Characterization of ZnO NPs
The XRD peaks were consistent as ZnO crystallite. The analysis showed no extra peaks, which is due to the purity of the material applied during the synthesis of ZnO NPs. The positions of the diffraction peaks showed the same pattern found in the Joint Committee on Powder Diffraction Standards: 36-1451 database (JCPDS).
Figure 1 shows the diffraction peaks of ZnO NPs at (100), (002), (101), (102), (110), (103) correspond respectively to the values in degrees (2θ) at 31.34°, 34.50°, 36.32°, 47.60°, 56.68°, 62.94°. High diffraction peaks indicated the crystalline nature of the material [21].
Table 1 shows the values of the structural parameters used to calculate the size of the ZnO crystallite by Equation (1) [22]. The high intensity peaks at (100), (002) and (101) were used to determine the lattice parameters.
\(D=\left[\frac{0.9\lambda }{\beta cos Ɵ}\right] x 100\) Equation (1)
Where D is the size of the ZnO crystallite; λ is the wavelength of Cu Kα radiation at 1.5418 Å; θ is the Bragg diffraction angle, and β is the full width at half maximum intensity of the diffraction peak of the sample.
Table 1
Structural parameters of ZnO crystallite.
Lattice parameters a (Å) | Lattice parameters c (Å) | c/a ratio | Volume of unit cell (Å3) | Average crystallite size (nm) | Microstrain ε (×103) |
3.24 | 5.21 | 1.608 | 47.48 | 82.38 | 0.47 |
The crystallite size can be measured more accurately by high resolution X-ray diffraction (HRXRD) using the Bond method, which increases peak resolution to find the values of the Lattice parameters [22-23]. In this study, we determined by XRD powder that most synthesized ZnO crystallites are around 80 nm in size. Similar results obtained through the synthesis of ZnO NPs by sonochemical-coprecipitation were shown by Khataee et al. [24].
The surface appearance and morphology of synthesized ZnO NPs were analyzed by SEM at 29.0 kx. Based on the images in Figure 2, the ZnO NP showed complex bead and rod morphology. In addition, the ZnO NPs have an irregular size with formation of aggregated nanocrystallite.
FT-IR spectra are like molecular fingerprints that provide a valuable insight into chemical structures and their changes due to interactions with other molecules [19]. FT-IR analyses detected the characteristic functional group associated with the ZnO NPs, shown in Figure 3. The peak at 575 cm−1 corresponds to bond metal-oxygen in Zn–O stretching vibrations. The peak at 3713 cm−1 was carbon residues identified during sample measurement; and 1210 cm−1 belongs to elongation of C–O. Hydrogen bond is displayed at 1690 and 2346 cm−1 is ascribed to the stretching vibration of hydroxyl compounds. The hydroxyl group influences photocatalytic reactions in ZnO by generating superoxide radicals, which act as an antimicrobial [25].
Antimicrobial activity
The bactericidal activity of ZnO NPs against E. coli, P. aeruginosa, S. aureus and B. subtilis was evaluated by monitoring the cell respiration. Polynomial regression applied to the dose-response data was used to extrapolate the IC100 values, which were expressed as mM. The decrease in cell numbers observed after treatment is shown in Figure 4 by the plot containing the concentration of ZnO NPs versus the inhibition of cell growth.
ZnO NPs showed growth inhibition against E. coli and P. aeruginosa in IC100 values at 0.6 mM for both strains. The IC100 values for B. subtilis and S. aureus were reached at concentrations of 0.8 and 1.0 mM, respectively.
Gram-negative bacteria have a thin layer of peptidoglycan between two membrane lipopolysaccharides and proteins, which provide cellular resistance [26]. In addition, dissociated carboxyl groups present in the cell membrane generate negative charges on the surface. ZnO NPs, on the other hand, have a positive charge, with a zeta potential of +24 mV [27]. As a result of electrostatic forces, damage to the cell membrane occurs due to electrostatic gradient differences across the negative membrane and the positive charges of the Zn2+ ions. Therefore, E. coli and P. aeruginosa cells achieved cell death at the lowest concentration of ZnO NPs. Although the present study did not observe a large difference in the IC values for Gram-negative and Gram-positive, it is noteworthy that Gram-positive exhibited IC100 values higher than Gram-negative. Similar inhibition in Gram-negative bacteria was previously reported by Yusof et al. [28], Saqib et al. [29] and Zubair and Akhtar [30]; however, with slight variations in the IC100 values due to differences in the synthesis of the nanomaterial, which yields unique characteristics to each one of them. Overall the results were close; however, our study not only investigated the percentage of inhibitory growth, but also the MOA target of ZnO NPs in clinical strains.
Effect of ZnO NPs on the bacteria cell
Bacterial cell division is a complex and dynamic process, which starts by the polymerization of the FtsZ protein in order to assemble the divisome, which will guide all the processes related to cell division and cell wall synthesis and remodelling [31]. FtsZ is the ancestral tubulin conserved in bacteria, which exerts its function dependent on the nucleotide guanosine triphosphate (GTP) [32-33]. Some bactericidal compounds act by preventing the GTPase activity of FtsZ, which will inhibit cell division and lead to cell death [16]. In addition, blockage of the cell division process generally leads to cell filamentation, which can be easily accessed by fluorescence microscopy. Alternatively, by using mutant cells expressing labelled division proteins, e.g. FtsZ-GFP, one can follow the dynamics of division and study the effects compounds might have on the process.
B. subtilis expressing FtsZ-GFP was exposed to ZnO NPs at its IC100 for 15 minutes, and after observed under the microscope (Figure 5). Note that even after exposure cells still have intact bars perpendicular to the long axis of the rods, which is the normal profile for the Z-ring. This cytological profile was comparable to the control and did not show any disruption of the divisional ring.
The integrity of the membranes of E. coli, P. aeruginosa, S. aureus, and B. subtilis cells was also investigated upon compound exposure using fluorescence microscopy. The results showed the disruption of cytoplasmic membranes in all strains after 15 min of exposure at IC100 (Figure 6). The filters Tx Red and DAPI Blue were applied together and used to visualize PI and DAPI. Cells with intact membranes are artificially stained in blue, while cells with damaged membranes are stained red [34]. Thus, an increase in red-stained cells by PI is related to the increase in cell permeability due to damaged membranes.
In this study, all species of bacteria had their cytoplasmic membrane affected within the first 15 minutes of exposure to ZnO NPs. Over 70% cells died with detectable membrane damage. This result shows the ZnO NPs efficacy towards bacterial surface acting at the very initial contact and targeting it’s as the first structure nanoparticles comes into contact with. These results were expected as ZnO NPs bactericidal activity was already known, and its predominant MOA is associated with the membrane cell [13]. Because ZnO is a transition metal oxide and semiconductor (which belongs to class II-VI) with wide band gap (3.3 eV), there is a general pattern expected. When the radiation has energy larger than the band gap of the ZnO, electron-hole pairs are formed. Electrons are promoted to the conduction band (CB). The hole generated in the valence band (VB) gets a strongly oxidizing character and oxidizing sites are created, which are capable of oxidizing water molecules or hydroxide anions and generate strong oxidizing species [12]. This reaction leads to the redox chain reaction with the generation of reactive oxygen species (ROS) formed by hydroxyl radical (•OH), hydroperoxyde radical (•HO2−) and superoxide radical anion (O2•−) as the pathways of bactericidal action [35].
Oxidative stress in the bacterial cell can be induced by ROS generation produced from ZnO NPs, which leads to the inhibition of protein synthesis and DNA replication [13]. In this situation, ZnO conductivity increases, close to the "band gap" of the UV-spectrum characterized by high emission energy. The electronic excitation can destabilize the charges present in the cytoplasmic membrane resulting in their rupture. ZnO can also damage the cytoplasmic membrane by releasing Zn2+ ions from the dissolution of ZnO in aqueous solution. The Zn2+ ion acts as an inhibitor of the glycolytic enzyme through the thiol group oxidation due to specific affinity for the sulphur group [3].
The MOA reported in this study are represented in schematic drawing shown in Figure 7.
ZnO NPs are attached in the surfaces of Gram-positive and Gram-negative bacteria through different patways. Teichoic acid in the peptidoglycan layer and lipoteichoic acid in the membrane are the source of negative charges from cell walls. Positive charges from ZnO NPs are attracted to the cell surface by electrostatic interactions, and the difference in electrostatic gradient, which leads to damage in the cell surface [36-37].
Teicoic and lipoteichoic acids act as a chelating agent with Zn2+ ions, which is carried by passive diffusion across membrane proteins (Figure 8). Moreover, the bactericidal action can occur by different mechanisms, such as adsorption in the bacterial surface, formation of different intermediates and electrostatic interactions.
The electrochemical gradient is generated by the movement of hydrogen ions across the cell membrane, which facilitates the diffusion of metallic ions [35]. This mechanism is associated with the size of the material, whose small particles would have better electrostatic interactions. Thus, the ZnO target for inhibitory action is dependent on different factors such as concentration, size and time of interaction.
Zanet et al. [6] showed that ZnO NPs affect the cell morphology and DNA. However, this can be a side effect, since the main target of ZnO NPs ends up being the first structure they have contact with and consequently act, such as the cytoplasmic membrane. Siddiqi et al. [12] through SEM and TEM analysis concluded that ZnO NPs damage the cell membrane, and right after go to the cytoplasm, where they interact with other cell structures. Our results also showed damage to the cell. Therefore, it can be concluded that ZnO NPs are multi-target compounds and affect several structures of bacteria cells, but their main mechanism of action is in the cytoplasmic membrane, being other structure effects a consequence/secondary effect after the membrane rupture.