubes. J Biomed Mater Res A, 102(6): 1774-1781.
25. Yazdani MR, Sheikhpour M, Siadat SD, Safarian P (2021). Overcoming the antibiotic resistance of Acinetobacter baumannii by using nanofluid containing functionalized carbon nanotubes. Nanomed Res J, 6(2): 179-187.
26. Brown C, Seidler RJ (1973). Potential Pathogens in the Environment: Klebsiella pneumoniae, a Taxonomic and Ecological Enigma. Appl Microbiol, 25(6): 900-904.
27. Holmes B, Willcox WR, Lapage SP (1978). Identification of Enterobacteriaceae by the API 20E system. J Clin Pathol, 31: 22-30.
28. Weinstein MP, Lewis JS, Bobenchick AM (2020). Clinical Labratory Standard Institute, performance standards for Antimicrobial Susceptibility Testing, M100 30th ed. pp. 31-37. https://www.nih.org.pk/wp-content/uploads/2021/02/CLSI-2020.pdf
29. Namivandi Zangeneh R, Sadrearhami Z, Dutta D, et al (2019). Synergy between Synthetic Antimicrobial Polymer and Antibiotics: A Promising Platform to Combat Multidrug-resistant Bacteria. ACS Infect Dis, 5(8): 1357–1365.
30. Kon K, Rai M (2016). Antibiotic Resistance Mechanisms and New Antimicrobial Approaches. 1st ed. Elsevier Inc,UK. pp.130-134.
IssueVol 50 No 11 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijph.v50i11.7585
Carbon nanotube Functionalized MWCNT Klebsiella pneumoniae Virulence genes

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Mehdizadeh M, Sheikhpour M, Salahshourifar I, Siadat S, Saffarian P. An In-Vitro Study of Molecular Effects of a Combination Treatment with Antibiotics and Nanofluid Containing Carbon Nano-tubes on Klebsiella pneumoniae. Iran J Public Health. 2021;50(11):2292-2301.