Bacterial toxicity of biomimetic green zinc oxide nanoantibiotic: insights into ZnONP uptake and nanocolloid-bacteria interface

Abstract

This study was aimed to fill the critical gap of knowledge regarding the interaction between green zinc oxide nanoparticles (ZnONPs) and bacterial interface. Wurtzite phase ZnONPs with a band gap energy of 3.28 eV were produced by exploiting a simple and green biosynthesis method using an inexpensive precursor of A. indica leaf extract and zinc nitrate. ZnONPs were characterized using UV-Vis spectroscopy, XRD, FTIR, SEM, EDX, DLS, TEM, and zeta-potential analysis. The primary size obtained was 26.3 nm (XRD) and 33.5 ± 6.5 nm (TEM), whereas, the secondary size was found to be 287 ± 5.2 nm with -32.8 ± 1.8 mV ζ-potential denoting the physical colloid chemistry of ZnONPs. Crystallinity and the spherical morphology of ZnONPs were also evident with some sort of particle agglomeration. ZnONPs retained plant functional groups endorsing their hydrophilic character. The antibacterial and antibiofilm activity of ZnONPs was significant (p ≤ 0.05) and the MIC/MBC against most frequent clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus ranged from 0.5 to 1.0 (MIC)/1.0 to 1.5 mg ml-1 (MBC). The dissolution of ZnONPs to Zn2+ ions in a nutrient medium increased as a result of interaction with the bacterial surface and metabolites. Substantial surface binding of ZnONPs followed by intracellular uptake disrupted the cell morphology and caused obvious injury to the cell membrane. Interrupted bacterial growth kinetics, loss of cell respiration, enhanced production of intracellular ROS, and the leakage of the cytoplasmic content unequivocally suggested a strong interaction of ZnONPs with the exterior cell surface and intracellular components, eventually leading to cell death and destruction of biofilms. Overall, the results elucidated eco-friendly production of ZnONPs expressing a prominent interfacial correlation with bacteria and hence, prospecting the use of green ZnONPs as effective nanoantibiotics.

Figures

Fig. 1. Characterization of ZnONPs; UV-visible spectrum (A) of green synthesized ZnO-NPs (0.1% w/v; solid line) and its comparison with commercially available ZnO-NPs (dashed line) which confirms the purity of ZnONPs. SEM analysis of ZnO-NPs at a direct magnification of ×10 000 (B) and ×50 000 (C). EDX spectrum of ZnONPs (D) and the TEM micrograph of at ×60 000 (E) with an average diameter of 33.5 ± 6.5. The inset in panel B depicts the morphology of ZnO-NPs. Panel F shows the X-ray diffractogram of ZnO nanopowder. The average crystal size by XRD was found to be 26.3 nm.

Fig. 2. Antibacterial activities of ZnONPs against the clinical isolates of bacteria. Comparison of the absorption spectra at 620 nm, as an index of the growth kinetics of clinical isolates, at an increasing ZnONP dose (0.125, 0.25, 0.5, 1, and 1.5 mg ml–1) and as a function of incubation time at 37 °C is shown in panels A–D. Panel A: Gram +ve S. aureus; panel B: Gram –ve E. coli; panel C: Gram –ve P. aeruginosa; and panel D: Gram –ve K. pneumoniae.

Fig. 3. Mean absorbance spectra of nucleic acid released from the membrane compromised cells of the clinical strains of E. coli (panel A), K. pneumoniae (panel B), P. aeruginosa (panel C), and S. aureus (panel D) after exposure with various concentrations of ZnONPs (0–1.5 mg ml–1). The inset linear plots show enhancement in the optical density at 260 nm as a function of ZnO-NP concentration. Panel E depicts the extracellular β-glycosidase activity of bacterial cells as a result of inner plasma membrane damage. The absorbance change of o-nitrophenol at 420 nm has been plotted against various ZnONP concentrations (0.125–1.5 mg ml–1) at 0 and 4 h of incubation at 37 °C. Values from three replicates are expressed as mean ± SD (*p ≤ 0.05 vs. control at 4 h).

Fig. 4. ZnONP induced cell death, reactive oxygen species, and inhibition of cellular respiration in treated clinical isolates. The ZnONP (1.0 mg ml–1) mediated increase in the number of dead cells as a result of increased membrane permeability. Panels A and C show the residual fluorescence of PI in K. pneumoniae and S. aureus cells. Panel C and D present an increase in PI fluorescence due to the damaged cell membrane. Panels E and G show the residual fluorescence of DCF in K. pneumoniae and S. aureus cells. Panel F and H show a significant increase in intracellular ROS when exposed to 1 mg ml–1 of ZnONPs. Panel I shows the inhibition of cellular respiration of E. coli, S. aureus, K. pneumoniae, and P. aeruginosa by green synthesized ZnONPs at 0.125–1.5 mg ml–1. The decrease in red color intensity in microtitre wells represents the loss of metabolic activity of bacterial cells.

Fig. 5. SEM images and EDX spectra indicating ZnONP induced morphological damage in bacteria. Representative SEM micrographs depict cellular damage and structural distortion in ZnONP (1 mg ml–1) exposed cells: Panels C: Damage to K. pneumoniae cells while Panel G has S. aureus cells. Panels A and E show the untreated cells of K. pneumoniae and S. aureus cells, respectively. EDX spectra and elemental mapping of treated K. pneumoniae and S. aureus cells in panels D and H show the presence of Zn as peak signals and dots in mapping along with C and O; Panel B and F represent untreated cells. Panel I shows the release of soluble Zn ions from NPs. Panel J represents the concentration of Zn in the cell supernatant and bacterial pellets of E. coli, P. aeruginosa, S. aureus, and K. pneumoniae grown in 1000 μg ml–1 of ZnONPs (for 24 h at 37 °C. The loss of ZnONPs is shown as the undetected percentage of Zn. Values from three replicates are expressed as mean ± SD (*p ≤ 0.05, **p ≤ 0.005 vs. control).

Fig. 6. Inhibition of biofilm formation by ZnONP treated bacterial isolates (Panel I). Micrographs A–E represent the biofilm formed on the glass surface; untreated K. pneumoniae biofilm (A), K. pneumoniae + 0.25 mg ml–1 ZnONPs (B), K. pneumoniae + 0.5 mg ml–1 ZnONPs (C), K. pneumoniae + 1 mg ml–1 ZnONPs (D), and K. pneumoniae + 1.5 mg ml–1 ZnONPs (E). Images F–J represent the biofilm of S. aureus formed on the glass surface; untreated S. aureus biofilm (A), S. aureus + 0.25 mg ml–1 ZnONPs (B), S. aureus + 0.5 mg ml–1 ZnONPs (C), S. aureus + 1 mg ml–1 ZnONPs (D), and S. aureus + 1.5 mg ml–1 ZnONPs (E). CLSM analysis of the bacterial biofilm: images K–O represent the biofilm formed by K. pneumoniae on the glass surface; untreated K. pneumoniae biofilm (A), K. pneumoniae + 0.25 mg ml–1 ZnONPs (B), K. pneumoniae + 0.5 mg ml–1 ZnONPs (C), K. pneumoniae + 1 mg ml–1 ZnONPs (D), and K. pneumoniae + 1.5 mg ml–1 ZnONPs (E). Images P–T represent the biofilm formed by S. aureus on the glass surface; untreated S. aureus biofilm (A), S. aureus + 0.25 mg ml–1 ZnONPs (B), S. aureus + 0.5 mg ml–1 ZnONPs (C), S. aureus + 1 mg ml–1 ZnONPs (D), and S. aureus + 1.5 mg ml–1 ZnONPs (E). Bar diagrams in panel II show the percent inhibition in the biofilm formation of K. pneumoniae (U) and S. aureus (V) under ZnONP stress (0.25–1.5 mg ml–1). The bars represent the mean values of three independent replicates ± SD (*p ≤ 0.05, **p ≤ 0.005 vs. control).

Fig. 7. Scanning electron microimage of the bacterial biofilm. Images A–D represent the biofilm formed on a solid surface; untreated K. pneumoniae biofilm (A), K. pneumoniae + 0.25 mg ml–1 ZnONPs (B), K. pneumoniae + 0.5 mg ml–1 ZnONPs (C), and K. pneumoniae + 1 mg ml–1 ZnONPs (D). A magnified view at ×15 000 is shown (Panel A and D insets). Micrographs E–H show the biofilm of untreated S. aureus (E), S. aureus + 0.25 mg ml–1 ZnONPs (F), S. aureus + 0.5 mg ml–1 ZnONPs (G), and S. aureus + 1 mg ml–1 ZnONPs (H). A magnified view at ×15 000 is shown in Panel E and H insets. Red arrows indicate the distortion of the normal bacterial cell.

Fig. 8. A proposed mechanistic illustration of the various steps of ZnONP action on the bacterial interface and initiation of the signaling cascade of bacterial cell death.