Comparative in vitro study of the antimicrobial activity of metal-ZnO nanoparticles against several bacterial and fungal pathogens

Nowadays, the use of nanoparticles (NPs) has become useful in the different application fields. The aim of this study was to investigate the in vitro antimicrobial potential of metalZnO nanoparticles (ZnO NPs) against several bacterial and fungal strains including; Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 13753), Staphylococcus aureus (ATCC 8095), Pseudomonas aeruginosa (ATCC10662), Candida albicans (ATCC10231) and Aspergillus niger (AUMC3663). Results obtained by X-ray diffraction analysis (XRD) showed that the NPs size was in the range of 35.143.7 nm. Images of the scanning electron microscopy (SEM) demonstrated the rod shape nature of the ZnO NPs, and the semi-spherical shapes of the Zn9.7TM0.3O NPs. The effect of different concentrations of ZnO NPs on the in vitro growth of the bacterial and fungal strains was evaluated using the agar well diffusion assay. Current results showed that Cd-ZnO NP recorded the highest antimicrobial potency; expressing inhibition zones diameter range of 1245 mm, while ZnO NPs demonstrated the least activity exhibiting inhibition zones diameter that ranged from 036 mm. Among all the examined ZnO-NPs, treatment of E. coli and Staph. aureus cells with Cd-ZnO proved to be the most effective in causing membrane leakage of reducing sugars, protein and DNA recording; 0.41 μg/ ml and 0.38 μg/ ml; 14.91 μg/ ml and 15.98 μg/ ml; 0.81 μg/ ml and 0.96 μg/ ml, respectively. This study emphasized that ZnO NPs could be used as alternative antimicrobial agents to control the bacterial and fungal pathogens. Manipulation of ZnO NPs is ecofriendly; as it reduces the use of the synthetic pesticides and chemical therapeutic agents, which pollute the environment. In the future, in vivo application of these NPs necessitates the proof that they have no phytotoxicity and\or cytotoxicity.


Introduction
Metal oxide nanoparticles (NPs) are widely applied in the different fields of research and development (Soosen et al., 2009;Kolodziejczak-Radzimska et al., 2014). Their properties are determined by the size; shape, composition and crystallinity. NPs are unique as they can be produced with high surface area to volume ratio. The ZnO NPs as antimicrobial agents have excellent stability and\or long shelf life as revealed by Kolodziejczak-Radzimska et al., (2014). In addition, they are stable under diverse environmental conditions and fabricate at low temperature (Dhage et al., 2005). Metal oxide NPs has distinguished antimicrobial activities, which have opened new frontiers to the biological sciences (Allahverdiyev et al., 2011). They have strong toxicity towards a wide range of micro-organisms including bacteria (Huang et al., 2008) and fungi (He et al., 2011). A previous study conducted by Raghunath and Perumal, (2017) reported that ZnO-NPs possess antimicrobial efficacy against Gram-positive (Guo et al., 2015, Gudkov et al., 2021 and Gram-negative (Liu et al., 2009;Reddy et al., 2014, Guo et al., 2015Tiwari et al., 2018;Gudkov et al., 2021) bacteria, and against bacterial spores (Makhluf et al., 2005;Wagner et al., 2016). The antibacterial potency of NPs against the pathogenic bacteria such as E. coli and Staph. aureus are well known (Brayner et al., 2006).
Growth of the fungal pathogens in plants is the main cause of considerable economic losses during postharvest handling of grains and fruits. The control of fungal growth is difficult by using the organic substrate, because fungi have developed resistance to many chemical fungicides (Elad et al., 1992).
Currently, the structure and composition of synthesized Zn 1-x TM x O (x= 0.03 %), where; TM= Fe, Cd and Mg) have been examined and evaluated. Treatment of microorganisms with ZnO NPs causes membrane leakage of reducing sugars, DNA, proteins and reduces microbial cell viability (Tiwari et al., 2018;Yusof et al., 2020). The objectives of this study were to synthesize ZnO NPs, check their potent antimicrobial potential against pathogenic bacteria and fungi, and detect their effects on bacterial cellular membrane leakage of reducing sugars, DNA and proteins. The outcomes of our study will help to solve the problem of the microbial chemical resistance, as these metal ZnO NPs will be used to control the pathogenic microorganisms.

Synthesis and preparation of metal-ZnO NPs
All preparation steps of the metal ZnO NPs (Fe, Cd and Mg) and pure ZnO including weighing; grinding, mixing and storage were carried out in an air-filled glove box; where the O 2 and H 2 O levels were less than 0.1 µg/ l. Preparation of Zn 1-x TM x O (x= 0.03 %) NPs, where TM= Fe, Cd and Mg was carried out using the co-precipitation technique of Abdel-Baset and Abdel-Hafiez, (2021). During this assay, ZnSO 4 and NaOH solutions were prepared individually and then mixed together (according to their molecular weights). The resulting Zn(OH) 2 solution was stirred for 2 h at room temperature; followed by heating at 70 °C for 24 h for drying. The dried ingot was heated again for 4 h at 400 °C, and then the resulting powder was left to cool to room temperature to get the pure ZnO NPs. The prepared samples were the oxide dilute magnetic semiconductors; where 25 ml from mixed solutions of ZnSO 4 and 3 % of TMs SO 4 at the desired ratio were prepared, and then 25 ml of NaOH solution was added slowly. The above steps were repeated to obtain Zn9.7TM0.3O NPs.

X-ray diffraction (XRD) analysis
The size of the obtained NPs was detected using the X-ray diffraction (XRD). Analysis of the samples was performed using a Rigaku miniflex diffractometer Novel Research in Microbiology Journal, 2021 with CuK α radiation (λ = 1.5406 Å), according to Abdel-Baset, (2020). The lattice parameters a and c were calculated using the following relation, according to Karyaoui et al., (2015):

[ ]
Where h, k and l are the Miller Indices, d is inter-planar distance and 'a' and 'c' are lattice parameters, and listed in Table (1). On the other hand, the average crystalline size (D) was calculated using Debye-Scherrer's equation (D= kλ⁄ (β cosθ ) (Abdel-Baset, 2021), where k= 0.94 (a constant), λ is wavelength of X-rays, β is full width at half maximum (FWHM) and θ is the diffraction angle.

Scanning electron microscopy (SEM)
High-resolution scanning electron microscopy (Carl Zeiss sigma 500 VP) was used to determine the NPs size and morphology, in reference to Abdel-Baset, (2021).

Agar well diffusion assay
Agar well diffusion assay was used as described by Wolf and Gibbones, (1996). Briefly, 20 ml of Luria-Bertani agar medium (Atlas and Parks, 1997) was seeded individually with 1% of tested bacterial and fungal suspensions (10 6 cells/ ml) after being cooled to 47˚C, poured into Petri plates and then allowed to solidify. Wells (6 mm diameter) were cut in the solidified agar using a sterile cork borer, and then filled individually with 50 µl of each NPs concentration. Plates were left at 4-5˚C for 2 h to allow diffusion of the NPs suspension and then incubated at 37 °C for 24 h for the bacterial strains, and for 5 d for the fungal strains. After incubation, absence or presence of inhibition zones was recorded, and then the inhibition zone diameters were measured using a calibrated ruler, compared to the control treatment (DMSO). Three replicate plates were used for each treatment.

Quantitative evaluation of bacterial cellular membrane leakage
The effect of ZnO NPs on causing membrane leakage of reducing sugars, proteins and DNA from the intracellular cytosol of the selected bacterial cells was tested. Flasks containing 100 ml LB broth were inoculated individually with bacterial suspensions (10 6 / ml) of E. coli (ATCC 25922) and Staph. aureus (ATCC 8095), and with 500 µl of ZnO-NPs (2 mM final concentration). The plates were then incubated at 37 ᴼC on an orbital shaker at 125 rpm for 24 h. After incubation, the cultures were centrifuged at 10,000 g for 30 min. at 4 ᴼC. The resulting supernatants were used for determination of the release of bacterial reducing sugars, proteins and DNA. Three replicates were used for each treatment. The reducing sugars were estimated calorimetrically using Di-nitrosalicylic acid assay in reference to Miller, (1959). A blank treatment consisted of dist. water instead of the bacterial filtrate. The absorbance of each sample was measured individually at 540 nm; using a Jasco V-530 UV/ VIS spectrophotometer (JASCO International CO., LTD., Japan). The total protein was measured using the Biorad Protein Kit II (Biorad) spectrometric assay, according to Bradford, (1976). Following the manufacturer's instruction, the reaction color was stable for one hour. The absorbance was recorded at 595 nm. The linearity was up to 10 grams per decilitre (g/ dl): Finally, the released DNA (μg/ μl) in each sample was estimated using NanoPhotometer Pearl® (IMPLEN, München, Germany) at 260 nm, according to Li et al., (2010); Thombre et al., (2016).

Statistical analysis
All data were expressed as mean values and analysed using general linear model of SPSS. (2007), version 16.0 SPSS company Inc., Chicago, 11, USA, p.444. Mean of the values was compared to the main effects by Duncan's multiple range tests (Duncan, 1955); when significant F values were obtained p≤ 0.05.

X-ray diffraction assay
The X-ray diffraction (XRD) pattern of the Zn9.7TM0.3O nanostructure compared to the pure ZnO nanostructure is demonstrated in Fig. (1). Diffraction peaks corresponding to (002) and (100) planes of ZnO as a hexagonal phase were also observed in all samples, but with a different intensity ratio. In addition, no characteristic peaks were observed other than the ZnO peaks, which indicate that the synthesized samples were well crystallized, as shown in Fig. (1). In this study, the recorded crystalline size (D), lattice parameters (a and c) are demonstrated in Table (1).  The average crystal size decreased with doping, which might be due to the decrease of nucleation of ZnO NPs by doping. Moreover, the slight variation in the lattice parameters probably resulted from substitution of the doped ions with the different ionic radii of Zn. Pure ZnO and Zn9.7TM0.3O NPs were successfully synthesized by the co-precipitation method. From XRD data, the hexagonal wurtzite planes displayed a smaller shift on addition of the metal ions. This effect might be attributed to the different ionic radii of the dopant ions (TMs ions) compared to the ionic radius of Zn. However, on addition of the metal ions the NP size decreased; as revealed by results of the XRD analysis, which are listed in Table (1). This is attributed to the decrease in nucleation; due to the difference between radii of the dopant ions and the Zn ions.

SEM characterization
High resolution scanning electron microscope (HR-SEM) images of the prepared ZnO NPs were used to check their shapes. As demonstrated in Fig. (2a), the SEM image showed the rod shape nature of the ZnO NPs. In addition, the morphology of Zn9.7TM0.3O NPS was studied from the SEM images ( Fig. 2: b-d), which showed the semi-spherical shapes of these Zn9.7TM0.3O NPs. The SEM image represents the agglomeration of NPs with narrow particle size distribution.

The antibacterial and antifungal efficacy of metal ZnO NPs
Results presented in Table (2) indicated that all the metal ZnO NPs had antimicrobial potential against the tested bacterial and fungal strains manifested through the formation of growth inhibition zones, compared to the control treated with DIMSO only. However, no inhibition zone was recorded for C. albicans treated with different concentrations of pure ZnO and Mg-ZnO NPs, compared to the control. Interestingly, the highest antimicrobial potency (inhibition zone diameter of 45 mm) was recorded by Cd-ZnO NP, while ZnO NP presented the least activity (12 mm). In addition, B. cereus and E. coli strains had moderate sensitivity to the NPs, compared to the other tested bacterial strains. In addition, results revealed that the most effective concentration of NPs was (20 µg/ ml) observed by Cd-ZnO against B. cereus, compared to Mg-ZnO and ZnO NPs, as clear in Fig. (3).  The current results of this in vitro assay showed that Mg-ZnO, Cd-ZnO, Fe-ZnO NPs and pure ZnO had noticeable antibacterial potency against P. aeruginosa, exhibiting inhibition zone diameters of; 20, 45, 32 and 30, respectively. All results of the metal ZnO NPs treatments were compared to the control treatment.
From all the above mentioned data, the tested metal ZnO NPs had more significant antibacterial efficacy compared to the antifungal one; especially with the tested strain of C. albicans. Table (3) and Fig. (4) demonstrate the effect of metal ZnO-NPs on membrane leakage of reducing sugars, protein and DNA from cells of E. coli and Staph. aureus. Treatment with Cd-ZnO proved to be the most effective among all the examined ZnO-NPs on causing membrane leakage of reducing sugars, protein, and DNA from E. coli and Staph. aureus. The recorded membrane leakage of reducing sugars was; 0.27 μg/ ml, 0.41 μg/ ml, 0.19 μg/ ml and 0.22 μg/ ml on treatment of E. coli with Fe-ZnO, Cd-ZnO, Mg-ZnO NPs and pure ZnO, respectively. However, leakage of reducing sugars was; 0.31 μg/ ml, 0.38 μg/ ml, 0.19 μg/ ml and 0.28 μg/ ml on treatment of Staph. aureus with (Fe-ZnO, Cd-ZnO, Mg-ZnO NPs and pure ZnO, respectively (Fig. 4-A).

Results presented in
Similarly, the protein membrane leakage was 13.98 μg/ ml, 14.91 μg/ ml, 12.87 μg/ ml and 13.92 μg/ ml on treatment of E. coli with Fe-ZnO, Cd-ZnO, Mg-ZnO NPs and pure ZnO, respectively. Treatment of Staph. aureus cells with Fe-ZnO, Cd-ZnO, Mg-ZnO NPs and pure ZnO; scored protein leakage of 14.84 μg/ ml, 15.98 μg/ ml, 12.65 μg/ ml and 13.78 μg/ ml, respectively (Fig. 4-B). The DNA leakage after membrane disruption of E. coli Novel Research in Microbiology Journal, 2021 cells was 0.76 μg/ ml, 0.81 μg/ ml, 0.43 μg/ ml and 0.57 μg/ ml on treatment with Fe-ZnO, Cd-ZnO, Mg-ZnO NPs and pure ZnO, respectively. On the other hand; Staph. aureus recorded cellular DNA leakage of 0.90 μg/ ml, 0.96 μg/ ml, 0.60 μg/ ml and 0.66 μg/ ml on treatment with Fe-ZnO, Cd-ZnO, Mg-ZnO NPs and pure ZnO, respectively (Fig. 4-C). The control represented the non-treated bacterial cultures. Table (3) showed that treatment of E. coli and Staph. aureus with NPs caused cellular bacterial permeability leakage of reducing sugars, proteins and DNA. Accordingly, the use of NPs will be an important factor in preventing growth of the different bacterial pathogens, and displace the use of the deleterious synthetic bactericides that pollute the environment.

Discussion
The current results indicated that the chemically synthesized ZnO NPs were well crystallized. The average crystal size of the NPs detected using the XRD analysis decreased with doping, due to the decrease of nucleation of ZnO NPs by doping, in accordance with Abdel-Baset and Abdel-Hafiez, (2021). The slight variation in the lattice parameters resulted from substitution of the doped ions with different ionic radii of Zn, which led to improving their properties without changing the host (ZnO) crystal structure over a wide dopant concentration, similar to results of the recent study conducted by Abdel-Baset and Abdel-Hafiez, (2021).
The XRD pattern suggested that pure ZnO exhibits a hexagonal wurtzite structure belonging to the C46v space group (P63mc). ZnO is indexed and uses a standard JCPDS file for NPs (JCPDS 36-1451) with a preferred (101) orientation (Abdel-Baset and Belhaj, 2021). In agreement with Abdel-Baset and Abdel-Hafiez, (2021), the hexagonal wurtzite planes displayed a small shift with the addition of metal ions, which might be attributed to the different ionic radii of the dopant ions with the ionic radius of Zn. On the other hand, on addition of the metal ions the particle size decreased, because of the decrease in the nucleation and subsequent growth rate related to the difference between the dopant ions and the Zn ions.
The diffraction peaks corresponding to (002) and (100) planes of ZnO as a hexagonal phase were also observed in all samples, but with a different intensity ratio. In addition, no characteristic peaks were observed other than the ZnO peaks, which indicate that the synthesized samples were well crystallized. On the other hand, the peaks were slightly shifted toward higher angle with doping, which might be attributed to the different ionic radii of the dopant ions with the ionic radius of Zn as proposed by Irshad et al., (2018). The SEM images showed the rod shape nature of pure ZnO; the presence of Zn9.7TM0.3O NPs samples in a semi-spherical shape, which represents the agglomeration of particles with narrow particle size distribution. The current results indicated that all metal ZnO NPs had antimicrobial activity against all the tested bacterial and fungal strains, compared to the control. Nanotechnology is increasingly used to control pathogens as an alternative to antibiotics (Wang et al., 2017). These NPs have significantly high antibacterial potential, as reported by Gudkov et al., (2021). In addition, the present results confirmed that bacterial treatment with Cd-ZnO was the most effective among all the examined ZnO-NPs; causing membrane leakage of reducing sugars, protein and DNA from cells of E. coli and Staph. aureus. The NPs are less prone to promoting resistance in microbial pathogens than the antibiotics (Wang et al., 2017). The ZnO-NPs could be developed as alternative therapeutics against the pathogenic microorganisms. Direct interaction of the ZnO NPs with the microbial cell cause damages of the cell membrane integrity, which leads to cell rupture and leakage of intracellular contents (Yusof et al., 2020). One mechanism of the antimicrobial action of ZnO NPs is the intracellular responses such as the cell membrane damage (Su-Eon Jin and Hyo-Eon Jin, 2021). The use of ZnO NPs as nano-microbicides is ecofriendly and will increase both the crop production and the national income. Moreover, NPs act as powerful and new drug substitutes against the multi-drug resistant bacteria (i.e. E. coli and Staph. aureus) and the pathogenic fungi (C. albicans), in agreement with results of the previous study of Abd Elkodous et al., (2019). However, the phytotoxicity and cytotoxicity of these NPs should be checked; respectively, before in vivo wide scale application in the field as nano-microbicides, and\or their use in treatments of multidrug resistant pathogenic microorganisms.

Conclusion
The use of ZnO NPs could be alternative therapeutics against the bacterial and fungal pathogens; as they decrease plant and animals infestation and increase crop production. Manipulation of metal ZnO NPs on a wide scale in the field preserve the environment, and provide safe food in larger quantities and at lower costs. However, the phytotoxicity and cytotoxicity of these metal ZnO NPs should be tested before their in vivo use on a wide scale in the fields.