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

Abstract

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.

Keywords: Carbon nanotube; Klebsiella pneumoniae; Virulence genes.

Fig. 1:

MBC results of the effect of ciprofloxacin and f-MWCNTs on K. pneumoniae. A) The effect of 8 μg/ml ciprofloxacin on the ES133 resistant strain did not inhibit the growth, but 16 μg/ml ciprofloxacin inhibited that. B) The effect of 8 μg/ml ciprofloxacin+ 400 μg/ml f- MWCNT completely killed the bacteria. As a conclusion, the effective dose of the antibiotic was reduced when combined with 400 μg/ml f-MWCNT

Fig. 2:

Transmission Electron Microscopy (TEM) image of K pneumoniae. A) Untreated resistant strain (control) B) The same strain treated with 8 μl/ml ciprofloxacin with 100 μg/ml f-MWCNTs. Arrows indicate the f-MWCNTs particles that have entered the bacteria, as well as the location of the wall destruction. The accumulation of f-MWCNTs on the bacterial wall, indicated by a yellow arrow

Fig. 3:

fimA, fimD, wza and wzi genes expression in K. pneumoniae resistant strain to antibiotic and f-MWCNT. From left to right respectively: the untreated K. pneumoniae (negative control), treated with 8 μg/ml antibiotic, 400 μg/ml f-MWCNT, 400 μg/ml f-MWCNT + 8 μg/ml antibiotics. After treatment with 400 μg/ml f-MWCNTs + 8 μg/ml ciprofloxacin in the resistant strain, fimA, fimD, wza and wzi genes expression significantly upregulated in comparison with the control, (P value <0.0001, <0.0001, <0.003, <0.0001 respectively) and downregulated compared to the antibiotic alone. Abbreviations: B; Bacteria, K: K. pneumoniae; Ab: Antibiotic; NF: Nanofluid. Expression levels were normalized against 16s rRNA as the reference housekeeping gene

Fig. 4:

fimA, fimD, wza and wzi genes expression in K. pneumoniae ATCC strain to antibiotic and f-MWCNT. From left to right respectively: untreated K. pneumoniae (negative control), treated with 2 μg/ml ciprofloxacin, 100 μg/ml f-MWCNT, 2 μg/ml ciprofloxacin + 100 μg/ml f-MWCNT. In the ATCC strain treated with 2 μg/ml ciprofloxacin + 100 μg/ml f-MWCNT, the expression of fimA, fimD and wza genes showed an upregulation compared to the control and showed a downregulation in comparison with ciprofloxacin treatment (P value <0.005, 0.005, 0.06), while wzi gene expression was downregulation compared to both the control and the ciprofloxacin treatment, although the rate of reduction was lower than the significant level (P value <0.37). Abbreviations: B: Bacteria; Ab: Antibiotic; NF: Nanofluid. Expression levels were normalized against 16s rRNA as the reference housekeeping gene