Is zinc oxide antibacterial

Is zinc oxide antibacterial DEFAULT

Mechanism of Anti-bacterial Activity of Zinc Oxide Nanoparticle Against Carbapenem-Resistant Acinetobacter baumannii

Introduction

Acinetobacter baumannii is a Gram-negative, strictly aerobic, catalase-positive, non-motile, non-fermenting, non-fastidious coccobacilli (Peleg et al., ), and mainly found in the hospital setups (Fournier and Richet, ) but rarely on human skin (Seifert et al., ; Berlau et al., ) and fingertips (Glew et al., ). This pathogen targets hospitalized patients who are critically ill and cracks in the skin and respiratory tract (Peleg et al., ). It can grow across a varying range of temperatures, pHs, and nutrient levels, making pathogen highly adapted to survival in both human or environmental vectors (Choi et al., ). Carbapenems (beta-lactams) are prescribed by doctors against it. Different mechanisms of resistance against carbapenem have been explained for A. baumannii, such as alteration of outer membrane proteins (Vashist et al., ), altered penicillin-binding proteins (Vashist et al., ), acquire carbapenemases (Tiwari et al., a,b; Tiwari and Moganty, , ), efflux pumps (Verma et al., ), enhanced metabolism (Tiwari et al., c; Tiwari, ), and biofilm formation (Tiwari et al., a, a; Roy et al., ). Therefore, there is an urgent need to design or develop an alternative drug to beta-lactams (carbapenem) that may be used to control A. baumannii. Different approaches have been investigated that includes screening of herbal compounds (Tiwari et al., b, ), in silico drug designing (Tiwari et al., a,b; Verma et al., ), nanomaterial-based approaches (Tiwari et al., a, b), etc. to find suitable alternative to the carbapenem.

The natural and synthetic polymers, metals, and metallic alloys offer several explicit properties that make them smart for biomedical applications (Fuku et al., ; Jesudoss et al., ; Kaviyarasu et al., a,b; Maria Magdalane et al., ; Matinise et al., ). Among the nanoparticles, the metal oxide such as zinc oxide (ZnO) has got much attention in the recently because it is stable under diverse environmental conditions, and fabrication at low temperature (Dhage et al., ). ZnO particles shown antimicrobial activity (Raghunath and Perumal, ) against both Gram-positive (Guo et al., ), Gram-negative bacteria (Liu et al., ; Reddy et al., ; Guo et al., ), and even antibacterial activity against spores (Makhluf et al., ; Wagner et al., ). ZnO NPs are believed to be nontoxic, bio-safe, and biocompatible (Hameed et al., ). The anti-microbial activity of ZnO NPs has not studied on the carbapenem-resistant strain of A. baumannii. The mechanisms of antibacterial activity of ZnO particles are not well understood, although some statements were proposed such as, generation of hydrogen peroxide could be the main factor of antibacterial activity (Xie et al., ; Sirelkhatim et al., ), or binding of ZnO particles on bacterial surface due to the electrostatic forces could be a mechanism (Stoimenov et al., ). Therefore, the present study is an attempt to synthesize ZnO and check its potent antimicrobial activity against carbapenem (beta-lactam) resistant strain of A. baumannii. The outcome of this study will help to find a suitable alternate to carbapenem, which is currently used to control the infection caused by A. baumannii.

Materials and Methods

In the present study, ZnO was prepared by chemical and green synthesis methods, and characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), and UV-Visible (UV-Vis) spectroscopy. This was followed by anti-microbial activity against three strains of A. baumannii using disk diffusion and growth kinetics. The mechanism of action of ZnO was also determined by different biochemical tests. All the methods used in the present study have been outlined in Figure 1.

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FIGURE 1. Flowchart representation of different steps used in the present study.

Bacterial Strain

Two carbapenem-resistant A. baumannii (RS and RS) and one carbapenem sensitive (ATCC) strains were used in the present study. These three bacterial strains were available in our laboratory at Central University of Rajasthan, Ajmer. These strains showed the similar pattern in the results hence result of RS is presented here to reduce the repetition.

Chemical Synthesis of ZnO and Its Characterization

Nanophasic ZnO was successfully synthesized by using acetate precursor-based sol-gel route (Kuberkar et al., ; Kansara et al., ). Zinc acetate [Zn (CH3COO)2 × 2H2O] was dissolved in 25 mL double distilled water by maintaining M solution with continuous stirring at 90°C with rpm for 30 min. After overlapped processes of polymerization, condensation, and gelation, the gel-based solution was preheated at °C for 1 h, to repel out water content from light thick solution. Brown colored powder was then calcined at °C for 2 h in high-temperature automated furnace, to remove the organic content from the material. This results in a black fine powder of ZnO. To identify the structural phase present in the ZnO nano-powder, XRD was performed at room temperature using Cu Kα radiation. To understand the size distribution, particle size analyzer was used for dispersed nanoparticles of ZnO. A FTIR spectrum was recorded in the range of to cm-1 for the presently studied ZnO to understand the optical properties of nanoparticles. Water dispersed ZnO nanoparticles (ZnO-NPs) were further analyzed for their optical band gap by performing UV-Vis spectroscopy at room temperature.

Synthesis of Green ZnO and Antibiotics Capping ZnO

g of zinc nitrate ( mM) was added to 50 mL water extract of leaf of Calotropis procera with constant magnet stirring until complete dissolution as per published protocol (Vijayakumar et al., , ). A beta-lactam antibiotics, i.e., ampicillin was tagged to ZnO NPs as per published protocol (Brown et al., ; Vijayakumar et al., ). The synthesized NPs were characterized using UV-Vis spectroscopy.

Determination of Anti-bacterial Activity Using Disk Diffusion Assay

Antibacterial assay was performed to study the effect of ZnO on the A. baumannii using our published protocol (Tiwari et al., ). Disks of chemically synthesized ZnO, conjugated chemically synthesized ZnO, green ZnO, and conjugated green ZnO were used. Disks of distilled water were used as a control. Plates were incubated overnight at 37°C. Antibacterial activity was evaluated by measuring the inhibition-zone diameter (Odebiyi and Sofowora, ).

Growth Kinetics Study of A. baumannii

Growth kinetics of A. baumannii was determined in the absence or presence of differently prepared ZnO. Bacteria were grown in Luria-Bertani broth in the incubator shaker at rpm, and OD was measured at nm at an interval of 30 min using UV-Vis spectrophotometer as per our published protocol (Tiwari et al., b). The experiment was performed in triplets for control, the presence of chemically synthesized ZnO-NPs, green synthesized ZnO-NPs, ampicillin conjugated ZnO-NPs (chemical or green synthesized), and ampicillin. Relative growth curves of growth kinetics of untreated and treated bacterial culture were prepared for comparison purpose.

Determination of IC50 of ZnO

Inhibitory concentrations of the ZnO-NPs were determined as per published protocol (Gattringer et al., ) with some modification. Ten microliters primary culture was mixed with 90 μL LB broth (taken in ELISA plate) and incubated for 2 h at 37°C. Bacterial cultures were treated with different concentrations of ZnO-NPs (1 to 50 mM) and incubated for 5 h at 37°C. This is followed by addition of 5 μL of MTT solution (5 mg/mL) in each well and incubated for 1 h in the dark at 37°C. This step is followed by addition of μL DMSO to each well and incubated again for 2 h at 37°C. After incubation, the OD was monitored at nm and viability rate of each well was determined. Viability percentage is the percentage of the ratio between absorbance of treated wells versus absorbance of control.

Quantification of Reactive Oxygen Species (ROS)

To estimate the reactive oxidative species produced in the microbial cell, the published protocol was followed (Choi et al., ). In brief, a mL bacterial culture was treated with μL of ZnO (final working concentration of 2 mM) and incubated at 37°C in an orbital shaker. After 6 h, bacteria pellet was collected by centrifuging at 10,g for 10 min at 4°C. 2% Nitro Blue Tetrazolium (NBT) solution was added to the pellet, mixed, and incubated for 1 h at room temperature in the dark. This step is followed by centrifugation and supernatant was discarded. Pellet was washed with PBS and centrifuge at g for 2 min. Pellet after centrifugation was washed again with methanol and centrifuge at g for 2 min. The pellet collected after centrifugation was suspended in 2 M KOH for cell membrane disruption. To this, 50% DMSO solution was added and incubated for 10 min at room temperature to dissolve formazan crystals. It was then centrifuged at 8,g for 2 min. After centrifugation, μL supernatant was transferred to 96 well plates and absorbance was recorded at nm using ELISA reader. Cultures without any treatment were taken as a control, and LB media was taken as blank.

Quantification of Membrane Lipid Peroxidation

Unstable lipid peroxides cause oxidative stress in microbial cells that decompose to form reactive compounds which lead to cellular damage. Thiobarbituric acid-reactive substances (TBARS) assay is used to detect lipid peroxidation (Joshi et al., ; Thombre et al., ). In this assay, malondialdehyde forms a complex with thiobarbituric acid, which can be quantified spectrophotometrically. In brief, the mL bacterial culture was treated with μL of chemically synthesized ZnO (final concentration of 2 mM) and incubated at 37°C in an orbital shaker. After 6 h, the culture was centrifuged at 10,g for 10 min at 4°C. The pellet was washed and redispersed in 10%-SDS ( μL). 20% acetic acid was added to this suspension and incubated for 10 min. μL TBA buffer (% TBA in 2 M NaOH) was added to the solution. This reaction mixture was incubated for 1 h at 95°C and then cooled to 25°C. To remove cell debris, the reaction mixture was again subjected to centrifugation at g for 15 min. Absorbance was recorded at nm using ELISA reader. Cultures without any treatment were taken as a control, and LB media was taken as blank.

Quantification of Membrane Leakage of Reducing Sugars, Proteins, and DNA

The effect of ZnO-NPs on membrane leakage of reducing sugars, proteins, and DNA released from the intracellular cytosol of the cells after treatment with ZnO-NPs was estimated. In the experiment, mL LB broth culture was treated with μL ZnO (2 mM final concentration) and incubated at 37°C in the orbital shaker at rpm. After 24 h, the culture was centrifuged at 10,g for 30 min at 4°C. The obtained supernatant was stored at °C. This sample is used for the estimation of reducing sugars, proteins, and DNA contents. Reducing sugar was estimated by Dinitrosalicylic acid assay, which is a colorimetric assay and absorbance was recorded at nm (Miller, ). Proteins were estimated by Bradford method and absorbance was recorded at nm (Bradford, ). DNA was estimated by absorption spectra at nm (Li et al., ; Thombre et al., ).

Transmission Electron Microscopy (TEM)

Acinetobacter baumannii was cultured in presence and absence of ZnO. TEM imaging was performed as per published protocol (Tiwari and Moganty, ).

Validation of Cell Viability Using MTT Assay

3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide is a light-sensitive dye, which gives purple color on reduction. It is used in colorimetric assay for assessing cell viability. In this assay, the oxidoreductase enzymes present in metabolically active cells reduce MTT in the cytosol of the cells. The cells with lower metabolic activity reduce very little MTT whereas rapidly dividing cells show higher MTT reduction. Therefore, only living and active cells reduce MTT and those affected cells after treatment unable to reduce MTT. In this assay, the bacterial pellet was suspended in LB media to which mg/mL MTT was added. This reaction mixture was incubated for 2 h and then solubilizing buffer was added. Absorbance was recorded at nm (Zhang and Liu, ).

Statistical Analysis

All the experiments were performed in triplicate and data were analyzed by a Student&#x;s t-test and a value of p was considered significant. Analyzes were performed using Microsoft excel.

Results

Beta-lactams (carbapenems) are commonly recommended by doctors against A. baumannii. The emergence of drug resistance in A. baumannii will lead to high mortality and morbidity. Therefore, it is high time to develop alternative molecule against carbapenem resistant A. baumannii. In the present study, we have used three strains (RS, RS, and ATCC) of A. baumannii. These strains showed the similar pattern in result hence result of RS is presented here to reduce the repetition. The RS strain has MIC > 64 μg/mL for the imipenem, i.e., carbapenem resistant strain.

Synthesis and Characterization of ZnO NPs

Figure 2 shows the XRD pattern recorded for sol-gel grown ZnO powder. It is clearly seen that ZnO, prepared possesses single phasic nature without any detectable impurity within the measurement range. ZnO possesses hexagonal wurtzite unit cell structure. Crystallite size (CS) was calculated using Scherer&#x;s formula: CS = K λ/B cos, where K is the shape factor, λ is the X-ray wavelength used, B is the peak broadening, and  is the angle of incidence. For the present case, K is considered equal to , by considering the stable spherical shape of the particles. Values of estimated CS are found to be nm for most intense peak ( o) and nm by carrying out the average contribution from all the peaks. Difference between these two values suggests the three-dimensional disorder in the nanoparticles.

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FIGURE 2.(A) XRD pattern of modified sol-gel grown ZnO nanoparticles (ZnO-NPs). (B) Particle size distribution with theoretical Gaussian fits for modified sol-gel grown ZnO-NPs.

To understand the size distribution over the presently grown ZnO, particle size analyzer was used at room temperature for dispersed ZnO-NPs. Figure 2B shows the particle size distribution for presently studied sample indicating a broad size distribution between the sizes 10&#x;60 nm with the mean size of nm. This mean size value of particles has been obtained by fitting theoretically the size distribution curve using the Gaussian function. A measurable mismatch between the CS ( and nm) and mean size ( nm) can be ascribed to the fact that particle size analyzer has provided result on dispersed ZnO-NPs where the possibility of agglomeration effect exists between the two or more smaller particles to form a larger one.

To understand the optical properties, purity, and nature of the presently studied ZnO-NPs, FT-IR spectrum was recorded at room temperature, as shown in Figure 3. Generally, metal oxides exhibit absorption bands well below cm-1 arising due to interatomic vibrations. As shown in Figure 3, the peaks below cm-1 ( and cm-1) correspond to the Zn-O bonds confirming the formation and purity of ZnO structure (Khana et al., ). Next peak cm-1 is also due to Zn-O bonds. The peak and cm-1 correspond to COO&#x; (carboxylate group) and C = C bond, respectively (Chithra et al., ). Last peak cm-1 can be ascribed to the O&#x;H stretching (Chithra et al., ).

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FIGURE 3. Fourier Transform Infra-Red (FT-IR) spectrum of modified sol-gel grown ZnO-NPs.

Inset of Figure 4 shows the UV&#x;Vis spectrum carried out for presently studied ZnO-NPs. To estimate the band gap for the presently studied nanoparticles, the Tauc relation can be employed as: αhν = B (hν-Eg)γ, where h is Plank&#x;s constant, Eg is band gap, ν is the frequency of incident photon, B is a constant known as band tailing parameter, and γ is the index. If γ = ½ then it is referring to indirect allowed band gap and if γ = 2 then it refers to direct allowed band gap. Figure 4 shows the Tauc [(αhν)2 vs hν] plot for the sample understudy revealing the straight-line fits with the X-axis intercept around eV which is the direct band gap of presently studied ZnO-NPs. Similarly, tagging of ampicillin to ZnO and characterization of tagged nanoparticles were done by recording absorption spectra using UV-Vis spectrophotometer.

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FIGURE 4. Tauc [(αhν)2 vs hν] plot for modified sol-gel grown ZnO-NPs. Inset: absorption spectrum of modified sol-gel grown ZnO-NPs.

Growth Kinetics of A. baumannii Under Different Conditions

The growth of RS strain of A. baumannii was analyzed in presence and absence of chemically synthesized ZnO, green synthesized ZnO, ampicillin tagged chemically or green synthesized ZnO, and ampicillin alone. Growth curves of treated bacterial culture experience a decline in comparison to that of the untreated one with time. This suggests that the ZnO, ampicillin, and ampicillin tagged ZnO have activity against the bacterial growth (Figure 5). The growth curves in Figure 5, shows that treatment of chemically synthesized ZnO inhibits the growth of A. baumannii more than the other nanoparticle variants. Green synthesized ZnO nanoparticle and its ampicillin-conjugated variant showed almost similar inhibition on the growth of A. baumannii. Ampicillin conjugated chemically synthesized ZnO nanoparticles showed a moderate effect on inhibition of bacterial growth. It is better than green synthesized nanoparticle and ampicillin but not as good as chemically synthesized ZnO nanoparticle (Figure 5).

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FIGURE 5. Comparative display of growth curves of Acinetobacter baumannii in absence and presence of differentially synthesized ZnO. Experiments were performed in triplicate and data are presented as mean ± SD.

Antibacterial Activity of ZnO-NPs by Disk Diffusion Assay

Disk diffusion assay was performed to analyze inhibition zone of ZnO variants against A. baumannii. Antimicrobial activity exhibited by all synthesized nanoparticles, which prevents the growth of bacteria, that can be seen in the form of the clear zone around the disks as seen in Figure 6. Here, all of the synthesized nanoparticles showed activity against the RS strain of A. baumannii but at different levels. The data shows that inhibition zone diameter for chemically synthesized ZnO nanoparticles is maximum as compared to others synthesized nanoparticles. A ring of mucoid growth immediately around disk has also been seen in the C-ZnO treated disks but its reason is unclear. Ampicillin conjugated chemically synthesized ZnO nanoparticle also shows a good inhibition zone. Based on inhibitory effect, the chemically synthesized ZnO nanoparticle has been selected for the study of its mechanism of action study. IC50 value of chemically synthesized ZnO NPs was found to be 2 mM for the RS strain of A. baumannii.

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FIGURE 6. Disk diffusion assay for analyzing the effect of ZnO-NPs on the carbapenem resistant strain of A. baumannii. (A) Comparative display of chemically synthesized (C-ZnO) and green synthesized (G-ZnO) zinc nanoparticles. (B) Comparative display of chemically synthesized (C-ZnO) and ampicillin tagged chemically synthesized (C-ZnO + Amp) zinc nanoparticles. (C) Comparative display of green synthesized (G-ZnO) and ampicillin tagged green synthesized (G-ZnO + Amp) zinc nanoparticles.

Effect of ZnO Treatment on ROS Production and Membrane Lipid Peroxidation

Treatment with chemically synthesized ZnO- NPs leads to increase in the formation of reactive oxygen species (ROS) that results into the destruction of the bacterial cells. Figure 7A showed that there is a fourfold increase in ROS production in ZnO treated A. baumannii as compared to untreated. These elevated ROS leads to have many effects in the bacteria and lipid peroxidation is one of them. The estimation showed that there is a twofold elevation in the lipid peroxidation after treatment with ZnO (Figure 7B). This lipid peroxidation effect the bacterial membrane integrity.

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FIGURE 7. Effect of chemically synthesized ZnO-NPs on the ROS generation (A) and lipid peroxidation (B) in carbapenem resistant strain of A. baumannii. Experiments were performed in triplicate and data are presented as mean ± SD.

Effect of ZnO on Membrane Leakage of Reducing Sugars, Proteins, and DNA

The effect of chemically synthesized ZnO-NPs on membrane leakage of reducing sugars, protein, and DNA was investigated and presented in Figure 8. The membrane leakage of reducing sugars was found to be times more in ZnO treated bacteria as compared to untreated (Figure 8A). Similarly, Figure 8B shows that the protein leakage via membrane is around times higher after ZnO treatment for 6 h as compared to control (Figure 8B). DNA leakage after membrane disruption was fold higher after treatment with ZnO (Figure 8C). Cultures without any treatment were taken as control.

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FIGURE 8. Membrane leakage of carbapenem resistant strain of A. baumannii after treatment by chemically synthesized ZnO-NPs. Comparative quantitative display of leakage of sugar (A), protein (B), and DNA (C). Cell viability of RS after treatment with ZnO using MTT assay (D). LB broth media is used as blank and untreated samples are used as control. Experiments were performed in triplicate and data are presented as mean ± SD.

Effect of ZnO Treatment on Cellular Viability by Using MTT

In addition to growth kinetics and disk diffusion assay, the cellular viability of A. baumannii was assessed using 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT). More metabolically active cells were present in the control (untreated) culture, but after treatment with chemically synthesized ZnO nanoparticle, the number of metabolically active cells in the bacterial culture decreases. Therefore, the lesser number of cells reduces MTT in the treated culture as compared to untreated (control) culture. The cellular viability of A. baumannii was found to be decreased by 15% in 2 h of ZnO treatment (Figure 8D).

TEM Results Confirm Membrane Disruption by ZnO-NPs

Transmission electron microscopy was performed to study the effect of ZnO on bacterial cell membrane integrity (Figure 9). TEM result showed that in the absence of ZnO (control), membrane remains intact (Figure 9A) while after ZnO treatment membrane rupture takes place that releases cell contents. After treatment, cells were empty, and cytoplasm is diffused out of cell membranes due to membrane rupture (Figure 9B). This suggests that ZnO kill bacteria via disrupting cell membrane. TEM results showed good correlation with our biochemical assays like estimation of ROS, lipid peroxidation, and release of cell contents.

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FIGURE 9. Transmission electron microscopy (TEM) image of A. baumannii cultured in the absence (A) and presence (B) of chemically synthesized ZnO-NPs.

Discussion

Acinetobacter baumannii is a pathogenic Gram-negative bacterium, which is one of the ESKAPE pathogens with multi-drug resistance. The emergence of resistance in A. baumannii causes high mortality and morbidity. Acinetobacter has developed an ability to accumulate diverse resistance mechanisms. The rise in the antibiotic resistance and emergence of antibiotic-resistant superbugs, stressing the need for strategies to developing new antimicrobial. Therefore, there is an urgent call for the discovery of new drugs or new target of the old molecules that are capable of enhancing the efficacy of current therapy. Therefore, there is a high time to develop antibiotic alternative therapeutics against carbapenem resistant A. baumannii. There are different approaches that can be employed which includes herbal-based (Tiwari et al., b, a; Roy et al., ), in silico based (Tiwari et al., a,b; Verma et al., ), nanomaterial-based (Tiwari et al., a, b; Wan et al., ), and combination therapy (Saballs et al., ; Song et al., ; Jain et al., ; Aydemir et al., ; Garnacho-Montero et al., ; Hiraki et al., ; Habash et al., ). These approaches are tried recently against A. baumannii and some of them shown very promising results.

Recently, ZnO NPs have shown antimicrobial activity on skin-specific bacteria (Aditya et al., ), Streptococcus mutans, Streptococcus pyogenes, Vibrio cholerae, Shigella flexneri, and Salmonella typhi (Soren et al., ). It has also shown antimicrobial activity against methicillin resistant Staphylococcus aureus (Kadiyala et al., ). Antimicrobial activity of metallic nanoparticle like PVP-capped AgNPs (Tiwari et al., , a, b) and citrate-capped AgNPs (Wan et al., ) were studied in A. baumannii. One study has been done so far where antibiotic-coated ZnO NPs showed anti-microbial activity against A. baumannii (Ghasemi and Jalal, ), but no study has been done so against the carbapenem-resistant strain of A. baumannii. Interaction of metallic NPs with different cell models and their cellular effect have been reviewed recently (Zhang et al., ) and showed the involvement of ROS during the interaction of NPs with different cell lines.

In the present study, the carbapenem resistant strain of A. baumannii was used to check the antimicrobial activity of synthesized ZnO compounds. ZnO nanoparticles were synthesized chemically and by the green method. Synthesized ZnO-NPs was characterized by XRD, FTIR, and UV-Vis spectroscopy. All of these nanoparticles were then tested for their antimicrobial activity that indicated that chemically synthesized ZnO nanoparticle shows the good inhibition in comparison to other synthesized nanoparticle variants. Furthermore, mechanism of effect of ZnO nanoparticles on A. baumannii were assessed on different parameters like ROS generation; lipid peroxidation; membrane leakage of reducing sugars, proteins, DNA, and cell viability. TEM result also confirms the membrane disruption after ZnO-NPs treatment. Based on all the results, the proposed mechanism of action of ZnO involves the production of ROS, which elevates membrane lipid peroxidation that causes membrane leakage of reducing sugars, proteins, DNA, and reduces cell viability.

Conclusion and Future Perspectives

Therefore, it can be concluded from the present study that chemically synthesized ZnO-NP can be developed as an alternative to carbapenem (beta-lactam), that inhibit the growth of carbapenem resistant A. baumannii by producing ROS and causing membrane damage. Therefore, chemically synthesized ZnO nanoparticles can be more favorable future hope as an alternative drug to carbapenem against this carbapenem-resistant strain of A. baumannii.

Similarly, pathogenicity of A. baumannii is influenced by its ability to survive in the human pulmonary cells. Therefore, it is also important to study effect of the ZnO in the interaction of A. baumannii with the human pulmonary host cell. The pulmonary cell-targeted delivery of ZnO in animal model need to be further validated to make it as a suitable drug against A. baumannii. Cell line and animal-based studies are also critical to have an improved mechanistic knowledge under in vivo setup. Detailed proteomic studies of A. baumannii in the presence of ZnO, is also required to identify the proteins involved in the mechanism of action of this molecule. Cytotoxicity of chemically synthesized ZnO-NP can be tested to determine the effective non-cytotoxic dose of the ZnO for cell line and human model.

Author Contributions

VT conceived and designed the experiments, wrote the manuscript, and proofread of final version. NM, MT, KG, and VT performed the experiments. VT, PS, and NS analyzed the data.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Keywords: Acinetobacter baumannii, microbial drug resistance, antimicrobial nanoparticles, mechanism of ZnO action, ROS

Citation: Tiwari V, Mishra N, Gadani K, Solanki PS, Shah NA and Tiwari M () Mechanism of Anti-bacterial Activity of Zinc Oxide Nanoparticle Against Carbapenem-Resistant Acinetobacter baumannii. Front. Microbiol. doi: /fmicb

Received: 10 February ; Accepted: 18 May ;
Published: 06 June

Reviewed by:

Younes Smani, Instituto de Biomedicina de Sevilla (IBIS), Spain
M. Oves, King Abdulaziz University, Saudi Arabia

Copyright © Tiwari, Mishra, Gadani, Solanki, Shah and Tiwari. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Vishvanath Tiwari, [email protected]; [email protected]

Sours: https://www.frontiersin.org/articles//fmicb/full

A Mini Review of Antibacterial Properties of ZnO Nanoparticles

Introduction

Today, antibiotics are the gold standard in treatment of many bacterial infections [1, 2]. However, microorganisms can develop antibiotic resistance. The majority of pathogenic microorganisms have an ability to develop resistance to at least some antimicrobial agents [3]. Antibiotic resistance in bacteria is achieved by several mechanisms: prevention of drug penetration into a cell [4, 5], changes in an antibiotic target [6, 7], enzymatic inactivation of antibiotics [8], active excretion of an antibiotic from a cell [4] and so on.

According to the data of the World Health Organization (WHO), lower respiratory infections and gastrointestinal infections are among the top ten factors of morbidity and mortality [9]. Appearance of antibiotic resistant strains significantly increased the number of deaths and severity of bacterial infections. Deaths of patients due to antibiotic resistant bacterial strains exceed the total number of global deaths due to cancer and diabetes mellitus [10, 11]. Despite the significant quantity of available antibiotics, resistance to almost all of them was confirmed. Antibiotic resistance emerges shortly after a new drug is approved for use [3, 12]. The indicated events urged WHO to endorse the Global action plan on antimicrobial resistance in [13]. Secondary bacterial infections can be a cause of increased lethality among patients in intensive care; in particular, bacterial co-infection and secondary infection are found in patients with COVID [14, 15]. All above mentioned make a search for new antimicrobial preparations a high priority task of public health in the world.

The number of scientific publications devoted to a search for new antimicrobial compounds is about only in &#x;; of them are devoted to a search for antibacterial compounds based on metal compounds [16].

Humans have been used antimicrobial properties of several metals and their ions since ancient times. For example, utensils from Cu and Ag were used in ancient Persia, Rome and Egypt [17]. It is known today that a wide range of metals has the antimicrobial activity: Ag, Al, As, Cd, Co, Cr, Cu, Fe, Ga, Hg, Mo, Mn, Ni, Pb, Sb, Te, Zn [18&#x;20].

The basis of the antimicrobial activity of metals is an ability of metal ions to inhibit enzymes [21, 22], facilitate generation of reactive oxygen species (the Fenton reaction) [23], cause the damage of cell membranes [24], prevent uptake of vitally important microelements by microbes [25]; moreover, several metals can exert the direct genotoxic activity [26&#x;28].

The use of nanoparticles based on metals and their oxides is of great interest. One of the well-studied metals affecting biological objects is zinc (Zn) and its oxide (ZnO). Zinc is an active element and exhibits strong reduction properties. It can easily oxidize to form zinc oxide. Zinc plays an important role in the human body, since it is one of the most important trace elements [29]. Zinc is found in all tissues of the human body, with the highest concentration found in myocytes (85% of the total zinc content in the body) [30]. Zinc has been shown to be critical for the proper functioning of a large number of macromolecules and enzymes, where it plays both a catalytic (coenzyme) and structural role. In turn, structures called Zincfinger provide a unique scaffold that allows protein subdomains to interact with either DNA or other proteins [31].

Zinc is also essential for the functioning of metalloproteins. Although zinc is considered relatively non-toxic, there is growing evidence that free zinc ions can cause negative effects on cells. To assess the toxicity of a test substance in vitro, animal cell cultures are usually used. It is known that nerve cells are the most sensitive to exogenous influences [32&#x;34]. It has been reported that exposure to zinc ions leads to neuronal degradation [35]. To eliminate the cytotoxic effect, zinc cations are bound with bioactive ligands (for example, proteins) and zinc oxide nanoparticles are synthesized. Nanostructured ZnO can have various morphological forms and properties.

At present, there is a growing interest to nanoparticles of metals and metal oxides as compounds with antibacterial potential: Ag [36, 37], Au [38], ZnO [10], TiO2 [39, 40], CuO [41, 42], Fe2O3 [43, 44]. ZnO has many applications in engineering and medicine. In engineering, ZnO nanoparticles are used in solar cells [45, 46], gas sensors, in particular, sensors for Liquefied petroleum gas (LPG) and EtOH [47], chemical sensors and biosensors, in LEDs, photodetectors [48, 49]. In biology and medicine, the cytostatic activity of ZnO nanoparticles (ZnO-NPs) against cancer cells [50], antimicrobial and fungicidal activities [51, 52], anti-inflammatory activity [53, 54], ability to accelerate wound healing [55], a possibility to use in bioimaging due to chemiluminescent properties of nanoparticles [56, 57], antidiabetic properties [58, 59] are of great interest.

ZnO nanoparticles have several advantages: high antibacterial effectiveness at low concentrations (&#x; mmol/L), activity against a wide range of strains, relatively low cost [43, 51, 60]. ZnO nanoparticles are synthesized by the physio-chemical sol-gel method from zinc salts [43, 61], sol-gel combustion method [62], solochemical method [63], chemical synthesis at low temperatures [64] and mechanical method [65]. In several cases, stabilizing agents, for example, chitosan are added [66, 67].

The mechanisms of action of zinc oxide nanoparticles can be reduced to the following: disruption of the cell membrane [68, 69], binding to proteins and DNA, generation of reactive oxygen species (ROS) [10, 70, 71], disturbance of the processes of bacterial DNA amplification, alteration (more often, down-regulation) of expression in a wide range of genes [72]. The direct bactericidal action of ZnO nanoparticles against both gram-negative and gram-positive bacteria and fungi was shown [73, 66, 74].

Nanoparticles of a number of metal oxides lead to the production of ROS upon interaction with bacteria [75]. The metal ions released by the nanoparticles affect the respiratory chain and inhibit some enzymes. This leads to the formation and accumulation of singlet oxygen, hydroxyl radical, hydrogen peroxide, superoxide anions, and other ROS. ROS can cause damage to the internal components of bacteria, such as proteins and DNA [76].

It has been shown that exposure to sublethal ROS concentrations can stimulate the manifestation of defense reactions. This process is called hormesis [77]. Hormesis induces defense mechanisms on two levels. The first level is enzymatic (short-term reaction). At this level, antioxidant enzymes are activated. The second level is long-term adaptation. Long-term adaptation consists of two sublevels: transcriptional and genomic. At the level of transcription, ROS induces adaptation due to the activation of antioxidant mechanisms within a few hours or days [78]. At the genomic level, ROS can cause damage to the DNA structure, which activates the mechanisms for repairing DNA damage. These mechanisms include homologous recombination and excisional repair. In these mechanisms, two of the DNA polymerases responsible for DNA synthesis have poor validation activity and may include abnormal bases in DNA strands, which leads to a high frequency of spontaneous mutations and genome plasticity under adverse influences [79]. Such plasticity of the genome can lead to the development of resistance to metals and metal oxide nanoparticles [80].

The adaptation mechanisms of bacteria in relation to nanoparticles also include overexpression of extracellular substances by bacterial cells, such as flagellin, which form an extracellular matrix that promotes agglomeration and deactivation of nanoparticles [81]. Despite the existing mechanisms of adaptation of bacteria to the impact, numerous studies have noted the high antibacterial potential of ZnO nanoparticles.

Literature Review

Despite the apparent wide range of strains, against which nanoparticles exert the antimicrobial activity, their effectiveness against particular strains can be significantly different. As a rule, gram-negative bacteria are less sensitive to ZnO nanoparticles than gram-positive bacteria [62, 66, 82]. Somewhat higher resistance of gram-negative bacteria can be explained by the peculiarities of their cell wall structure. In contrast to gram-positive bacteria, the cell wall of gram-negative bacteria includes the additional outer membrane containing lipopolysaccharides (LPS) [83]. It is shown that LPS can improve the barrier properties of the outer membrane and, therefore, increase bacterial resistance, in particular, to antibiotics [84]. Epidemiologically significant microorganisms deserve a special attention, for example, Mycobacterium tuberculosis, against which ZnO nanoparticles exert the bacteriostatic effect but not bactericidal [85].

On the contrary, several microorganisms (for instance, Campylobacter jejuni) have an increased sensitivity to ZnO nanoparticles, which make them a convenient model for studying molecular mechanisms of the antimicrobial effect of nanoparticles [24]. ZnO nanoparticles (ZnONPs) disturb the processes of bacterial DNA amplification, reduce expression of a wide range of genes of C. jejuni that are responsible for virulence, significantly alter expression of genes of oxidative and general stress [24]. An important feature of ZnO nanoparticles used in one of the studies is the antibacterial activity against resistant bacterial strains, for example, carbapenem-resistant Acinetobacter baumannii (RS and RS) [86]. The dependence of effectiveness on a bacterial growth phase was shown for ZnO nanoparticles. In particular, ZnO nanoparticles are effective against gram- negative and gram-positive bacteria at the exponential growth phase; however, the antibacterial properties of nanoparticles are significantly decreased at the lag and stationary phases [52]. A range of bactericidal concentrations of ZnO nanoparticles is usually significantly less than a range of 4 [62]. At present, an active search for methods to increase the antimicrobial action of nanoparticles is carried out. Below we present the literature search. Nanoparticles are classified by the method for synthesis, size, structure, form, absence or presence of the envelope or nucleus. The objects, on which nanoparticles influenced, are classified by types, biological effect of nanoparticles, concentration of nanoparticles, duration of exposure, temperature and environment. The data are presented in table 1.

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TABLE 1. Main characteristics, physicochemical and biological parameters of ZnO nanoparticles presented in the review.

Let us consider proposed methods for increasing antibacterial properties of ZnO nanoparticles. The first method for increasing antibacterial properties of ZnO nanoparticles is to use a combination of different metal compounds [52, 90]. For example, the CuO and ZnO have comparable effectiveness against gram-negative Escherichia coli and gram-positive Staphylococcus aureus at the exponential growth phase. ZnO nanoparticles were practically inactive at the lag and stationary phases, while CuO nanoparticles retained the significant activity [52]. Ag and Zn nanoparticles in different ratios inhibit the growth of antibiotic resistant Mycobacterium tuberculosis strains but did not lead to bacterial death [85]. ZrO2-ZnO nanoparticles have the pronounced antimicrobial action in contrast to ZrO2 nanoparticles, but the antimicrobial effect of ZrO2-ZnO nanoparticles does not exceed that of ZnO nanoparticles [94]. However, combinations of metal oxides not always give the synergetic effect. In particular, CdO-ZnO nanoparticles have the antimicrobial action comparable with that of CdO nanoparticles [90]. Doping of ZnO nanoparticles with the Fe ions enables achieving a significant antibacterial effect against E. coli, Pseudomonas aeruginosa []. TiO2/ZnO nanoparticles have more pronounced bactericidal effect against E. coli compared to ZnO nanoparticles. Ag/TiO2/ZnO nanoparticles are more effective than TiO2/ZnO nanoparticles []. Compared to ZnO nanoparticles, ZnO-Mn nanoparticles have higher antimicrobial activity against K. pneumoniae, Shigella dysenteriae, S. enterica Typhimurium, P. aeruginosa and other bacteria [].

The second method for increasing antimicrobial effectiveness is to use combinations of ZnO nanoparticles and carbon nanoparticles, in particular, spindle-shaped graphene oxide (GO) nanoparticles [68, , ]. It is shown that GO-ZnO nanoparticles effectively inhibit the growth of gram-negative (E. coli, S. typhimurium) and gram-positive (Bacillus subtilis, Enterococcus faecalis) bacteria [68]. With that, the antibacterial effectiveness of the mixture of GO-ZnO nanoparticles turned to be nearly twice as high as that of ZnO nanoparticles and almost four times higher than that of GO nanoparticles [82].

The third method is coating ZnO nanoparticles with modifying agents. Gelatin-coated ZnO nanoparticles showed higher inhibition of the growth of gram-negative bacteria compared to gram-positive bacteria [91]. As was mentioned above, overcoming antibiotic resistance in gram-negative bacteria is a more difficult task. Gelatin-coated ZnO nanoparticles inhibit biofilm formation of C. albicans (an additional resistance factor) [91]. These nanoparticles also inhibit angiogenesis in chick embryos, which makes them candidates for the development of preparations preventing undesirable angiogenesis [91]. The chemical surface modification of nanoparticles using (3-glycidyloxypropyl) trimethoxysilane (GPTMS) and decrease in a size up to 5 nm lead to an increase in antimicrobial effectiveness of nanoparticles against S. aureus [62]. Treatment with polystyrene increased the bacteriostatic effect of ZnO nanoparticles against E. coli and Listeria monocytogenes; with that, uncoated ZnO nanoparticles did not have the bacteriostatic effect against L. monocytogenes []. Modification of ZnO nanoparticles with polyethylene glycol or starch also alters properties of nanoparticles []. Modification with polyethylene glycol increased the bacteriostatic effect of ZnO nanoparticles against E. coli &#x; S. aureus; with that, effectiveness against gram-negative bacteria was higher. Polyethylene enhanced cytotoxicity of ZnO nanoparticles toward the cancer cell line (MG) by induction of apoptosis. Modification with starch allowed retention of antibacterial properties of ZnO nanoparticles and reduction of cytotoxicity compared to modification with polyethylene glycol []. Treatment with thioglycerol, contrary to the expectations, did not increase the bacteriostatic and bactericidal activity of ZnO nanoparticles [71]. Polymer films from sodium alginate/polyvinyl alcohol gained bacteriostatic properties after incorporation of ZnO nanoparticles, which can be used in the development of more durable materials [].

The fourth method is modification of the synthesis method leading to changes in the geometrical characteristics of nanoparticles. ZnO nanoparticles synthesized by the sonochemical method have more pronounced inhibitory properties against Bacillus cereus, S. aureus, S. Typhimurium and Pseudomonas aeruginosa than ZnO nanoparticles synthesized by the classical physio-chemical methods [63]. Nanoparticles synthesized at comparatively low temperatures are flower-shaped and have the comparable antimicrobial activity against gram-positive (S. aureus) and gram-negative (E. coli) bacteria and, to a lesser extent, fungi (C. albicans) [64]. When using ROS photocatalytic generation and release of Zn2 , flower-shaped ZnO nanoparticles show more pronounced antimicrobial activity against E. coli than more lacunary hexagon-shaped ZnO-NPs [70].

Antibacterial properties of nanoparticles depend on their size []. For several nanoparticles, the highest antibacterial activity is achieved at the smallest size [, , ]; however, we have not found in the literature a clear dependence of antibacterial effectiveness on a nanoparticle size. We had to analyze literature by ourselves and build a graph reflecting a dependence of an inhibition zone size on a size of ZnO nanoparticles (Figure 1). Analysis of literature allows stating that the highest potential antimicrobial effectiveness of nanoparticles against both E. coli, and S. aureus is observed at a nanoparticle size of about  nm. It is necessary to note that green chemistry not always leads to synthesis of effective nanoparticles. For example, in studies on S. aureus, only two types of nanoparticles out of six (33%) had the antibacterial activity at a level higher than average. When studying on E. coli, only one of five (20%) types of nanoparticles generated using green chemistry exerted the antibacterial activity at a level higher than average. Therefore, it can be suggested that nanoparticles generated by green chemistry still have insufficient effectiveness.

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FIGURE 1. Dependence of the minimum inhibitory capacity on the size of NP-ZnO, as well as the dependence of the size of the zone of inhibition on the size of NP-ZnO against gram-negative bacteria by the example of E. coli(A and B) and gram-positive bacteria by the example of S. aureus(C and D). Gray dots are samples synthesized by methods without using green synthesis , green dots are samples obtained using green synthesis .

As can be seen in Figures 1B,D, quite high dispersion of effectiveness is seen in the region of small sizes of nanoparticles (1&#x;50 nm). Therefore, we studied the dependence of the minimum inhibitory concentration (MIC) on sizes of ZnO nanoparticles (Figures 1A,C). It is shown that the use of nanoparticles with sizes of up to 10 nm is not effective. Usually, at these average sizes of nanoparticles, distribution of nanoparticles by sizes is rather complex and not always narrow. Nanoparticles with small sizes are quite prone to aggregation. Apparently, high dispersion of antibacterial activity at small sizes of nanoparticles can be explained by this fact.

Flower-shaped ZnO nanoparticles can reach large sizes (up to 3 µm) and demonstrate the antimicrobial activity against both gram-positive (S. aureus) and gram-negative (E. coli) bacteria [99]. For spherical ZnO nanoparticles, the antimicrobial activity practically does not depend on the type of a targeted organism. Hexagonal ZnO nanoparticles have higher bactericidal activity against antibiotic resistant Staphylococcus epidermidis, B. subtilis, Klebsiella pneumoniae and P. aeruginosa strains compared to ZnO-NPs with the triangular shape []. Thorn-like ZnO nanoparticles cause significant reduction in the growth of B. subtilis, E. coli and C. albicans colonies demonstrating the antibacterial and antifungal activities [].

The fifth method is modification by physio-chemical methods, for example, by annealing in the Ar environment at high temperatures, or plasma oxidation. With that, the effects of modification can be different: Ar annealing decreases the antibacterial activity of ZnO nanoparticles, while plasma oxidation improves antibacterial properties of ZnO nanoparticles against E. coli and S. aureus []. The sixth method is the use of additives causing photocatalysis of reactive oxygen species (ROS). This modification enables a significant increase in antibacterial properties of ZnO nanoparticles [70, 93]. The seventh method is the so-called green synthesis [&#x;]. ZnO nanoparticles generated by green synthesis have the antimicrobial activity against gram-negative and gram-positive bacteria, as well as several fungi of the genus Candida [88]. In turn, nanoparticles synthesized using the Tabernaemontana divaricata extract demonstrated the antibacterial activity against S. aureus, E. coli and lower activity against S. enterica Paratyphoid [98]. The eight method is a change in the environment conditions. At acidic pH levels, ZnO nanoparticles had higher bacteriostatic action against S. aureus and E. coli than at neutral pH []. The combination of all approaches described above can be most promising, for example, the use of Ag-ZnO nanoparticles synthesized in the Cannabis sativa extract. The generated nanoparticles can be used in combination with photocatalysis and have the antibacterial and antifungal activities.

Conclusion

Zinc oxide nanoparticles have significant antibacterial potential. The use of various methods of synthesis, chemical modification, as well as joint use with other nanomaterials affects the physical and morphological characteristics of nanoparticles, which, in turn, leads to a change in their antibacterial properties. As a result, nanoparticles based on zinc oxide are increasingly used not only in nanoelectronics and optics, but also in such industrial areas as cosmetic, food, rubber, pharmaceutical, household chemicals, etc. The use of packaging with incorporated zinc oxide nanoparticles is possible will allow in the future to prevent the growth of microorganisms and spoilage of food. In turn, the use of medical dressing materials containing ZnO nanoparticles will allow avoiding microbial contamination of the wound and promotes its early healing. Thus, zinc oxide nanoparticles can be considered as a promising new generation antimicrobial agent.

Author Contributions

SG designed this topic. DB, MR contributed to collecting related references. DB made a table. DS, SG, MR wrote most of the manuscript. AS and AL were involved in discussing the manuscript and translating it into English.

Funding

This work was supported by the Ministry of Science and Education of the Russian Federation (Grant Agreement ).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

Authors acknowledge the immense help received from the scholars whose articles are cited and included in references to this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

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Keywords: nanoparticles, zinc oxide, antibiotics, antibacterial, bacteriostatic, bactericidal, fungicidal, green synthesis

Citation: Gudkov SV, Burmistrov DE, Serov DA, Rebezov MB, Semenova AA and Lisitsyn AB () A Mini Review of Antibacterial Properties of ZnO Nanoparticles. Front. Phys.9 doi: /fphy

Received: 15 December ; Accepted: 19 January ;
Published: 11 March

Copyright © Gudkov, Burmistrov, Serov, Rebezov, Semenova and Lisitsyn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Correspondence: Sergey V. Gudkov, [email protected]

Sours: https://www.frontiersin.org/articles//fphy/full
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Review on Zinc Oxide Nanoparticles: Antibacterial Activity and Toxicity Mechanism

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Microbial synthesis of zinc oxide nanoparticles and their potential application as an antimicrobial agent and a feed supplement in animal industry: a review

Over the last decade, nanotechnology has emerged as a technology that has revolutionized every field of applied science. The field of nanoparticles (NPs) is one of the avenues to nanotechnology that is associated with nanoscale materials with very small particles size ranging from 1 to  nm. NPs exhibit distinctive properties owing to their extremely small size and high surface area to volume ratio, which have attributed to the significant differences in the properties over their bulk counterparts [1]. In this regard, NPs have been integrated into various industries by providing innovative solutions.

There are various types of metal oxide including titanium dioxide (TiO2), indium (III) oxide (In2O3), zinc oxide (ZnO), tin (IV) oxide (SnO2) and silicon dioxide (SiO2), where ZnO is one of the abundantly produced metal oxides after SiO2 and TiO2 [2]. ZnO is an inorganic material that exhibits unique properties including semiconductor, a wide range of radiation absorption, piezoelectric, pyroelectric and possesses high catalytic activity [3]. In addition, ZnO has been listed as “Generally Recognized as Safe” (GRAS) by the US Food and Drug Administration (FDA 21CFR) [4] due to its non-toxic properties [5]. Consequently, this makes it safe to be used on human and animals. In recent years, there has been increased interest in zinc oxide nanoparticles (ZnO NPs). This is mainly due to their smallest particles size, which enhances their chemical reactivity. Consequently, this has extended the wide application of ZnO NPs in electronics, optics, biomedicine and agriculture [6,7,8,9].

Zinc are an important nutrient in living organisms [9,10,11]. Evidence has indicated that ZnO NPs have a great potential in biological applications, particularly as the antimicrobial agents [12, 13]. Moreover, numerous studies have been reported on the efficiency of ZnO NPs in inhibiting the growth of broad-spectrum of pathogens [14,15,16], which potentially could replace the conventional antibiotic. Furthermore, zinc is an important trace mineral that plays a vital role in many physiological functions in the body [9, 11, 17, 18]. As such, the integration of NPs in feed would increase the absorption and efficient use of zinc in the body, hence, result in improved health and productivity [19]. Moreover, evidence has indicated that ZnO NPs exhibit potential applications in the poultry and livestock industries, particularly as a feed supplement in the animal’s diet [9]. Numerous studies have been carried out to verify the potential use of ZnO NPs as dietary supplement in improving the growth performances [20,21,22], increase in the bioavailability of zinc [23, 24], enhancing the immune response [18, 25, 26], enhancing the antioxidative property [25, 27] and also improving the egg qualities and productions of layer chicken [24, 25, 28]. Nevertheless, to date, data on the use of ZnO NPs produced by microbial synthesis for the applications in animal feed has been scant.

Traditionally, ZnO NPs are synthesized using physical and chemical processes, which offer higher production rate and produce the better-controlled size of NPs. Nonetheless, these methods are considered unfavourable due to high capital cost, high energy requirements and involve the use of toxic and hazardous chemicals. Consequently, these features result in secondary pollution to the environment. Moreover, a previous study demonstrated that the chemical synthesis of NPs is toxic and less biocompatible [29]. Hence, this has limited their clinical and biomedical applications. Therefore, there is a need to explore and develop cleaner, environmentally safe, economical and biocompatible alternatives to synthesize NPs.

In recent years, the green process of NPs has emerged as an alternative to conventional physical and chemical methods by using biological mediated approaches. The biological synthesis of metal and metal oxide NPs involves unicellular and multicellular biological entities including bacteria [30], yeast [14], fungi [31], virus [32] and algae [33]. These methods are cheap, non-toxic and eco-friendly. The microbes act as a tiny nano-factory in reducing the metal ions into metal NPs with the involvement of enzymes and other biomolecule compounds secreted or produced by the microbes. Nevertheless, only a few microbes are reported to have the capability to synthesise ZnO NPs. Hence, there is a need to explore more potential microbes for the synthesis of ZnO NPs. Therefore, the current paper reviews the microbes mediated synthesis of ZnO NPs, the mechanisms of NPs synthesis and optimization parameters and their potential application as an antimicrobial agent and feed supplement in animal industry as well as their toxicological hazards on animal.

Microbial mediated synthesis of ZnO NPs

NPs have been synthesized by using various conventional physical and chemical methods such as vapour condensation, interferometric lithography, physical fragmentation, sol-gel process, solvent evaporation process and precipitation from microemulsion method [34, 35]. The physical method involves the use of high energy consumption, pressure and temperature, whereas, the chemical method involves the use of perilous and toxic chemicals which contributing in environmental contaminants and hazardous to the person handling it [36]. The toxic chemical that frequently employed in chemical methods is triethyl amine [37], oleic acid [38], thioglycerol [39], and polyvinyl alcohol (PVA) [40] and ethylenediaminetetraacetic acid (EDTA) [41] which is typically used as a capping and stabilizing agent to control the size of NPs and preventing it from agglomeration. Furthermore, some of these hazardous chemicals may reside or bound in the final product of NPs. As such, these may interfere with the biological application as well as limit their usage on animals and human [34]. Collectively, the biological method has gained much interest in the synthesis of metal and metal oxide NPs due to the usage of less toxic chemical, eco-friendly nature and are energy efficient.

The biological synthesis methods of ZnO NPs is performed by using biologically active products from plants and microbes including bacteria, fungi, and yeast. This method is promising owing to its effectiveness, eco-friendly techniques, inexpensive, simple and mass productivity [42]. The biological synthesis using plant extracts is performed using compounds, which are extracted from different parts of the plant such as leaves, roots, stem, fruit and flowers. Some of the plant extracts tend to have complex phytochemical compounds that act as reducing and capping agent in the synthesis process such as phenol, alcohol, terpenes, saponins and protein [43]. Notably, the biological synthesis of metal and metal oxide using plants have been extensively reviewed [34, 44,45,46]. Hence, this paper emphasizes the biological synthesis of ZnO NPs using microbes.

Microbes such as bacteria, fungi, and yeast play an important role in the biological synthesis of metal and metal oxide NPs. In the last decade, the use of microbes has gained increased interest in which there have been many studies conducted using various microorganisms’ models. Nevertheless, the biological synthesis of ZnO NPs using microbes still remains unexplored. Table 1 summarizes several of microbes that mediate the synthesis of ZnO NPs including their size, shape and special applications. Biological synthesis using microbes offers an advantage over plants since microbes are easily reproduced. Nonetheless, there are many drawbacks pertaining to the isolation and screening of potential microbes. The main drawback includes cost-effective of the synthesis processes as it is time-consuming and involves the use of chemical for growth medium. The presence of various enzymes, protein and other biomolecules from microbes plays a vital role in the reduction process of NPs. These multiple organic components secreted in the suspension or growth medium attributed to the formation of multiple sizes, shape with mono- and polydispersed NPs [66]. Moreover, the protein secreted from microbes could act as a capping agent that confers stability of NPs formation.

Full size table

In general, not all microbes are able to synthesize NPs because each microbe has a different metabolic process and enzymes activities. Thus, in this regard, the selection of appropriate microbes (regardless of their enzyme activities and biochemical pathway) is crucial to forming NPs. Generally, the cultures are allowed to grow in the culture medium. Besides, the biological synthesis of metal and metal oxide NPs requires metal precursors, which are usually supplied in the form of soluble salts and precipitated in the suspension containing microbial cells or biological compounds extracts from the microbes. The synthesis reaction is usually completed within minutes or hours depending on the culture conditions, which results in the white deposition in the bottom flasks or changes in the colour of suspensions. Thus, this indicates a successful transformation. Furthermore, several parameters are important to determine the rate of production, yield and morphologies of NPs including the temperature, pH, concentration of metal precursor and reaction time. Figure 1 illustrates the process of a biological method utilizing microbes in the synthesis of metal and metal oxide. The NPs produced are characterized physicochemically to determine their properties including size, shape, surface charge, functional group, and the purity [67].

Microbe-mediated synthesis of metal and metal oxide NPs. Microbial synthesis of ZnO NPs requires the selection of microbes, optimal conditions for cell growth, and route of biosynthesis (intra- or extracellular). The ZnO NPs precipitates are washed repeatedly with distilled water followed by ethanol and afterwards dried at 60 °C overnight to obtain a white powder of ZnO NPs. Various physicochemical techniques are used to characterize the properties of NPs, including size, shape, surface charge, functional groups, and purity, by using ultraviolet–visible spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and dynamic light scattering (DLS)

Full size image

The synthesis of metal and metal oxide NPs depends on the ability of microbes to tolerate heavy metals. Moreover, it is well-known that high metal stress may have an effect on the various microbial activities [68]. Under stress condition, the microbes tend to reduce ions to respective metals. As such, this demonstrates their capability to act as natural nano-factory [69]. Generally, microbes that inhabit ecological niches rich in metal exhibit high metal resistance due to adsorption of metals and their chelation by intra- and extracellular proteins [70]. Therefore, mimicking the natural biomineralization process could be a promising approach for the synthesis of metal and metal oxide NPs. A number of metal-reducing microbes have been isolated to synthesise the metal NPs. A previous study isolated the soil fungus, Aspergillus aeneus from mine in India, which demonstrated a high zinc metal tolerance ability and exhibited the potential for the extracellular synthesis of ZnO NPs [59]. Similarly, another study isolated Cladosporium oxysporum AJP03 from the metal-rich soil in India [71]. The fungi were found to have high gold metal tolerance ability and secreted high enzymes and protein. Hence, the fungi was identified as a potential candidate for the extracellular synthesis of gold NPs.

Numerous microbes have been exploited to synthesise ZnO NPs in which bacteria are preferred due to the ease of handling and genetic manipulative attributes compared to other eukaryotic microorganisms [72]. The reproducible bacteria such as lactic acid bacteria (LAB) have attracted increased attention in bacteria mediated synthesis of NPs due to their non-pathogenic properties and high production of various enzymes. Moreover, LAB also recognized as the health beneficial bacteria, which are abundant in the food products [52]. Furthermore, the LAB are facultative anaerobic bacteria that are known to have negative electrokinetic potential. This causes LAB to be easily attracted to the metal ions for the NPs synthesis under both oxidizing and reducing conditions [50, 52]. Apart from that, LAB are Gram-positive bacteria that have a thick cell wall layer consisting of peptidoglycan, teichoic acid, lipoteichoic acid, protein, and polysaccharides [73]. This layer acts as a site for biosorption and bioreduction of metal ions. Additionally, LAB are able to produce exopolysaccharides, which serve as a compound to protect the cell against metal ions and may act as an additional site for biosorption of metal ions [74]. Selvarajan and Mohanasrinivasan [51] demonstrated the intracellular synthesis using Lactobacillus plantarum VITES07 that produced a pure crystalline and spherical shape of ZnO NPs with the size ranged from 7 to 19 nm. The authors reported that NPs produced were moderately stable in which the biomolecules secreted by the LAB acted as a capping agent in the synthesis process. Moreover, studies by Mishra et al. [53] and Prasad and Jha [52] demonstrated that using Lactobacillus sporogens to synthesis ZnO NPs could produce a similar hexagonal shape with different sizes.

The biological synthesis of ZnO NPs using fungi is a promising approach due to their high tolerance to higher metal concentration, high binding capacity and their ability in metal bioaccumulation over bacteria [75]. Moreover, the fungi exhibited the ability to secrete a large number of extracellular redox proteins and enzymes. As such, this contributed to the reduction of the metal ions into NPs in larger amounts, which is suitable for the large-scale production [66]. The higher amount of protein secreted in the medium by the fungi acted as capping protein that further bound and encapsulated the NPs surface and conferred to the stability. For instance, Raliya and Tarafdar [61] demonstrated the synthesis of ZnO NPs by using Aspergillus fumigates TFR-8 that resulted in the formation of NPs with the average diameter size of  nm and high monodispersity particles (uniformly distributed) without any agglomeration. Moreover, the authors suggested that the protein secreted by the fungi was bound and encapsulated the spherical NPs and prevented the NPs from agglomerate. Subsequently, the stability of NPs was examined for  days by measuring the size using particle size analyzer. The results demonstrated that NPs were stable until day 90 and the size increased thereafter due to the agglomeration. This concludes that protein could act as a capping agent to stabilize the NPs up to 90 days. In addition, filtrate-cell free supernatant (FCF) of Alternaria alternate was used in the synthesis of ZnO NPs. On that account, the FCF was found to produce NPs after the precipitation of zinc sulfate solution with the size of 75 ± 5 nm. In addition, the FTIR absorption spectra analysis demonstrated the presence of protein and other organic compounds on the ZnO NPs produced. This results corroborated with the previous study that suggested the fungi can generate a high extracellular protein, which bound on the surface of NPs in order to stabilize and prevent it from the aggregation [58]. Therefore, the use of fungi for the synthesis of NPs is favourable as the fungi are efficient in secreting of extracellular enzymes and protein.

Similar to fungi, yeast has been proven to synthesize metallic NPs due to their higher tolerance to the toxic metal. A study conducted by Moghaddam et al., [14] demonstrated that a new isolated Pichia kudriavzevii yeast strain was able to synthesize ZnO NPs with ~ 10–61 nm of the size range of NPs produced. The formation of NPs were reported to depend on the reaction duration, which was found to play an important role in the size, shape and distribution of ZnO NPs. Moreover, Chauhan et al. [65] demonstrated an extracellular synthesis of silver NPs and ZnO NPs using Pichia fermentans JA2 isolated from the pulp of spoiled fruits. Moreover, the UV-vis spectra results indicated a strong and broad peak at  nm and  nm implying the successful formation of silver and ZnO NPs, respectively.

The microbes mediated synthesis of ZnO NPs seems to be eco-friendly and safe as it does not involve the use of any toxic and hazardous chemical in the synthesis process. In addition, the biologically active compounds secreted by the microbes were acted as a reducing and capping agents. Thus, this approach is more advantageous than the conventional methods. Furthermore, fungal mediated synthesis seems to be a promising candidate for the synthesis as it produces more biologically active compounds than the other microbes. Nevertheless, in term of the cells growth activity, the bacteria are promising compared to the other alternatives. Moreover, the mechanisms of biological synthesis of ZnO NPs among the microbes are different and are not fully understood yet, hence, further investigation is needed.

Mechanisms of microbes mediated synthesis of NPs

Evidence has shown that enzymes, protein and other compounds produced by microbes play a vital role in the synthesis process. Nonetheless, to date, the data on the identification of chemical components responsible for the synthesis of NPs has been scant. Microbes exhibit the intrinsic potential to synthesise NPs of inorganic materials, which may be routed either by the intracellular or extracellular pathway. Extracellular synthesis is more advantageous and has been widely applied compared to the intracellular route. This is mainly due to the fact that it could be used to synthesise large quantities and involves simple downstream processing that eliminates various steps of synthesis, easy separation and industrialization. While the recovery process of NPs in the intracellular synthesis requires additional step such as harvesting the cell biomass by centrifugation and subjected to several cycle ultra-sonication for cells disruption to obtain the purified NPs [76]. Nonetheless, the specific mechanism with regard to this has not been completely elucidated.

Intracellular mechanisms of microbial synthesis

In the intracellular synthesis pathway, the cell walls of microbes and ions charge play an important role in the synthesis of NPs. This involves distinctive ion transportation in the microbial cell in the presence of enzymes, coenzymes and others. The cell wall of microbes consists of a variety of polysaccharides and protein, which provides active sites for binding of the metal ions [77]. Moreover, not all microbes are able to synthesize metal and metal oxide NPs. Evidence has shown that heavy metal ions exhibit great threat to the microbes in which when there is a threat, the microbes will react by gripping or trapping the ions on the cell wall through the electrostatic interactions [51]. This is due to the fact that metal ion is attracted to the negative charge from the carboxylate groups (specific enzymes, cysteine, polypeptides) that is present on the cell wall [78]. Furthermore, the trapped ions are reduced into the elemental atom initiated by the electron transfer from NADH by NADH-dependent reductase that acts as an electron carrier, which is embedded in the plasma membrane. Finally, the nuclei grow to form NPs and accumulate in the cytoplasm or in the cell wall (periplasmic space). On the other hand, the protein or peptides and amino acids such as cysteine, tyrosine and tryptophan exist inside the cells are responsible for providing stabilization of NPs [79, 80]. Figure 2 demonstrates the mechanisms of the microbes mediated intracellular synthesis of ZnO NPs.

Schematic representation of intracellular synthesis mechanisms of ZnO NPs. The intracellular mechanisms involve the transportation of metal ions into the cell wall by electrostatic attraction. The metal ions are reduced to a metal atom by the enzymes found in the cell wall and then initiate the nuclei growth to form NPs in periplasmic space and cytoplasm. The intracellular synthesis requires ultrasonication to obtain the purified NPs

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Mukherjee et al. [81] demonstrated the intracellular mechanisms using Verticillium sp. for the synthesis of NPs involving three steps, which are trapping, bioreduction and capping. The interaction forces between metal ions and the enzymes present on the cell wall reduce the metal ion within the cell wall leading to the aggregation of metal atoms and formation of metal NPs. The authors also reported the presence of metal NPs on the cytoplasmic membrane by transmission electron microscopy (TEM) analysis, which suggested that the formation of NPs occur in both the cell wall and the cytoplasm of the cell. The small metal ions diffused across the cell wall and entered the cytoplasmic membrane. The bioreduction of metal ions into NPs occurred with the presence of local enzymes. Similarly, the intracellular synthesis of gold NPs by utilizing Rhodococcus sp. was reported to occur on the cell wall and cytoplasmic membrane that produced 5 to 15 nm size of NPs with better monodispersity form [82].

The intracellular, extracellular route or surface production of NPs have been reported to be pH-dependent. A slower rate of intracellular synthesis of silver NPs synthesis by using Meyerozyma guilliermondii KX was observed at pH 3. Moreover, TEM image analysis revealed that small NPs population existed in the cytoplasms in a cluster or nanoaggregates form. Additionally, the NPs were also observed to be located away from the cell wall. The authors suggested that the biomolecules found on the cell such as protein and polysaccharides that are involved in the bioreduction of NPs were inactivated under an extremely acidic condition. Consequently, this caused the metal ion to move into cytoplasms [83]. In another study, the deposition of gold NPs synthesis using Shewanella alga cells was reported to depend on the pH conditions. At pH 7, the NPs were deposited in the periplasmic space of the cells whereas, at pH 2, the NPs were observed to be deposited in the cytoplasm and larger NPs were deposited extracellularly (outside the cell) [84]. While in other cases, some bacteria species are pH dependent, the membrane-bound oxidoreductases of L. sporogens that were used for the synthesis of ZnO NPs were activated under a low pH. This suggests that lower pH ambient is a prerequisite for the synthesis of NPs [52]. Similarly, this was in agreement with a previous study that reported the biological synthesis of ZnO NPs by membrane-bound oxidoreductases of L. plantarum VITES07, which was pH-sensitive [51].

Extracellular mechanisms of microbial synthesis

Numerous studies reported that extracellular synthesis is a nitrate reductase-mediated synthesis, which is responsible for the reduction of metal ions into metal NPs [30, 65, 85,86,87]. The extracellular synthesis pathway involves enzyme-mediated synthesis which located on the cell membrane or the releasing of the enzyme to the growth medium as an extracellular enzyme. Nitrate reductase is an enzyme in the nitrogen cycle that catalyses the conversion of nitrate to nitrite. For instance, the bioreduction of Zn2+ was initiated by the electron transfer from NADH by NADH-dependent reductase that acts as an electron carrier [88]. Consequently, the Zn2+ obtained electron and reduced to Zn0. Subsequently, this resulted in the formation of ZnO NPs. The schematic of the extracellular synthesis mechanisms is illustrated in Fig. 3.

Schematic representation of extracellular synthesis mechanisms of ZnO NPs. The extracellular mechanisms involve enzyme-mediated synthesis such as nitrate reductase enzyme, which is secreted in the growth medium, to reduce the metal ions to their respective metal atoms and lead to nucleation and growth of NPs. The extracellular protein secreted by the microbes acts as a capping agent for NPs stabilization. The formation of white precipitation in the medium shows the production of NPs

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Kundu et al. [30] conducted an experiment to determine the involvement of the secreted protein or enzyme by the multi-metal tolerant bacteria, Rhodococcus pyridinivorans NT2 for the synthesis of ZnO NPs. The bacteria biomass were exposed to zinc ion and millipore water as a control. The extracellular protein from the supernatant was determined by a protein expression profile. The results of Lowry’s assay demonstrated that the concentration of extracellular protein secreted by the bacteria biomass that was exposed to zinc ions was twice higher than in the control, which was  ±  μg/mL and  ±  μg/mL for control. Moreover, the electrophoretic profile by one-dimensional SDS-PAGE revealed that the presence of a molecular mass of 43 kDa, which was an NADH-dependent reductase. The findings indicated that NADH-dependent reductase resulted in the formation of ZnO NPs.

Studies have shown that protein produced and secreted by microbes play an important role in the NPs synthesis [57, 89,90,91]. Nevertheless, some studies suggested that the native form of the protein is not compulsory for the NPs synthesis process. A study by Jain et al., [59] revealed that amino acids present in the protein were found to interact with the Zn2+ ions to form NPs. The study also investigated the ability of denatured (heat treated) and native (untreated) protein present in the fungal cell-free filtrate suspension for the synthesis of ZnO NPs. The results demonstrated that both heats treated and untreated samples were able to synthesize ZnO NPs. Notably, the absorbance spectra result by UV-Vis demonstrated a higher reaction rate on the heat treated protein compared to the untreated. This indicates that the synthesis of ZnO NPs was higher in the heat-treated samples. Hence, the results confirmed that the presence of the native form of the protein is not mandatory for the synthesis process. This may attribute to the fact that, the interaction between hydrogen bond and non-polar hydrophobic was disrupted during the heating process. Consequently, this resulted in the exposed contact of amino acids with zinc ions that led to the formation of ZnO NPs [59]. Moreover, the authors also suggested that the biosynthesis of metal NPs was non-enzymatic due to the denaturation of the structure of the enzyme during heat treatment.

In some cases, the non-enzymatic mediated synthesis depends on the certain organic functional groups present on the microbial cell wall, which facilitates the reduction of metal ions. The live cell biomass and dead cell (heat killed by autoclaving) of Corynebacterium glutamicum were used to synthesize silver NPs. After sonication of the cell, the UV-Vis spectra results demonstrated a strong plasmon resonance between and  nm for both samples. Both samples were further incubated for a few days. The results indicated that the peak area and height of the UV-Vis spectrum for the dead cell were comparatively higher compared to live cell samples. This indicates a higher productivity of silver NPs [92]. The authors also validated the results by investigating the total organic content (TOC) for both samples and revealed that the TOC in dead cells was twice higher than the live cell. This indicates the release of organic molecules (reducing agent) from the cell due to rupturing of the cell walls during the heat-killed in which the silver ions obtained the access to more organic molecules and hence a higher amount of reduction occurred [92]. Therefore, the study confirmed that the formation of NPs could occur without the involvement of biological enzymes or metabolites compounds. Nevertheless, this was in contrast to Korbekandi et al. [74], which suggested that the NPs synthesis was an enzymatic reaction. The study demonstrated that the boiling of the Lactobacillus casei biomass killed the bacterial cells and denatured the enzymes. The results demonstrated the presence of NPs in the reaction mixture with active biomass, whereas, no absorbance was observed in the reaction mixture with boiled biomass. This indicates that the enzymes found in the medium were denatured while the dead cells were unable to secrete enzymes resulting in absence of synthesis of NPs. Thus, it can be speculated that the inconsistent finding was due to variations in the methods and species of microbes used for the synthesis of NPs.

The protein secreted by microbes could also act as a capping agent despite acting as a reducing agent. As such, this facilitated the higher stabilization and dispersion of NPs [30]. As such, several studies demonstrated the involvement of protein as a capping agent. For instance, Velmurugan et al., [64] investigated the role of protein in live, dried and dead biomass of Fusarium spp. as a capping agent in the synthesis of ZnO NPs. The SEM-EDS results on the NPs produced by live and dried biomass demonstrated signals of Na and K. This indicated the bound of proteins on the surface of zinc crystallites. This result was supported by the FTIR results, which demonstrated clear peaks. The findings revealed the presence of protein and amide, I and II bands at , , , and  cm− 1, respectively. Nevertheless, there was no protein signal detected in zinc crystal produced by the dead biomass. Similarly, Bao et al. [93] evaluated the chemical composition of the ligands capping on the NPs. The FTIR results demonstrated two absorption bands at and  cm− 1, which indicated the typical amide I and II absorptions of protein molecules, respectively. Further verification was carried out via protein purification by using high-performance liquid chromatography (HPLC) to analyze the molecular mass of the capped protein. The results demonstrated the presence of two proteins with molecular mass of kDa and kDa. Additionally, the authors suggested that the yeast used in the experiment facilitated the synthesis of NPs and generated protein ligands to act as a capping agent, which inhibited the aggregation of NPs.

Effect of various parameters on the optimization process of NPs synthesis

Microbes-mediated synthesis of NPs has the potential to be a great alternative to chemical and physical methods, despite the main drawback in applying biological synthesis of NPs, which refers to the difficulty in controlling both the size and the shape of NPs. The main major concerns in using microbes are to increase yield production for industrial scale, which demand further investigation. It has been widely reckoned that the physicochemical properties of NPs are highly dependent on their size and morphology structures. Studies have proven the direct effects of NPs size and shape on their performance. Sadeghi et al., [94] revealed that the nano-plate-shaped NPs exhibited good antibacterial activity due to their large surface area, in comparison to those with nano-rod shape. In another study, ZnO NPs at 12 nm effectively inhibited the growth of pathogenic bacteria, when compared to those at  nm [95]. Therefore, in order to generate effective size distribution, morphologies, and yield production of NPs, it is necessary to optimize both the cultural condition and the varied physical parameters, including pH, temperature, metal ions concentration, microbial age, and reaction time. The biological synthesis of NPs seems to gain better commercial acceptance if the NPs are produced in high yield with the desired size and shape. The schematic representation of parameters for producing the desired NPs is portrayed in Fig. 4.

Strategies for optimizing the synthesis of ZnO NPs. The synthesis of NPs is associated with different physicochemical parameters including pH, temperature, precursor concentration, microbe age, reaction times, irradiation, and stirring. Each of these parameters contributes to variations in size, shape, monodispersity, and yield of NPs

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Effect of pH

Generally, pH is a key factor that has a major role in the synthesis of metal NPs, mainly because pH has the ability to alter the shape of biomolecules that is responsible in capping and stabilizing the NPs [96]. Gericke and Pinches [97] assessed the biosynthesis of gold NPs using Verticillium luteoalbum by varying the pH level to determine its impact on the size and shape of the generated NPs. The outcomes displayed a majority smaller size of NPs with spherical shape obtained at pH 3, in comparison to those retrieved for pH values 7 and 9 that predominantly produced larger NPs with irregular and undefined shapes. Optimization of pH may also be influenced by the species of microbes applied in the synthesis. For instance, an acidophilic bacterium, Lactobacillus casei, resulted in increased absorbance of silver NPs production as the pH value was reduced; indicating that growth and enzymes activity of L. casei are better in weak acidic environment [74]. In the case of alkaline condition, Gurunathan et al., [98] asserted that hydroxide ion is essential to decrease metal ions. The authors observed rapid increment in silver conversion within less than 30 min’ reaction time at pH 10; signifying that the protein, which served as a reducing agent, was present in the supernatant and was active in reducing power under alkaline conditions. Additionally, the authors confirmed their results by observing the NPs with TEM analysis that recorded smaller size of NPs ranging between 10 and 15 nm. The finding is in agreement with that reported by Ma et al., [99], which recorded increase in absorbance peak of silver NPs with increment of pH value. The synthesis of silver NPs using Fusarium oxysporum at pH 6 resulted in the smallest size, whereas higher pH generated the biggest size, which indicated the catalytic activity of enzymes involved in the synthesis of NPs that appeared to be deactivated under alkaline condition, thus causing an increase in the size of NPs [29].

Effect of temperatures

Numerous researches have investigated the impacts of various temperatures on the size and yield production of NPs. Mohammed Fayaz et al., [] assessed the effects of temperatures on NPs size produced by Trichoderma viride at 10 °C, 27 °C, and 40 °C. The UV-Vis spectra outcomes showed that lower wavelength regions at  nm were obtained at 40 °C and higher wavelength regions at  nm and  nm were obtained at 27 °C and 10 °C, respectively, indicating increment in NPs size at higher wavelength regions. The author also verified their results with TEM analysis that showed a high temperature of 40 °C generated smaller monodisperse NPs size ranging between 2 and 4 nm, while at a lower temperature, larger NPs were produced. In another study, maximum production of silver NPs synthesized by Sclerotinia sclerotiorum was obtained at 80 °C with 10–15 nm size range. This postulated that higher temperature increased the kinetic energy, thus leading to rapid synthesis rate and maximum NPs with a smaller size []. The decrease in particle size with increased temperature is normally due to increment in reaction rate at higher temperature. This causes the metal ion to be consumed rapidly in forming nuclei, while the size is reduced initially due to reduction in the aggregation of the growing NPs [98].

Effect of precursor concentration

The impact of various precursor salt concentrations on the synthesis of metal NPs using soil fungus Cladosporium oxysporum revealed that the optimum concentration of precursor salt at  × 10− 3 mol/L gave maximum NPs yield. Nonetheless, at concentrations  × 10− 3 and  × 10− 3 mol/L, no NPs was generated due to the insufficient biomolecules in minimizing the high amount of metal ions present [71]. This finding is in agreement with that reported by Jamdagni et al. [12], who discovered that absorbance of ZnO NPs by UV-Vis spectra increased with increment of precursor concentration ( × 10− 5 to  × 10− 4 mol/L). They added that further increment in concentration ( × 10− 4 mol/L) resulted in broad peak, while decrease in absorbance signified reduction in the synthesis of ZnO NPs. The influence of metal ion concentration on the synthesis of silver NPs using Penicillium aculeatum Su1 suggested that high concentration of metal ions increased the aggregation of NPs, which resulted in the formation of larger NPs size. The authors reported that maximum production of NPs yield was obtained at absorbance peak of  nm by UV-vis spectra, whereby as the concentration increased to  × 10− 3 mol/L, the absorbance peak shifted to  nm; signifying the increased size of NPs formation [99]. Meanwhile, another study reported that increment in metal ions concentration to a certain point generated NPs with smaller size. The study of silver NPs synthesis by using extracellular supernatant of Escherichia coli revealed that increment in silver ion concentration up to 5 × 10− 3 mol/L minimized the size of NPs by about 15 nm, in which the authors speculated that the silver ions bound on the growing particles to form a coat that prevented them from aggregation [98].

Effects of microbial age and reaction time

The growth phase of cell is essential for the synthesis of NPs. Since microbes generate various enzymes at different growth phases, controlling the cell age may be useful in producing high yield of NPs. Gericke and Pinches [97] reported that the biomass of Verticillium luteoalbum harvested at 24 h produced a high yield of gold NPs, when compared to biomass harvested at 72 h. This may be attributed to the fact that cell at the early exponential stage actively generated high concentrations of enzymes and protein, which resulted in high reduction of metal NPs. In a study pertaining to ZnO NPs synthesis that employed Pichia kudriavzevii, prolonged reaction time was discovered upon exposure to metal ions at 36 h that produced aggregate with irregular-shaped NPs, whereas reaction time of 12 and 24 h generated the smallest size of NPs [14]. On the other hand, ZnO NPs synthesis that employed Lactobacillus sp. yielded NPs with an average size of 7 nm for 5 to 10 min of reaction time [51].

In summary, microbes have been reckoned to generate NPs. Nevertheless, optimization process is essentially required to produce the desired NPs size, shape, yield, and homogeneous particles (monodispersity), mainly because these NPs have a significant role in determining their unique properties for specific applications. The study is still ongoing because each microbe has a wide range of abilities in producing NPs and further investigation is required to improve the synthesis process for implementation in practice.

The potential application of ZnO NPs in animal industry

ZnO NPs is one of the largest produced metals oxide [2] which has been extensively studied due to its unique properties of semiconductor characterized by broad direct band gap width ( eV) with high excitation binding energy (60 meV) and deep borderline ultraviolet (UV) absorption []. In this regard, its unique and attractive properties have made it a promising tool for application in many industrial areas including pharmaceutical [], cosmetic [], photocatalyst [], UV light emitting devices [] and agriculture industries [9, ]. Zinc plays a significantly important role in a variety of physiological processes in human, animals as well as plants. It is extensively present in all body tissues including muscles, bones, and skin [], In addition, zinc is an integral component in numerous enzyme structures [11] and has a crucial part in hormone secretion, growth, reproduction, body immune system, antioxidant defence system and many other biochemical processes in the body [17]. Figure 5 illustrates the role of zinc in poultry and livestock.

Role of zinc supplementation in poultry and livestock. Zinc is an important trace element for physiological and biological functions of the body. The utilization rate of zinc in the animal’s body is low and therefore the addition of ZnO NPs to animal feeds is believed to increase zinc uptake and bioavailability in the body

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ZnO NPs possess many valuable features including their eco-friendly materials, biocompatibility, biodegradability and most importantly their bio-safety traits which have been graded by the US Food and Drug Administration [4]. Furthermore, ZnO NPs have been found to exhibit non-toxic properties in human cells at a certain concentration level []. In fact, biological mediated synthesis of ZnO NPs does not involve any hazardous chemical and material, thus making their application in living organisms safe. The efficiency of ZnO NPs is greater than their counterparts due to their high surface to volume ratios. Therefore, the use of bulk zinc oxide has been widely replaced with ZnO NPs in many aforementioned applications. With the onset of biological mediated synthesis of NPs, the application of ZnO NPs has extended into the next level of application particularly in the field of biomedical and nutrition in human and animals. In the recent year, ZnO NPs have been extensively investigated for use in animal husbandry and production as an antimicrobial agent for disease prevention and as a feed supplement in animals diet to improve the utilization efficiency of trace elements in the animal’s body.

Potential role as an antimicrobial agent in animal industry

The continuous usage of conventional antibiotic has led to the growth and spread of multidrug-resistant strains []. Thus, the discovery and development of new approaches as an alternative to a conventional antibiotic is necessary. ZnO NPs produced by the biological enzymatic process have varied application and have been prominently studied recently on their excellent antimicrobial activities such as antibacterial [13] and antifungal [12]. The distinctive features of NPs such as their small size in relation to a large surface area, composition and morphology allow the NPs to interact with the bacterial cell surface and penetrate the cell’s core and subsequently exhibit bactericidal mechanisms []. Moreover, the inorganic antibacterial properties of NPs’ materials have the ability to withstand extremely harsh conditions and high temperatures compared to organic materials []. Microbes mediated synthesis of ZnO NPs could become potential antimicrobial agents as some of the bacterial species are able to produce a variety of compounds that exhibit antimicrobial properties which are known as bacteriocin. Bacteriocin is a small heat-stable peptide which has a bactericidal effect on pathogenic microorganisms []. The bacteriocin derived from the microbes could act as a reducing agent for the synthesis of metal NPs []. In addition, previous study has proved that this small peptide could also bind to the surface of NPs as a capping agent which in turn enhance the antimicrobial effects of metal NPs [,,,,]. Nonetheless, there is a lack of study on employing the biological synthesized of ZnO NPs by microbes on animal production which possibly due to their limitation on mass production for large scale application. Furthermore, many in vitro studies have been conducted on the antibacterial ability of ZnO NPs [14,15,16, 67], however, the exact mechanism of antibacterial activity of ZnO NPs remains elusive.

Antibacterial mechanisms of ZnO NPs

Scientists have suggested a few possible bactericidal mechanisms, some proposed that smaller NPs have greater surface reactivity and easier cell penetration that released the Zn2+. The release of Zn2+ from ZnO NPs is one of the main propositions in antibacterial mechanisms which are known to inhibit several bacterial cells activities including active transport, bacteria metabolism and enzymes activity. Subsequently, the toxicity properties of Zn2+ on the bacterial cell biomolecules induced the cell to death []. Moreover, the release of Zn2+ is size and morphology dependent. For instance, the release of Zn2+ in smaller size spherical structures of NPs is higher than in rod structures due to its smaller surface causing equilibrium solubility []. While the other proposed antibacterial activity is caused by the formation of reactive oxygen species (ROS) which leads to oxidative stress and subsequent cell damage or death. The formation of ROS is a common antibacterial activity adopted by ZnO NPs [34] which are generated under UV exposure and consist mainly of reactive species such as superoxide anion (O2), hydroxyl ion (OH) and hydrogen peroxide (H2O2). These reactive species are generated from the surface of the NPs that react with the hydroxyl groups and absorb water (H2O) to create hydroxyl radicals (OH) and H+ and consequently creates a superoxide anion (O2) with the presence of O2 [67]. The O2 will then react with H+ to produce HO2 and generate into H2O2 in the presence of electrons and H+ [13]. Eventually, the H2O2 penetrates the bacterial membrane and damage the cellular components such as lipid, protein and DNA resulting in injuries and cells death []. However, OH and O2 are unable to enter the membrane of bacteria cell due to their negative charge and may be found on the outer surface except for H2O2 [].

Another possible mechanism for the antimicrobial activity of ZnO NPs is through the attachment of NPs to the bacteria cell membrane via electrostatic forces. The positive zeta potential of ZnO NPs promotes the attachment to the negatively charged bacterial cell which leads to the penetration of ZnO NPs into the cells []. This interaction may distort the membrane plasma structure and damage the bacterial cell integrity, resulting in the leakage of intracellular contents and ends with cell death [16]. In addition, the accumulation of ZnO NPs in the cell also interfered with the metabolic functions of the bacteria that leads to death. The mechanism of ZnO NPs antibacterial activity is illustrated in Fig. 6. Therefore, the aforesaid bactericidal mechanisms provide better action modes compared to the conventional therapeutic agents tendency to develop multidrug-resistant microorganism.

Schematic illustration of the antimicrobial mechanism of ZnO NPs against bacterial cells. ZnO NPs act as an antimicrobial agent through the following mechanisms: (1) the formation of reactive oxygen species (ROS), which induces oxidative stress and membrane and DNA damage, resulting in bacterial death; (2) dissolution of ZnO NPs into Zn2+, which interferes with enzyme, amino acid, and protein metabolisms in bacterial cells; and (3) direct interaction between ZnO NPs and cell membrane through electrostatic forces that damages the membrane plasma and causes intracellular content leaks

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Antimicrobial activity of ZnO NPs in animal industry

In the poultry and livestock industry, Salmonella and Campylobacter are common enteric foodborne pathogens which can be found in the gut and skin of the animals [, ]. These pathogenic bacteria can be transmitted from animals to human through the handling of animals and the consumption of contaminated undercooked meat and egg products []. Staphylococcus aureus a pathogenic bacteria present in meat product cause food poisoning and is also responsible for bovine mastitis and bumblefoot disease in poultry [, ]. Furthermore, S. aureus has the ability to build a resistance rapidly with the prolonged use of antibiotics []. Escherichia coli is another pathogenic bacteria commonly colonized in human and domestic animal gut microbiota. In poultry, E. coli is the major factor of mortality in newly hatched young chicks [] which contribute to economic losses in the poultry industry. Therefore, NPs has arisen to be a new approach in the reduction of these pathogenic bacteria colonization in animals without the risk of developing multi-drug resistance.

The well-diffusion test using a biologically synthesized of ZnO NPs against various Gram-positive and Gram-negative bacteria and fungus was carried out to evaluate their antimicrobial activity. The results showed a maximum zone of inhibition was observed in Pseudomonas aeruginosa and Aspergillus flavus, 22 ±  mm and 19 ±  mm respectively [16]. Similarly, the extracellular synthesis of ZnO NPs employing the endophytic bacteria Sphingobacterium thalpophilum

Sours: https://jasbsci.biomedcentral.com/articles//sz

Oxide is antibacterial zinc

A systematic review on antibacterial activity of zinc against Streptococcus mutans

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GREEN SYNTHESIS OF ZINC OXIDE NANOPARTICLES BY USING BANANA PEEL EXTRACT

Properties of Zinc Oxide Nanoparticles and Their Activity Against Microbes

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Abstract

Zinc oxide is an essential ingredient of many enzymes, sun screens, and ointments for pain and itch relief. Its microcrystals are very efficient light absorbers in the UVA and UVB region of spectra due to wide bandgap. Impact of zinc oxide on biological functions depends on its morphology, particle size, exposure time, concentration, pH, and biocompatibility. They are more effective against microorganisms such as Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Sarcina lutea, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Pseudomonas vulgaris, Candida albicans, and Aspergillus niger. Mechanism of action has been ascribed to the activation of zinc oxide nanoparticles by light, which penetrate the bacterial cell wall via diffusion. It has been confirmed from SEM and TEM images of the bacterial cells that zinc oxide nanoparticles disintegrate the cell membrane and accumulate in the cytoplasm where they interact with biomolecules causing cell apoptosis leading to cell death.

Background

Nanotechnology deals with the manufacture and application of materials with size of up to  nm. They are widely used in a number of processes that include material science, agriculture, food industry, cosmetic, medical, and diagnostic applications [1,2,3,4,5,6,7,8,9,10]. Nanosize inorganic compounds have shown remarkable antibacterial activity at very low concentration due to their high surface area to volume ratio and unique chemical and physical features [11]. In addition, these particles are also more stable at high temperature and pressure [12]. Some of them are recognized as nontoxic and even contain mineral elements which are vital for human body [13]. It has been reported that the most antibacterial inorganic materials are metallic nanoparticles and metal oxide nanoparticles such as silver, gold, copper, titanium oxide, and zinc oxide [14, 15].

Zinc is an essential trace element for human system without which many enzymes such as carbonic anhydrase, carboxypeptidase, and alcohol dehydrogenase become inactive, while the other two members, cadmium and mercury belonging to the same group of elements having the same electronic configuration, are toxic. It is essential for eukaryotes because it modulates many physiological functions [16, 17]. Bamboo salt, containing zinc, is used as herbal medicine for the treatment of inflammation by regulating caspase-1 activity. Zinc oxide nanoparticles have been shown to reduce mRNA expression of inflammatory cytokines by inhibiting the activation of NF-kB (nuclear factor kappa B cells) [18].

Globally, bacterial infections are recognized as serious health issue. New bacterial mutation, antibiotic resistance, outbreaks of pathogenic strains, etc. are increasing, and thus, development of more efficient antibacterial agents is demand of the time. Zinc oxide is known for its antibacterial properties from the time immemorial [19]. It had been in use during the regime of Pharaohs, and historical records show that zinc oxide was used in many ointments for the treatment of injuries and boils even in BC [20]. It is still used in sun screen lotion, as a supplement, photoconductive material, LED, transparent transistors, solar cells, memory devices [21, 22], cosmetics [23, 24], and catalysis [25]. Although considerable amount of ZnO is produced every year, very small quantity is used as medicine [26]. The US Food and Drug Administration has recognized (21 CFR ) zinc oxide as safe [27]. It is characterized by photocatalytic and photooxidizing properties against biochemicals [28].

Zinc oxide has been classified by EU hazard classification as N; R (ecotoxic). Compounds of zinc are ecotoxic for mammals and plants in traces [29, 30]. Human body contains about 2–3 g of zinc, and the daily requirement is 10–15 mg [29, 31]. No report has demonstrated carcinogenicity, genotoxicity, and reproduction toxicity in humans [29, 32]. However, zinc powder inhaled or ingested may produce a condition called zinc fever, which is followed by chill, fever, cough, etc.

Morphology of zinc oxide nanoparticles depends on the process of synthesis. They may be nanorods, nanoplates [33,34,35], nanospheres [36], nanoboxes [35], hexagonal, tripods [37], tetrapods [38], nanowires, nanotubes, nanorings [39,40,41], nanocages, and nanoflowers [42, 43]. Zinc oxide nanoparticles are more active against gram-positive bacteria relative to other NPs of the same group of elements. Ready to eat food is more prone to infection by Salmonella, Staphylococcus aureus, and E. coli which pose a great challenge to food safety and quality. The antimicrobial compounds are incorporated in the packed food to prevent them from damage. Antimicrobial packaging contains a nontoxic material which inhibits or slows down the growth of microbes present in food or packaging material [44]. An antimicrobial substance for human consumption must possess the following properties.

  1. a)

    It should be nontoxic.

  2. b)

    It should not react with food or container.

  3. c)

    It should be of good taste or tasteless.

  4. d)

    It should not have disagreeable smell.

Zinc oxide nanoparticle is one such inorganic metal oxide which fulfills all the above requirements, and hence, it can safely be used as medicine, preservative in packaging, and an antimicrobial agent [45, 46]. It easily diffuses into the food material, kill the microbes, and prevent human being from falling ill. In accordance with the regulations //EC and //EC of the European Union, active packaging is defined as active material in contact with food with ability to change the composition of the food or the atmosphere around it [47]. Therefore, it is commonly used as preservative and incorporated in polymeric packaging material to prevent food material from damage by microbes [48]. Zinc oxide nanoparticles have been used as an antibacterial substance against Salmonella typhi and S. aureus in vitro. Of all the metal oxide nanoparticles studied thus far, zinc oxide nanoparticles exhibited the highest toxicity against microorganisms [49]. It has also been demonstrated from SEM and TEM images that zinc oxide nanoparticles first damage the bacterial cell wall, then penetrate, and finally accumulate in the cell membrane. They interfere with metabolic functions of the microbes causing their death. All the characteristics of the zinc oxide nanoparticles depend on their particle size, shape, concentration, and exposure time to the bacterial cell. Further, biodistribution studies of zinc oxide nanoparticles have also been examined. For instance, Wang et al. [50] have investigated the effect of long-term exposure of zinc oxide nanoparticle on biodistribution and zinc metabolism in mice over 3 to 35 weeks. Their results showed minimum toxicity to mice when they were exposed to 50 and  mg/kg zinc oxide nanoparticle in diet. At higher dose of  mg/kg, zinc oxide nanoparticle decreased body weight but increased the weight of the pancreas, brain, and lung. Also, it increased the serum glutamic-pyruvic transaminase activity and mRNA expression of zinc metabolism-related genes such as metallothionein. Biodistribution studies showed the accumulation of sufficient quantity of zinc in the liver, pancreas, kidney, and bones. Absorption and distribution of zinc oxide nanoparticle/zinc oxide microparticles are largely dependent on the particle size. Li et al. [51] have studied biodistribution of zinc oxide nanoparticles fed orally or through intraperitoneal injection to 6 weeks old mice. No obvious adverse effect was detected in zinc oxide nanoparticles orally treated mice in 14 days study. However, intraperitoneal injection of  g/kg body weight given to mice showed accumulation of zinc in the heart, liver, spleen, lung, kidney, and testes. Nearly ninefold increase in zinc oxide nanoparticle in the liver was observed after 72 h. Zinc oxide nanoparticles have been shown to have better efficiency in liver, spleen, and kidney biodistribution than in orally fed mice. Since zinc oxide nanoparticles are innocuous in low concentrations, they stimulate certain enzymes in man and plants and suppress diseases. Singh et al. [52] have also been recently reviewed the biosynthesis of zinc oxide nanoparticle, their uptake, translocation, and biotransformation in plant system.

In this review, we have attempted to consolidate all the information regarding zinc oxide nanoparticles as antibacterial agent. The mechanism of interaction of zinc oxide nanoparticles against a variety of microbes has also been discussed in detail.

Antimicrobial Activity of Zinc Oxide Nanoparticles

It is universally known that zinc oxide nanoparticles are antibacterial and inhibit the growth of microorganisms by permeating into the cell membrane. The oxidative stress damages lipids, carbohydrates, proteins, and DNA [53]. Lipid peroxidation is obviously the most crucial that leads to alteration in cell membrane which eventually disrupt vital cellular functions [54]. It has been supported by oxidative stress mechanism involving zinc oxide nanoparticle in Escherichia coli [55]. However, for bulk zinc oxide suspension, external generation of H2O2 has been suggested to describe the anti-bacterial properties [56]. Also, the toxicity of nanoparticles, releasing toxic ions, has been considered. Since zinc oxide is amphoteric in nature, it reacts with both acids and alkalis giving Zn2+ ions.

The free Zn2+ ions immediately bind with the biomolecules such as proteins and carbohydrates, and all vital functions of bacteria cease to continue. The toxicity of zinc oxide, zinc nanoparticles, and ZnSO4·7H2O has been tested (Table 1) against Vibrio fischeri. It was found that ZnSO4·7H2O is six times more toxic than zinc oxide nanoparticles and zinc oxide. The nanoparticles are actually dispersed in the solvent, not dissolved, and therefore, they cannot release Zn2+ ions. The bioavailability of Zn2+ ions is not always % and may invariably change with physiological pH, redox potential, and the anions associated with it such as Cl or SO42−.

Full size table

Solubility of zinc oxide (– mg/L) in aqueous medium is higher than that of zinc oxide nanoparticles (– mg/L) in the same medium [57] which is toxic to algae and crustaceans. Both nano-zinc oxide and bulk zinc oxide are 40–fold less toxic than ZnSO4 against V. fischeri. The higher antibacterial activity of ZnSO4 is directly proportional to its solubility releasing Zn2+ ions, which has higher mobility and greater affinity [58] toward biomolecules in the bacterial cell due to positive charge on the Zn2+ and negative charge on the biomolecules.

Since zinc oxide and its nanoparticles have limited solubility, they are less toxic to the microbes than highly soluble ZnSO4·7H2O. However, it is not essential for metal oxide nanoparticles to enter the bacterial cell to cause toxicity [59]. Contact between nanoparticles and the cell wall is sufficient to cause toxicity. If it is correct, then large amounts of metal nanoparticles are required so that the bacterial cells are completely enveloped and shielded from its environment leaving no chance for nutrition to be absorbed to continue life process. Since nanoparticles and metal ions are smaller than the bacterial cells, it is more likely that they disrupt the cell membrane and inhibit their growth.

A number of nanosized metal oxides such as ZnO, CuO, Al2O3, La2O3, Fe2O3, SnO2, and TiO2 have been shown to exhibit the highest toxicity against E. coli [49]. Zinc oxide nanoparticles are externally used for the treatment of mild bacterial infections, but the zinc ion is an essential trace element for some viruses and human beings which increase enzymatic activity of viral integrase [45, 60, 61]. It has also been supported by an increase in the infectious pancreatic necrosis virus by % when treated with 10 mg/L of Zn [46]. It may be due to greater solubility of Zn ions relative to ZnO alone. The SEM and TEM images have shown that zinc oxide nanoparticles damage the bacterial cell wall [55, 62] and increase permeability followed by their accumulation in E. coli preventing their multiplication [63].

In the recent past, antibacterial activity of zinc oxide nanoparticle has been investigated against four known gram-positive and gram-negative bacteria, namely Staphylococcus aureus, E. coli, Salmonella typhimurium, and Klebsiella pneumoniae. It was observed that the growth-inhibiting dose of the zinc oxide nanoparticles was 15 μg/ml, although in the case of K. pneumoniae, it was as low as 5 μg/ml [63, 64]. It has been noticed that with increasing concentration of nanoparticles, growth inhibition of microbes increases. When they were incubated over a period of 4–5 h with a maximum concentration of zinc oxide nanoparticles of 45 μg/ml, the growth was strongly inhibited. It is expected that if the incubation time is increased, the growth inhibition would also increase without much alteration in the mechanism of action [63].

It has been reported that the metal oxide nanoparticles first damage the bacterial cell membrane and then permeate into it [64]. It has also been proposed that the release of H2O2 may be an alternative to anti-bacterial activity [65]. This proposal, however, requires experimental proof because the mere presence of zinc oxide nanoparticle is not enough to produce H2O2. Zinc nanoparticles or zinc oxide nanoparticles of extremely low concentration cannot cause toxicity in human system. Daily intake of zinc via food is needed to carry out the regular metabolic functions. Zinc oxide is known to protect the stomach and intestinal tract from damage by E. coli [65]. The pH in the stomach varies between 2 to 5, and hence, zinc oxide in the stomach can react with acid to produce Zn2+ ions. They can help in activating the enzyme carboxy peptidase, carbonic anhydrase, and alcohol dehydrogenase which help in the digestion of carbohydrate and alcohol. Premanathan et al. [66] have reported the toxicity of zinc oxide nanoparticles against prokaryotic and eukaryotic cells. The MIC of zinc oxide nanoparticles against E. coli, Pseudomonas aeruginosa, and S. aureus were found to be and  μg/ml, respectively. Two mechanisms of action have been proposed for the toxicity of zinc oxide nanoparticles, namely (1) generation of ROS and (2) induction of apoptosis. Metal oxide nanoparticles induce ROS production and put the cells under oxidative stress causing damage to cellular components, i.e., lipids, proteins, and DNA [67,68,69]. Zinc oxide nanoparticles, therefore, induce toxicity through apoptosis. They are relatively more toxic to cancer cells than normal cells, although they cannot distinguish between them.

Recently, Pati et al. [70] have shown that zinc oxide nanoparticles disrupt bacterial cell membrane integrity, reduce cell surface hydrophobicity, and downregulate the transcription of oxidative stress-resistance genes in bacteria. They enhance intracellular bacterial killing by inducing ROS production. These nanoparticles disrupt biofilm formation and inhibit hemolysis by hemolysin toxin produced by pathogens. Intradermal administration of zinc oxide nanoparticles was found to significantly reduce the skin infection and inflammation in mice and also improved infected skin architecture.

Solubility and Concentration-Dependent Activity of Zinc Oxide Nanoparticle

Nanoparticles have also been used as a carrier to deliver therapeutic agents to treat bacterial infection [1, 9]. Since zinc oxide nanoparticles up to a concentration of  μg/ml are harmless to normal body cells, they can be used as an alternative to antibiotics. It was found that 90% bacterial colonies perished after exposing them to a dose of – μg/ml of zinc oxide nanoparticles only for 6 h. Even the drug-resistant S. aureus, Mycobacterium smegmatis, and Mycobacterium bovis when treated with zinc oxide nanoparticles in combination with a low dose of anti-tuberculosis drug, rifampicin ( μg/ml), a significant reduction in their growth was observed. These pathogens were completely destroyed when incubated for 24 h with  μg/ml of zinc oxide nanoparticles. It is, therefore, concluded that if the same dose is repeated, the patient with such infective diseases may be completely cured. It was also noted that the size of zinc oxide nanoparticles ranging between 50 and  nm have identical effect on bacterial growth inhibition.

Cytotoxicity of zinc oxide has been studied by many researchers in a variety of microbes and plant systems [71,72,73,74]. Toxicity of zinc oxide nanoparticles is concentration and solubility dependent. It has been shown that maximum exposure concentration of zinc oxide ( mg/l) suspension released  mg/l of Zn2+ ions. Toxicity is a combined effect of zinc oxide nanoparticles and Zn2+ ions released in the aqueous medium. However, minimal effect of metal ions was detected which suggests that the bacterial growth inhibition is mainly due to interaction of zinc oxide nanoparticles with microorganisms. The cytotoxic effect of a particular metal oxide nanoparticle is species sensitive which is reflected by the growth inhibition zone for several bacteria [75].

It has been suggested that growth inhibition of bacterial cells occurs mainly by Zn2+ ions which are produced by extracellular dissolution of zinc oxide nanoparticles [76]. Cho et al. [77] have concluded from their studies on rats that zinc oxide nanoparticles remain intact at around neutral or biological pH but rapidly dissolve under acidic conditions (pH ) in the lysosome of the microbes leading to their death. This is true because in acidic condition, zinc oxide dissolves and Zn2+ ions are produced, which bind to the biomolecules inside the bacterial cell inhibiting their growth.

The zinc oxide nanoparticles have been shown to be cytotoxic to different primary immune-competent cells. The transcriptomics analysis showed that nanoparticles had a common gene signature with upregulation of metallothionein genes ascribed to the dissolution of the nanoparticles [78]. However, it could not be ascertained if the absorbed zinc was Zn2+ or zinc oxide or both, although smaller sized zinc oxide nanoparticles have greater concentration in the blood than larger ones (19 and >  nm). The efficiency of zinc oxide nanoparticles depends mainly on the medium of reaction to form Zn2+ and their penetration into the cell.

Chiang et al. [79] have reported that dissociation of zinc oxide nanoparticles results in destruction of cellular Zn homeostasis. The characteristic properties of nanoparticles and their impact on biological functions are entirely different from those of the bulk material [80]. Aggregation of nanoparticles influences cytotoxicity of macrophages, and their concentration helps in modulation of nanoparticle aggregation. Low concentration of zinc oxide nanoparticles is ineffective, but at higher concentration ( μg/ml), they exhibited cytotoxicity which varies from one pathogen to another.

The inadvertent use of zinc oxide nanoparticles may sometime adversely affect the living system. Their apoptosis and genotoxic potential in human liver cells and cellular toxicity has been studied. It was found that a decrease in liver cell viability occurs when they are exposed to 14–20 μg/ml of zinc oxide nanoparticles for 12 h. It also induced DNA damage by oxidative stress. Sawai et al. [56] have demonstrated that ROS generation is directly proportional to the concentration of zinc oxide powder. ROS triggered a decrease in mitochondria membrane potential leading to apoptosis [81]. Cellular uptake of nanoparticles is not mandatory for cytotoxicity to occur.

Size-Dependent Antibacterial Activity of Zinc Oxide Nanoparticles

In a study, Azam et al. [82] have reported that the antimicrobial activity against both gram-negative (E. coli and P. aeruginosa) and gram-positive (S. and Bacillus subtilis) bacteria increased with increase in surface-to-volume ratio due to a decrease in particle size of zinc oxide nanoparticles. Moreover, in this investigation, zinc oxide nanoparticles have shown maximum (25 mm) bacterial growth inhibition against B. subtilis (Fig. 1).

Antibacterial activity and/or zone of inhibition produced by zinc oxide nanoparticles against gram-positive and gram-negative bacterial strains namely aEscherichia coli, bStaphylococcus aureus, cPseudomonas aeruginosa, and dBacillus subtilis [82]

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It has been reported that the smaller size of zinc oxide nanoparticles exhibits greater antibacterial activity than microscale particles [83]. For instance, Au55 nanoparticles of nm size have been demonstrated to interact with the major grooves of DNA which accounts for its toxicity [84]. Although contradictory results have been reported, many workers showed positive effect of zinc oxide nanoparticles on bacterial cells. However, Brayner et al. [63] from TEM images have shown that zinc oxide nanoparticle of 10–14 nm were internalized (when exposed to microbes) and damaged the bacterial cell membrane. It is also essential that the zinc/zinc oxide nanoparticles must not be toxic to human being since they are toxic to T cells above 5 mM [85] and to neuroblastoma cells above  mM [86]. Nair et al. [87] have exclusively explored the size effect of zinc oxide nanoparticles on bacterial and human cell toxicity. They have studied the influence of zinc oxide nanoparticles on both gram-positive and gram-negative bacteria and osteoblast cancer cell lines (MG).

It is known that antibacterial activity of zinc oxide nanoparticle is inversely proportional to their size and directly proportional to their concentration [88]. It has also been noticed that it does not require UV light for activation; it functions under normal or even diffused sunlight. Cytotoxic activity perhaps involves both the production of ROS and accumulation of nanoparticles in the cytoplasm or on the outer cell membrane. However, the production of H2O2 and its involvement in the activation of nanoparticles cannot be ignored. Raghupathi et al. [88] have synthesized zinc oxide nanoparticles from different zinc salts and observed that nanoparticles obtained from Zn(NO3)2 were smallest in size (12 nm) and largest in surface area (). Authors have shown that the growth inhibition of S. aureus at a concentration of 6 mM of zinc oxide nanoparticles is size dependent. It has also been indicated from the viable cell determination during the exposure of bacterial cells to zinc oxide nanoparticles that the number of cells recovered decreased significantly with decrease in size of zinc oxide nanoparticles. Jones et al. [89] have shown that zinc oxide nanoparticles of 8-nm diameter inhibited the growth of S. aureus, E. coli, and B. subtilis. Zinc oxide nanoparticles ranging between 12 and  nm were selected and confirmed the relationship between antibacterial activity and their size. Their toxicity to microbes has been ascribed to the formation of Zn2+ ions from zinc oxide when it is suspended in water and also to some extent to a slight change in pH. Since Zn2+ ions are scarcely released from zinc oxide nanoparticles, the antibacterial activity is mainly owing to smaller zinc oxide nanoparticles. When the size is 12 nm, it inhibits the growth of S. aureus, but when the size exceeds  nm, the inhibitory effect is minimal [89].

Shape, Composition, and Cytotoxicity of Zinc Oxide Nanoparticles

Zinc oxide nanoparticles have shown cytotoxicity in concentration-dependent manner and type of cells exposed due to different sensitivity [90, 91]. Sahu et al. [90] have highlighted the difference of cytotoxicity between particle size and different sensitivity of cells toward the particles of the same composition. In another recent study, Ng et al. [91] examined the concentration-dependent cytotoxicity in human lung MRC5 cells. Authors have reported the uptake and internalization of zinc oxide nanoparticles into the human lung MRC5 cells by using TEM investigation. These particles were noticed in the cytoplasm of the cells in the form of electron dense clusters, which are further observed to be enclosed by vesicles, while zinc oxide nanoparticles were not found in untreated control cells. Papavlassopoulos et al. [92] have synthesized zinc oxide nanoparticle tetrapods by entirely a novel route known as “Flame transport synthesis approach”. Tetrapods have different morphology compared to the conventionally synthesized zinc oxide nanoparticles. Their interaction with mammalian fibroblast cells in vitro has indicated that their toxicity is significantly lower than those of the spherical zinc oxide nanoparticles. Tetrapods exhibited hexagonal wurtzite crystal structure with alternating Zn2+ and O2− ions with three-dimensional geometry. They block the entry of viruses into living cells which is further enhanced by precisely illuminating them with UV radiation. Since zinc oxide tetrapods have oxygen vacancies in their structure, the Herpes simplex viruses are attached via heparan sulfate and denied entry into body cells. Thus, they prevent HSV-1 and HSV-2 infection in vitro. Zinc oxide tetrapods may therefore be used as prophylactic agent against these viral infections. The cytotoxicity of zinc oxide nanoparticles also depends on the proliferation rate of mammalian cells [66, 93]. The surface reactivity and toxicity may also be varied by controlling the oxygen vacancy in zinc oxide tetrapods. When they are exposed to UV light, the oxygen vacancy in tetrapods is readily increased. Alternatively, the oxygen vacancy can be decreased by heating them in oxygen-rich environment. Thus, it is the unique property of zinc oxide tetrapods that can be changed at will which consequently alter their antimicrobial efficiency.

Animal studies have indicated an increase in pulmonary inflammation, oxidative stress, etc. on respiratory exposure to nanoparticles [94]. Yang et al. [95] have investigated the cytotoxicity, genotoxicity, and oxidative stress of zinc oxide nanoparticles on primary mouse embryo fibroblast cells. It was observed that zinc oxide nanoparticles induced significantly greater cytotoxicity than that induced by carbon and SiO2 nanoparticles. It was further confirmed by measuring glutathione depletion, malondialdehyde production, superoxide dismutase inhibition, and ROS generation. The potential cytotoxic effects of different nanoparticles have been attributed to their shape.

Polymer-Coated Nanoparticles

Many bacterial infections are transmitted by contact with door knobs, key boards, water taps, bath tubs, and telephones; therefore, it is essential to develop and coat such surfaces with inexpensive advanced antibacterial substances so that their growth is inhibited. It is important to use such concentrations of antibacterial substances that they may kill the pathogens but spare the human beings. It may happen only if they are coated with a biocompatible hydrophilic polymer of low cost. Schwartz et al. [96] have reported the preparation of a novel antimicrobial composite material hydrogel by mixing a biocompatible poly (N-isopropylacrylamide) with zinc oxide nanoparticles. The SEM image of the composite film showed uniform distribution of zinc oxide nanoparticles. It exhibited antibacterial activity against E. coli at a very low zinc oxide concentration ( mM). Also, the coating was found to be nontoxic toward mammalian cell line (N1H/3T3) for a period of 1 week. Zinc oxide/hydrogel nanocomposite may safely be used as biomedical coating to prevent people from contracting bacterial infections.

Although zinc oxide nanoparticles are stable, they have been further stabilized by coating them with different polymers such as polyvinyl pyrolidone (PVP), polyvinyl alcohol (PVA), poly (α, γ, l-glutamic acid) (PGA), polyethylene glycol (PEG), chitosan, and dextran [97, 98]. The antibacterial activity of engineered zinc oxide nanoparticles was examined against gram-negative and gram-positive pathogens, namely E. coli and S. aureus and compared with commercial zinc oxide powder. The polymer-coated spherical zinc oxide nanoparticles showed maximum bacterial cell destruction compared to bulk zinc oxide powder [99]. Since nanoparticles coated with polymers are less toxic due to their low solubility and sustained release, their cytotoxicity can be controlled by coating them with a suitable polymer.

Effect of Particle Size and Shape of Polymer-Coated Nanoparticles on Antibacterial Activity

E. coli and S. aureus exposed to different concentrations of poly ethylene glycol (PEG)-coated zinc oxide nanoparticles (1–7 mM) of varying size ( nm– μm) showed that the antimicrobial activity increases with decreasing size and increasing concentration of nanoparticles. However, the effective concentration in all these cases was above 5 mM. There occurs a drastic change in cell morphology of E. coli surface which can be seen from the SEM images of bacteria before and after their exposure to zinc oxide nanoparticles [84]. It has been nicely demonstrated by Nair et al. [87] that PEG-capped zinc oxide particles and zinc oxide nanorods are toxic to human osteoblast cancer cells (MG) at concentration above  μM. The PEG starch-coated nanorods/nanoparticles do not damage the healthy cells.

In Vivo and In Vitro Antimicrobial Activity for Wound Dressing

Of all natural and synthetic wound dressing materials, the chitosan hydrogel microporous bandages laced with zinc oxide nanoparticles developed by Kumar et al. [] are highly effective in treating burns, wounds, and diabetic foot ulcers. The nanoparticles of approximately 70– nm are dispersed on the surface of the bandage. The degradation products of chitosan were identified as d-glucosamine and glycosamine glycan. They are nontoxic to the cells because they are already present in our body for the healing of injury. The wound generally contains P. aeruginosa, S. intermedicus, and S. hyicus which were also identified from the swab of mice wound and successfully treated with chitosan zinc oxide bandage in about 3 weeks [].

Effect of Doping on Toxicity of Zinc Oxide Nanoparticles

Doping of zinc oxide nanoparticles with iron reduces the toxicity. The concentration of Zn2+ and zinc oxide nanoparticles is also an important factor for toxicity. The concentration that reduced 50% viability in microbial cells exposed to nano- and microsize zinc oxide is very close to the concentration of Zn2+ that induced 50% reduction in viability in Zn2+-treated cells [, ].

Coating of zinc oxide nanoparticles with mercaptopropyl trimethoxysilane or SiO2 reduces their cytotoxicity []. On the contrary, Gilbert et al. [] showed that in BEAS-2B cells, uptake of zinc oxide nanoparticles is the main mechanism of zinc accumulation. Also, they have suggested that zinc oxide nanoparticles dissolve completely generating Zn2+ ions which are bonded to biomolecules of the target cells. However, the toxicity of zinc oxide nanoparticles depends on the uptake and their subsequent interaction with target cells.

Interaction Mechanism of Zinc Oxide Nanoparticles

Nanoparticles may be toxic to some microorganisms, but they may be essential nutrients to some of them [55, ]. Nanotoxicity is essentially related to the microbial cell membrane damage leading to the entry of nanoparticles into the cytoplasm and their accumulation [55]. The impact of nanoparticles on the growth of bacteria and viruses largely depends on particle size, shape, concentration, agglomeration, colloidal formulation, and pH of the media [,,]. The mechanism of antimicrobial activity of zinc oxide nanoparticles has been depicted in Fig. 2.

Mechanisms of zinc oxide nanoparticle antimicrobial activity

Full size image

Zinc oxide nanoparticles are generally less toxic than silver nanoparticles in a broad range of concentrations (20 to  mg/l) with average particle size of  nm [55, 62, 63]. Metal oxide nanoparticles damage the cell membrane and DNA [63, ,,] of microbes via diffusion. However, the production of ROS through photocatalysis causing bacterial cell death cannot be ignored []. UV-Vis spectrum of zinc oxide nanoparticle suspension in aqueous medium exhibits peaks between and  nm []. It has been shown that it produces ROS (hydroxyl radicals, superoxides, and hydrogen peroxide) in the presence of moisture which ostensibly react with bacterial cell material such as protein, lipids, and DNA, eventually causing apoptosis. Xie et al. [] have examined the influence of zinc oxide nanoparticles on Campylobacter jejuni cell morphology using SEM images (Fig. 3). After a h treatment ( mg/ml), C. jejuni was found to be extremely sensitive and cells transformed from spiral shape to coccoid forms. SEM studies showed the ascendency of coccoid forms in the treated cells and display the formation of irregular cell surfaces and cell wall blebs (Fig. 3a). Moreover, these coccoid cells remained intact and possessed sheathed polar flagella. However, SEM image of the untreated cells clearly showed spiral shapes (Fig. 3b). In general, it has been demonstrated from SEM and TEM images of bacterial cells treated with zinc oxide nanoparticles that they get ruptured and, in many cases, the nanoparticles damage the cell wall forcing their entry into it [, ].

SEM images of Campylobacter jejuni. a Untreated cells from the same growth conditions were used as a control. bC. jejuni cells in the mid-log phase of growth were treated with  mg/ml of zinc oxide nanoparticles for 12 h under microaerobic conditions []

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Zinc oxide nanoparticles have high impact on the cell surface and may be activated when exposed to UV-Vis light to generate ROS (H2O2) which permeate into the cell body while the negatively charged ROS species such as O22− remain on the cell surface and affect their integrity [, ]. Anti-bacterial activity of zinc oxide nanoparticles against many other bacteria has also been reported [1, 5, , ]. It has been shown from TEM images that the nanoparticles have high impact on the cell surface (Fig. 4).

a TEM images of untreated normal Salmonella typhimurium cells. b Effects of nanoparticles on the cells (marked with arrows). c, d Micrograph of deteriorated and ruptured S. typhimurium cells treated with zinc oxide nanoparticles []

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Sinha et al. [] have also shown the influence of zinc oxide nanoparticles and silver nanoparticles on the growth, membrane structure, and their accumulation in cytoplasm of (a) mesophiles: Enterobacter sp. (gram negative) and B. subtilis (gram positive) and (b) halophiles: halophilic bacterium sp. (gram positive) and Marinobacter sp. (gram negative). Nanotoxicity of zinc oxide nanoparticles against halophilic gram-negative Marinobacter species and gram-positive halophilic bacterial species showed 80% growth inhibition. It was demonstrated that zinc oxide nanoparticles below 5 mM concentration are ineffective against bacteria. The bulk zinc oxide also did not affect the growth rate and viable counts, although they showed substantial decrease in these parameters. Enterobacter species showed dramatic alterations in cell morphology and reduction in size when treated with zinc oxide.

TEM images shown by Akbar and Anal [] revealed the disrupted cell membrane and accumulation of zinc oxide nanoparticles in the cytoplasm (Fig. 4) which was further confirmed by FTIR, XRD, and SEM. It has been suggested that Zn2+ ions are attached to the biomolecules in the bacterial cell via electrostatic forces. They are actually coordinated with the protein molecules through the lone pair of electrons on the nitrogen atom of protein part. Although there is significant impact of zinc oxide nanoparticles on both the aquatic and terrestrial microorganisms and human system, it is yet to be established whether it is due to nanoparticles alone or is a combined effect of the zinc oxide nanoparticles and Zn2+ ions [55, , , ]. Antibacterial influence of metal oxide nanoparticles includes its diffusion into the bacterial cell, followed by release of metal ions and DNA damage leading to cell death [63, ,,]. The generation of ROS through photocatalysis is also a reason of antibacterial activity [62, ]. Wahab et al. [] have shown that when zinc oxide nanoparticles are ingested, their surface area is increased followed by increased absorption and interaction with both the pathogens and the enzymes. Zinc oxide nanoparticles can therefore be used in preventing the biological system from infections. It is clear from TEM images (Fig. 5a, b) of E. coli incubated for 18 h with MIC of zinc oxide nanoparticles that they had adhered to the bacterial cell wall. The outer cell membrane was ruptured leading to cell lysis. In some cases, the cell cleavage of the microbes has not been noticed, but the zinc oxide nanoparticles can yet be seen entering the inner cell wall (Fig. 5c, d). As a consequence of it, the intracellular material leaks out leading to cell death, regardless of the thickness of bacterial cell wall.

TEM images of Escherichia coli (a), zinc oxide nanoparticles with E. coli at different stages (b and inset), Klebsiella pneumoniae (c), and zinc oxide nanoparticles with K. pneumoniae (d and inset) []

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Mechanism of interaction of zinc oxide nanoparticles with bacterial cells has been outlined below []. Zinc oxide absorbs UV-Vis light from the sun and splits the elements of water.

Dissolved oxygen molecules are transformed into superoxide, O2, which in turn reacts with H+ to generate HO2 radical and after collision with electrons produces hydrogen peroxide anion, HO2. They subsequently react with H+ ions to produce H2O2.

It has been suggested that negatively charged hydroxyl radicals and superoxide ions cannot penetrate into the cell membrane. The free radicals are so reactive that they cannot stay in free and, therefore, they can either form a molecule or react with a counter ion to give another molecule. However, it is true that zinc oxide can absorb sun light and help in cleaving water molecules which may combine in many ways to give oxygen. Mechanism of oxygen production in the presence of zinc oxide nanoparticles still needs experimental evidence.

Zinc oxide at a dose of 5 μg/ml has been found to be highly effective for all the microorganisms which can be taken as minimum inhibitory dose.

Conclusions

Zinc is an indispensable inorganic element universally used in medicine, biology, and industry. Its daily intake in an adult is 8–15 mg/day, of which approximately 5–6 mg/day is lost through urine and sweat. Also, it is an essential constituent of bones, teeth, enzymes, and many functional proteins. Zinc metal is an essential trace element for man, animal, plant, and bacterial growth while zinc oxide nanoparticles are toxic to many fungi, viruses, and bacteria. People with inherent genetic deficiency of soluble zinc-binding protein suffer from acrodermatitis enteropathica, a genetic disease indicated by python like rough and scaly skin. Although conflicting reports have been received about nanoparticles due to their inadvertent use and disposal, some metal oxide nanoparticles are useful to men, animals, and plants. The essential nutrients become harmful when they are taken in excess. Mutagenic potential of zinc oxide has not been thoroughly studied in bacteria even though DNA-damaging potential has been reported. It is true that zinc oxide nanoparticles are activated by absorption of UV light without disturbing the other rays. If zinc oxide nanoparticles produce ROS, they can damage the skin and cannot be used as sun screen. Antibacterial activity may be catalyzed by sunlight, but hopefully, it can prevent the formation of ROS. Zinc oxide nanoparticles and zinc nanoparticles coated with soluble polymeric material may be used for treating wounds, ulcers, and many microbial infections besides being used as drug carrier in cancer therapy. It has great potential as a safe antibacterial drug which may replace antibiotics in future. Application of zinc oxide nanoparticles in different areas of science, medicine, and technology suggests that it is an indispensable substance which is equally important to man and animals. However, longtime exposure with higher concentration may be harmful to living system.

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ANTIBACTERIAL ACTIVITY OF ZINC OXIDE NANOPARTICLES SYNTHESIZED BY SOLOCHEMICAL PROCESS

Kinetics and Catalysis, Reaction Engineering, and Materials Science • Braz. J. Chem. Eng. 36 (2) • Apr-Jun  • https://doi.org//scopy

ZnO-NPs can be obtained through various methods, resulting in nanoparticles with different size and morphology, which directly influences their antimicrobial potential. The objective of this work was to evaluate the antibacterial activity of ZnO-NPs obtained by a solochemical process against important human foodborne pathogens: Staphylococcus aureus, Salmonella Typhimurium, Bacillus cereus and Pseudomonas aeruginosa. ZnO-NPs were identified as nanorods with the length between and nm ( % frequency), the diameter between and 90 nm (21 % frequency), and wurtzite type crystalline structure. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were equal to mg mL-1 and mg mL-1 for S. aureus and S. Typhimurium, respectively, lower than previous results related in the literature. ZnO-NPs produced by solochemical method had a superior antibacterial activity. For instance, they can be incorporated in packaging materials for increasing microbial safety and food shelf-life by inhibiting bacterial growth.

Keywords:
Foodborne pathogens; Bacillus cereus; Staphylococcus aureus; Salmonella Typhimurium; Pseudomonas aeruginosa

The increasing concern about resistant microorganisms stimulates the study of new and more effective antimicrobial agents (Raghunath and Perumal, Raghunath, A., Perumal, E., Metal oxide nanoparticles as antimicrobial agents: a promise for the future. International Journal of Antimicrobial Agents, 49, (). https://doi.org//j.ijantimicag
https://doi.org//j.ijantimicag ; Tang and Lv, Tang, Z. X., Lv, B. F., MgO nanoparticles as antibacterial agent: Preparation and activity. Brazilian Journal of Chemical Engineering , 31, (). https://doi.org//s
https://doi.org// ). New agents can be obtained from biological sources, such as bacteriocins and essential oils, or synthesized organic/inorganic compounds (Han, Han, J. H., Antimicrobial food packaging. In Ahvenainen, R. (Ed.), Novel Food Packaging Techniques, p. , CRC Press, Boca Raton (). https://doi.org//
https://doi.org// ; Medeiros et al., Medeiros, G. R., Ferreira, S. R. S., Carciofi, B. A. M., High pressure carbon dioxide for impregnation of clove essential oil in LLDPE films. Innovative Food Science & Emerging Technologies, 41, (). https://doi.org//j.ifset
https://doi.org//j.ifset ; Medeiros et al., Medeiros, G. R., Guimarães, C., Ferreira, S. R. S., Carciofi, B. A. M., Thermomechanical and transport properties of LLDPE films impregnated with clove essential oil by high-pressure CO2. The Journal of Supercritical Fluids, , (). https://doi.org//j.supflu
https://doi.org//j.supflu ). Good antimicrobial effects have been obtained from metal oxide nanoparticles (Raghunath and Perumal, ), such as MgO (Tang et al., Tang, Z. X., Fang, X. J., Zhang, Z. L., Zhou, T., Zhang, X. Y., Shi, L. E., Nanosize MgO as antibacterial agent: Preparation and characteristics. Brazilian Journal of Chemical Engineering, 29, (). https://doi.org//S
https://doi.org//S ), Cu2O, CuO, ZnO, TiO2, and WO3 (Duffy et al., Duffy, L. L., Osmond-McLeod, M. J., Judy, J., King, T., Investigation into the antibacterial activity of silver, zinc oxide and copper oxide nanoparticles against poultry-relevant isolates of Salmonella and Campylobacter. Food Control, 92, (). https://doi.org//j.foodcont
https://doi.org//j.foodcont ; Vargas-Reus et al., Vargas-Reus, M. A., Memarzadeh, K., Huang, J., Ren, G. G., Allaker, R. P., Antimicrobial activity of nanoparticulate metal oxides against peri-implantitis pathogens. International Journal of Antimicrobial Agents , 40, (). https://doi.org//j.ijantimicag
https://doi.org//j.ijantimicag ), which present distinct behavior and properties from micrometric or millimetric particles (Azeredo, Azeredo, H. M. C., Antimicrobial nanostructures in food packaging. Trends in Food Science and Technology, 30, (). https://doi.org//j.tifs
https://doi.org//j.tifs ; Martinez-Gutierrez et al., Martinez-Gutierrez, F., Olive, P. L., Banuelos, A., Orrantia, E., Nino, N., Sanchez, E. M., Av-Gay, Y., Synthesis, characterization, and evaluation of antimicrobial and cytotoxic effect of silver and titanium nanoparticles. Nanomedicine: Nanotechnology, Biology, and Medicine, 6, (). https://doi.org//j.nano
https://doi.org//j.nano ; Morais and Durán, ).

Zinc oxide nanoparticles (ZnO-NPs) have been reported as an antimicrobial agent against both pathogenic and spoilage microorganisms (Ann et al., Ann, L. C., Mahmud, S., Bakhori, S. K. M., Sirelkhatim, A., Mohamad, D., Hasan, H., Rahman, R. A., Antibacterial responses of zinc oxide structures against Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pyogenes, Ceramics International, 40, (). https://doi.org//j.ceramint
https://doi.org//j.ceramint ; Duffy et al., Duffy, L. L., Osmond-McLeod, M. J., Judy, J., King, T., Investigation into the antibacterial activity of silver, zinc oxide and copper oxide nanoparticles against poultry-relevant isolates of Salmonella and Campylobacter. Food Control, 92, (). https://doi.org//j.foodcont
https://doi.org//j.foodcont ; Pasquet et al., Pasquet, J., Chevalier, Y., Couval, E., Bouvier, D., Noizet, G., Morlière, C., Bolzinger, M. A., Antimicrobial activity of zinc oxide particles on five micro-organisms of the Challenge Tests related to their physicochemical properties. International Journal of Pharmaceutics, , (). https://doi.org//j.ijpharm
https://doi.org//j.ijpharm ; Raghunath and Perumal, Raghunath, A., Perumal, E., Metal oxide nanoparticles as antimicrobial agents: a promise for the future. International Journal of Antimicrobial Agents, 49, (). https://doi.org//j.ijantimicag
https://doi.org//j.ijantimicag ; Raghupathi et al., Raghupathi, K. R., Koodali, R. T., Manna, A. C., Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir, 27, (). https://doi.org//lau
https://doi.org//lau ; Savi et al., Savi, G. D., Bortoluzzi, A. J., Scussel, V. M., Antifungal properties of Zinc-compounds against toxigenic fungi and mycotoxin. International Journal of Food Science and Technology, 48, (). https://doi.org//ijfs
https://doi.org//ijfs ; Vargas-Reus et al., Vargas-Reus, M. A., Memarzadeh, K., Huang, J., Ren, G. G., Allaker, R. P., Antimicrobial activity of nanoparticulate metal oxides against peri-implantitis pathogens. International Journal of Antimicrobial Agents , 40, (). https://doi.org//j.ijantimicag
https://doi.org//j.ijantimicag ; Xie et al., Xie, Y., He, Y., Irwin, P. L., Jin, T., Shi, X., Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Applied and Environmental Microbiology, 77, (). https://doi.org//AEM
https://doi.org//AEM ). ZnO-NPs application as antimicrobial agent stands out in comparison to other metallic nanoparticles (Jones et al., Jones, N., Ray, B., Ranjit, K. T., Manna, A. C. (). Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiology Letters, , (). https://doi.org//jx
https://doi.org//j ). Their main antimicrobial mechanisms have been attributed to the induction of oxidative stress due to the formation of reactive oxygen species, membrane disruption due to the accumulation of ZnO-NPs therein, and internalization of nanoparticles followed by the release of antimicrobial ions (Zn+2) (Raghunath and Perumal, ; Sirelkhatim et al., Sirelkhatim, A., Mahmud, S., Seeni, A., Kaus, N. H. M., Ann, L. C., Bakhori, S. K. M., Mohamad, D., Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Letters, 7, (). https://doi.org//sx
https://doi.org//s ). In addition to its unique antibacterial properties, ZnO is classified as a Generally Recognized as Safe (GRAS) compound by the U.S. Food and Drug Administration (FDA, FDA (Food and Drug Administration), Electronic Code of Federal Regulations. Title 21, Chapter I, Subchapter E, Part , Subpart F, § (). Last seen: October 10, , from Last seen: October 10, , from http://www.ecfr.gov/cgi-bin/text-idx?SID=a55fcd5afc41aaacddd5e5f57mc=truenode=se_rgn=div8
http://www.ecfr.gov/cgi-bin/text-idx?SID ).

ZnO-NPs can be synthesized through various methods by controlling the synthesis conditions, such as sol-gel (Kolekar et al., Kolekar, T. V, Yadav, H. M., Bandgar, S. S., Raskar, A. C., Rawal, S. G., Mishra, G. M., Synthesis By Sol-Gel Method And Characterization Of ZnO Nanoparticles. Indian Streams Research Journal, 1, ().), hydrothermal (Hu and Chen, Hu, Y., Chen, H.-J., Preparation and characterization of nanocrystalline ZnO particles from a hydrothermal process. Journal of Nanoparticle Research, 10, (). https://doi.org//s
https://doi.org//s ), co-precipitation (Zhong Matijević, Zhong, Q., Matijević, E., Preparation of uniform zinc oxide colloids by controlled double-jet precipitation. Journal of Material Chemistry, 6, (). https://doi.org//jm
https://doi.org//jm ) and solochemical (Vaezi, Vaezi, M. R., Two-step solochemical synthesis of ZnO/TiO2 nano-composite materials. Journal of Materials Processing Technology, , (). https://doi.org//j.jmatprotec
https://doi.org//j.jmatprotec ) methods. The solochemical process produces nanostructures of ZnO through the reaction between a precursor solution containing zinc and an alkaline solution. This method has significant advantages, such as synthesis under low temperatures, no addition of a stabilizing agent, short reaction time, low cost, and nanoparticles with controlled morphology and size (Gusatti et al., Gusatti, M., Rosário, J. A., Campos, C. E. M., Kuhnen, N. C., Carvalho, E. U., Riella, H. G., Bernardin, A. M., Production and Characterization of ZnO Nanocrystals Obtained by Solochemical Processing at Different Temperatures. Journal of Nanoscience and Nanotechnology , 10, (). https://doi.org//jnn
https://doi.org//jnn ). The synthesis method directly influences the morphology and size of NPs. In turn, functional activities (chemical, catalytic and biological) of NPs are significantly affected by the combination of their size, morphology, surface area, electronic states and surface charge (Jones et al., Jones, N., Ray, B., Ranjit, K. T., Manna, A. C. (). Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiology Letters, , (). https://doi.org//jx
https://doi.org//j ; Pasquet et al., Pasquet, J., Chevalier, Y., Couval, E., Bouvier, D., Noizet, G., Morlière, C., Bolzinger, M. A., Antimicrobial activity of zinc oxide particles on five micro-organisms of the Challenge Tests related to their physicochemical properties. International Journal of Pharmaceutics, , (). https://doi.org//j.ijpharm
https://doi.org//j.ijpharm ; Ramani et al., Ramani, M., Ponnusamy, S., Muthamizhchelvan, C., Marsili, E., Amino acid-mediated synthesis of zinc oxide nanostructures and evaluation of their facet-dependent antimicrobial activity. Colloids and Surfaces B: Biointerfaces , , (). https://doi.org//j.colsurfb
https://doi.org//j.colsurfb ). Therefore, the synthesis method must be selected to produce NPs with optimum functional activities for the desired application (Fan and Lu, Fan, Z., Lu, J. G., Zinc Oxide Nanostructures: Synthesis and Properties. Journal of Nanoscience and Nanotechnology, 5, (). https://doi.org//jnn
https://doi.org//jnn ; Gusatti et al., ; Sirelkhatim et al., Sirelkhatim, A., Mahmud, S., Seeni, A., Kaus, N. H. M., Ann, L. C., Bakhori, S. K. M., Mohamad, D., Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Letters, 7, (). https://doi.org//sx
https://doi.org//s ).

Controlling the growth of pathogenic microorganisms is of foremost importance for food safety and public health. Among others, some foodborne pathogenic bacteria have been receiving particular attention in the last decades. Pseudomonas aeruginosa is an ubiquitous environmental bacterial that is the major cause of opportunistic human infections. It is a common soil and water bacteria, widely distributed among fresh foods (Jay et al., Jay, J. M., Loessner, M. J., Golden, D. A. Modern Food Microbiology, 7a ed. New York: Springer. ().; Stover et al., Stover, C.-K., Pham, X.-Q., Erwin, A.-L., Mizoguchi, S.-D., Warrener, P., Hickey, M. J., Olson, M. V., Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature, , (). https://doi.org//
https://doi.org// ). Bacillus cereus is a pathogenic bacterium commonly isolated from soil and easily spread in the environment (Granum and Lindbäck, Granum, P. E., Lindbäck, T., Bacillus cereus. In Doyle, M. P., Buchanan, R. L. (Eds.), Food Microbiology: Fundamentals and Frontiers. 4th ed., p. , ASM Press, Washington, D.C (). ). Humans are the primary reservoir of S. aureus, and food contaminated during its preparation is the most significant source of staphylococcal food poisoning (Seo and Bohach, Seo, K. S., Bohach, G. A., Staphylococcus aureus. In Doyle, M. P., Buchanan, R. L. (Eds.), Food Microbiology: Fundamentals and Frontiers, 4th ed., p. , ASM Press, Washington, D.C ().). Salmonella spp. is a resilient bacterium capable of adapting to temperature, pH, and water activity beyond their normal growth range, posing high risks to safety (Li et al., Li, H., Wang, H., D’Aoust, J.-Y., Maurer, J. Salmonella Species. In Doyle, M. P., Buchanan, R. L. (Eds.), Food Microbiology: Fundamentals and Frontiers, 4th ed., p. , ASM Press Washington, D.C. ().).

The present study aimed to characterize ZnO-NPs synthesized via a solochemical technique and to evaluate their antibacterial activity against the above mentioned foodborne Gram-positive and Gram-negative pathogenic bacteria: S. aureus, B. cereus, S. Typhimurium and P. aeruginosa, and to obtain both minimum bactericidal and inhibitory concentrations for each of them by a proposed broth dilution method.

ZnO-NPs

ZnO-NPs were synthesized the by the solochemical method as described by Gusatti et al. (Gusatti, M., Rosário, J. A., Campos, C. E. M., Kuhnen, N. C., Carvalho, E. U., Riella, H. G., Bernardin, A. M., Production and Characterization of ZnO Nanocrystals Obtained by Solochemical Processing at Different Temperatures. Journal of Nanoscience and Nanotechnology , 10, (). https://doi.org//jnn
https://doi.org//jnn ) and kindly provided by Kher Nanotecnologia Química Ltda. (Santa Catarina, Brazil). Immediately before each test, ZnO-NPs suspensions in different concentrations were prepared by dispersing the ZnO-NPs in Milli-Q water using an ultrasonic bath (A, Unique) for 30 min followed by vortex mixing (AP56, Phoenix) for 5 s.

Characterization of ZnO-NPs

ZnO-NPs were characterized by transmission electron micrographs (TEM) (JEM TEM, kV) and X-ray diffraction (XRD) (X´Pert, Philips, The Netherlands). TEM analyses used an aqueous ZnO-NPs dispersion with mg mL-1 placed on a grid and kept at room temperature until complete solvent evaporation. A TEM image with whole ZnO-NPs was used to estimate the particle’s length and diameter with the help of specific software (ImageJv, Wayne Rasband, USA) for digital measurements. XRD analyses were done at 40 kV and 30 mA with CuK at Å wavelengths. The samples were analyzed in an interval of 2θ between 20° and 80° with increments of °/s.

Antibacterial activity

The antibacterial activity of ZnO-NPs was tested against Gram-positive bacteria, Bacillus cereus (ATCC ) and Staphylococcus aureus (ATCC ), and Gram-negative bacteria, Pseudomonas aeruginosa (ATCC ) and Salmonella Typhimurium (ATCC ). Stock cultures were prepared by inoculating the strains in Tryptone Soya Agar (TSA) (Himedia, India), followed by incubation at 35 °C for 24 h. These cultures were kept at 4 °C until the preparation of the working cultures. Working cultures were obtained by transferring a loopful from the stock culture into 5 mL of Brain Heart Infusion broth (BHI) (Oxoid, England) and incubating at 35 °C for 24 h.

Diffusion methods

Disk diffusion and agar well diffusion methods were accomplished aiming at a qualitative screening for bacteria susceptibility and to select the ZnO-NPs concentration for the broth dilution method. Disc diffusion tests started by swabbing the working cultures on the agar surface. Then, sterile discs of filter paper (9 mm diameter) were impregnated with 10 µL of sterile ZnO-NPs suspensions and placed onto the inoculated agar surface. For the well diffusion, the working cultures were pour plated. After the agar solidification, wells (5 mm diameter) were aseptically made and filled with 32 µL of sterile ZnO-NPs suspensions.

For both methods, Müeller-Hinton Agar (Kasvi, Italy) was used. Initial cell concentration was around 109 CFU mL-1, and aqueous ZnO-NPs suspensions ranged between and mg mL-1. Positive and negative control tests were ciprofloxacin ( mg mL-1) and Milli-Q water, respectively. After incubation at 35 °C for 48 h, the presence or absence of inhibition zones around the discs and wells were observed.

Broth dilution

Quantitative tests were performed in tubes with Nutrient Broth (NB) (Acumedia, USA) (5 mL) and ZnO-NPs at final concentrations of , , , and mg mL-1. Positive and negative control tests were ciprofloxacin ( mg mL-1) and Milli-Q water, respectively. The test tubes were inoculated with the working culture to reach an initial cell concentration around to 105CFU mL-1 and then incubated under shaking conditions (TECNAL, TE, Brazil) at 35 °C. After 0, 24 and 48 h of incubation, cell concentration was determined by serial dilution in peptone water ( %) followed by spread plating in Plate Count Agar (PCA) (Kasvi, Italy). Plates were incubated at 35 °C for 24 h. The Minimum Bactericidal Concentration (MBC) was defined as the lowest concentration at which no bacterial colonies were detected in the 10-1 dilution after 48 h incubation, while the Minimum Inhibitory Concentration (MIC) was the concentration at which the bacterial concentration after 48 h incubation was equal to the initial cell concentration.

Microbial growth curve

The bacterial growth curve of each strain was determined in media containing the respective MIC and MBC of ZnO-NPs suspensions. Sterile bottles with 50 mL of NB were inoculated with a loopful of the working culture and incubated at 35 °C for h to reach an initial cell concentration of 105CFU mL-1. Then, ZnO-NPs dispersions were added to the bottles at levels equal to the MIC and MBC of each bacterial strain. Positive and negative control tests were ciprofloxacin ( mg mL-1) and Milli-Q water, respectively. The bottles were incubated under agitation at 35 °C. After pre-determined incubation periods, cell concentration was determined by serial dilution in peptone water (%), followed by spread plating in Plate Count Agar (PCA) (Kasvi, Italy) and incubation at 35°C for 24 h.

ZnO-NPs characterization

XRD diffraction results (Figure 1) demonstrated a pattern that matched with the standard of ZnO provided by the Joint Committee on Powder Diffraction Standards (JCPDS). This result showed the hexagonal wurtzite structure of the studied ZnO-NPs, with spatial group P63mc and network parameters a = Å and c = , as specified in the card number. The nanoparticles showed a high purity level, as no peaks of any other phase were detected.

Figure 1
XRD patterns of present ZnO-NPs (top) and for standard ZnO (bottom) provided by Joint Committee on Powder Diffraction Standards (JCPDS), International Centre for Diffraction Data, card number

ZnO-NPs were predominantly rod-like shaped as depicted by the TEM image (Figure 2a), with varying length and diameter according to the frequency distribution histograms (Figures 2b and 2c). The nanoparticle average length and diameter were and nm, respectively. The histograms show that, for the established ranges, the nanoparticles were higher from to nm in length ( % frequency) and between and 90 nm in diameter (21 % frequency).

Figure 2
Characterization of the ZnO-NPs: (a) TEM images and frequency distribution histograms of the (b) length and (c) diameter.

Antibacterial activity

Diffusion in agar

For S. aureus, the inhibition halo was present in concentrations equal to and higher than and mg mL-1 for the disk and the agar well diffusion methods, respectively. For S. Typhimurium, the inhibition halo was present in concentrations equal to and higher than mg mL-1 for both methods. For P. aeruginosa, the inhibition halo was observed in concentrations equal to and higher than 1 mg mL-1 for the disk diffusion, and no evident inhibition was found in the agar well method, for which only some spots with an absence of growth were observed in concentrations equal to and higher than mg mL-1. For B. cereus, no inhibition zone was observed, even for the highest tested concentration of mg mL-1. This result suggests that either ZnO-NPs had no antibacterial effect against this bacterium or these methods are not suitable to detect the antibacterial effect of ZnO-NPs against B. cereus. The following analyses in broth media did not include tests for B. cereus. For all strains, negative controls showed no antibacterial effect, while positive controls had a clear inhibition zone.

Broth dilution

ZnO-NPs MIC was equal to mg mL-1 ( mM) for S. aureus (Table 1). A minor bactericidal effect was observed at the same concentration for S. Typhimurium. Despite this effect, mg mL-1 was considered as the MIC for S. Typhimurium since lower ZnO-NPs concentrations were not tested. The MBC for both S. Typhimurium and S. aureus was equal to mg mL-1 ( mM) as no viable cells were detected after 48 h.

Table 1
Log (N)/Log (N0)* for broth dilution tests at different ZnO-NPs concentrations after 48h incubation at 35°C. Positive control and negative controls were ciprofloxacin ( mg mL-1) and Milli-Q water, respectively.

P. aeruginosa growth was not affected by the maximum tested concentration of 2 mg mL-1 ( mM), and the bacterial concentration increased similarly to the negative control for all tested ZnO-NPs concentrations. Concentrations higher than 2 mg mL-1 were not tested as much ZnO precipitation was observed, hampering the results.

Microbial growth curve

After 24h, no viable cells of S. aureus (Figure 3) and S. Typhimurium (Figure 4) were detected when at MBC, while the negative control reached the maximum cell concentration of approximately 109 CFU mL-1. After 6h of incubation, ZnO-NPs at MBC reduced bacterial count in 38 and 61 % when comparing to the negative control for S. aureus and S. Typhimurium, respectively. It indicates that ZnO-NPs can be more effective against S. Typhimurium compared to S. aureus. The growth curves at MIC presented a decrease in the cell concentration in the first 24 h, followed by an increase, reaching concentrations close to the initial cell concentration for both microorganisms. This decrease in cell concentration was also observed for S. Typhimurium during the MIC estimation (Table 1).

Figure 3
S. aureus cell concentration [Log (N)] over time for (○) ZnO-NPs MIC ( mg mL-1), (▲) ZnO-NPs MBC ( mg mL-1), (♦) negative control (Milli-Q water) and (□) positive control (ciprofloxacin, mg mL-1). N is the cell concentration in CFU mL-1.

Figure 4
S. Typhimurium cell concentration [Log (N)] over time for (○) ZnO-NPs MIC ( mg mL-1), (▲) ZnO-NPs MBC ( mg mL-1), (♦) negative control (Milli-Q water) and (□) positive control (ciprofloxacin, mg mL-1). N is the cell concentration in CFU mL-1.

P. aeruginosa growth was not inhibited in ZnO-NPs concentrations up to 2 mg mL-1 (Figure 5). A minor effect of the ZnO-NPs can be observed at 48h as bacterial cell concentrations were lower than the negative control for all time points. After h, the cultures containing ZnO-NPs reached a cell concentration approximately 3 logs higher than the initial concentration, while the negative control was about times higher.

Figure 5
P. aeruginosa cell concentration [Log (N)] over time for ZnO-NPs at (▲) mg mL-1 (○) mg mL-1 and (■) mg mL-1, (♦) negative control (Milli-Q water) and (□) positive control (ciprofloxacin, mg mL-1). N is the cell concentration in CFU mL-1.

Agar diffusion tests were performed as a qualitative test to observe and predict the ZnO-NPs antibacterial behavior. These methods have many advantages over other methods, such as simplicity, low cost, the ability to test a high number of microorganisms and antimicrobial agents. However, it is not able to determine the MIC or MBC, as it is impossible to determine the diffusion of the antimicrobial agent in the agar (Balouiri et al., Balouiri, M., Sadiki, M., Ibnsouda, S. K., Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis, 6, (). https://doi.org//j.jpha
https://doi.org//j.jpha ). The broth media assay can be considered as confirmative and more accurate than the agar diffusion assay as the chances of nanoparticle-bacteria interactions are higher in the liquid phase (Negi et al., Negi, H., Agarwal, T., Zaidi, M. G. H., Goel, R., Comparative antibacterial efficacy of metal oxide nanoparticles against Gram negative bacteria. Annals of Microbiology, 62, (). https://doi.org//s
https://doi.org//s ). It is essential for ZnO particles to contact or penetrate into microbial cells to express the antibacterial activity (Mirhosseini and Firouzabadi, Mirhosseini, M., Firouzabadi, F. B., Antibacterial activity of zinc oxide nanoparticle suspensions on food-borne pathogens. International Journal of Dairy Technology, 66, (). https://doi.org//
https://doi.org// ). Therefore, MIC and MBC values were accurately estimated by the broth dilution methods, which were equal to mg mL-1 ( mM) and mg mL-1 ( mM) for both S. aureus and S. Typhimurium, respectively, while no significant bactericidal effect was observed for P. aeruginosa in concentrations up to 2 mg mL-1 ( mM) (Table 1). Agar diffusion tests revealed that B. cereus was highly resistant to ZnO-NPs, the reason why broth media tests were not done for this bacterium.

Many studies have reported that ZnO-NPs antimicrobial activity is significantly affected by different particle morphologies (Stanković et al., Stanković, A., Dimitrijević, S., Uskoković, D., Influence of size scale and morphology on antibacterial properties of ZnO powders hydrothermally synthesized using different surface stabilizing agents. Colloids and Surfaces B: Biointerfaces , , (). https://doi.org//j.colsurfb
https://doi.org//j.colsurfb ; Talebian et al., Talebian, N., Amininezhad, S. M., Doudi, M., Controllable synthesis of ZnO nanoparticles and their morphology-dependent antibacterial and optical properties. Journal of Photochemistry and Photobiology B: Biology, , (). https://doi.org//j.jphotobiol
https://doi.org//j.jphotobiol ). This shape-dependent activity can be explained regarding the percent of active facets on the NPs. Thus, NPs research has been motivated to achieve selective nanostructured ZnO for antibacterial tests (Sirelkhatim et al., Sirelkhatim, A., Mahmud, S., Seeni, A., Kaus, N. H. M., Ann, L. C., Bakhori, S. K. M., Mohamad, D., Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Letters, 7, (). https://doi.org//sx
https://doi.org//s ). Particle size and concentration also have an essential influence on the antimicrobial activity. Studies have revealed that the smaller the NP size, the higher their toxic effect on microorganisms (Nair et al., Nair, S., Sasidharan, A., Divya Rani, V. V., Menon, D., Nair, S., Manzoor, K., Raina, S., Role of size scale of ZnO nanoparticles and microparticles on toxicity toward bacteria and osteoblast cancer cells. Journal of Materials Science: Materials in Medicine, 20, (). https://doi.org//s
https://doi.org//s ; Yamamoto, Yamamoto, O., Influence of particle size on the antibacterial activity of zinc oxide. International Journal of Inorganic Materials, 3, (). https://doi.org//S(01)
https://doi.org//S(01) ). Smaller nanoparticles have relatively large interfacial area and can easily penetrate bacterial membranes, increasing their antibacterial effectiveness (Ramani et al., Ramani, M., Ponnusamy, S., Muthamizhchelvan, C., Marsili, E., Amino acid-mediated synthesis of zinc oxide nanostructures and evaluation of their facet-dependent antimicrobial activity. Colloids and Surfaces B: Biointerfaces , , (). https://doi.org//j.colsurfb
https://doi.org//j.colsurfb ). The ZnO-NPs used in the present work were synthesized by the solochemical process and presented a nanorod shape with a wurtzite crystalline structure with average length and diameter of and nm, respectively.

The results of the antibacterial effect of the ZnO-NPs synthesized via the solochemical method evaluated in the present study indicate that the MIC and MBC were smaller than the values from previous studies with ZnO-NPs synthesized by different methods, even for smaller particle sizes, or evaluated by other methodologies. For instance, ZnO-NPs with an average size of 50 nm had MIC values for P. aeruginosa, S. Typhimurium and S. aureus of 26, 22, and 10 mM, respectively (Tayel et al., Tayel, A. A., El-Tras, W. F., Moussa, S., El-Baz, A. F., Mahrous, H., Salem, M. F., Brimer, L. Antibacterial action of zinc oxide nanoparticles against foodborne pathogens. Journal of Food Safety, 31, (). https://doi.org//jx
https://doi.org//j ), which are much bigger than the values observed in the present work. A comparative scheme is presented in Table 2, showing literature results for the MIC and MBC of ZnO obtained from different methods against Salmonella and S. aureus.

Table 2
Literature results for S. Typhimurium and S. aureus inhibition, and MIC and MBC values of aqueous ZnO-NPs suspensions.

ZnO-NPs did not affect the growth of P. aeruginosa in the range evaluated in the present study. This result corroborates Jan et al. (Jan, T., Iqbal, J., Ismail, M., Zakaullah, M., Haider Naqvi, S., Badshah, N., Sn doping induced enhancement in the activity of ZnO nanostructures against antibiotic resistant S. aureus bacteria. International Journal of Nanomedicine, 8, (). https://doi.org//IJN.S
https://doi.org//IJN.S ), who observed an antibacterial effect of ZnO-NPs more effective against S. aureus than P. aeruginosa. Lee et al. (Lee, J.-H., Kim, Y.-G., Cho, M. H., Lee, J., ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production. Microbiological Research, , (). https://doi.org//j.micres
https://doi.org//j.micres ) found that ZnO-NPs (< 50 nm) at 10 mM only slightly decrease the growth of P. aeruginosa planktonic cells, while successfully inhibiting biofilm formation. These authors suggested a MIC of about mM against the planktonic cells.

A remarkable result was observed by following bacterial growth up to 9 days when the MIC concentration was applied to S. aureus (Figure 3) and S. Typhimurium (Figure 4). In both, an initial decrease was observed followed by the latest increase of the microbial population under this non-lethal condition. Initially, a population sensitive to ZnO-NPs dies, consequently decreasing the total concentration. Then, a resistant population can persist for a long time or even start to grow. This behavior occurs due to phenotypic heterogeneity in the microbial population, resulting in distinct subpopulations. Microbial populations benefit from the creation of variant subpopulations that have the potential to be better prepared to persist during stress conditions (Avery, Avery, S. V., Microbial cell individuality and the underlying sources of heterogeneity. Nature Reviews. Microbiology, 4, (). https://doi.org//nrmicro
https://doi.org//nrmicro ), such as the presence of ZnO. This bacterial behavior reveals the time dependency of the MIC methodology; experiments with longer incubation times will most probably result in different MIC results. This fact, among many others, should be taken into account when designing products containing new antimicrobial compounds.

ZnO-NPs obtained by the solochemical method showed a strong antimicrobial effect against both Gram-negative S. aureus and S. Typhimurium. On the other hand, the effect was minor against P. aeruginosa for the tested concentrations, whereas no apparent effect was observed for B. cereus. The antibacterial activity observed was superior to nanoparticles obtained by other processes, even when the latter presented smaller particle sizes. It can result from both nanoparticle properties and the evaluation methodology. From any of them, the result impacts the amount of required ZnO-NPs, showing potential cost savings for reaching similar antibacterial effect. Importantly, the solochemical process has many advantages over other methods of nanoparticle synthesis, such as low cost and synthesis under low temperatures. The growth and survival curves obtained in this study enhance our understating of the ZnO-NPs antibacterial action over time, often neglected in the literature. Finally, the results obtained in this study suggest that the use of ZnO-NPs as an antibacterial agent in food systems can successfully inhibit some of the most dangerous and frequent foodborne pathogens.

The authors are thankful for the financial support from the Brazilian governmental agencies: National Council for Scientific and Technological Development (CNPq), Foundation to Support Research and Innovation in Santa Catarina State (FAPESC), and Coordination for the Improvement of Higher Education Personnel (CAPES). The authors also thank the laboratories Central de Análises and Laboratório Central de Microscopia Eletrônica (LCME) from the Federal University of Santa Catarina for the technical support with characterization analyses.

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  • Publication in this collection
    30 Sept 
  • Date of issue
    Apr-Jun 
  • Received
    19 Jan 
  • Reviewed
    22 May 
  • Accepted
    23 Sept 
Roberta C. de Souza

Universidade Federal de Santa Catarina, Centro Tecnológico, Departamento de Engenharia Química e Engenharia de Alimentos, Florianópolis, SC, Brasil. E-mail: [email protected]

Leticia U. Haberbeck

Technical University of Denmark, National Food Institute, Kongens Lyngby - Copenhagen, Denmark.

Humberto G. Riella

Universidade Federal de Santa Catarina, Centro Tecnológico, Departamento de Engenharia Química e Engenharia de Alimentos, Florianópolis, SC, Brasil. E-mail: [email protected]

Deise H. B. Ribeiro

Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Departamento de Ciência e Tecnologia de Alimentos, Florianópolis, SC, Brasil.

Bruno A. M. Carciofi

Universidade Federal de Santa Catarina, Centro Tecnológico, Departamento de Engenharia Química e Engenharia de Alimentos, Florianópolis, SC, Brasil. E-mail: [email protected]

* Corresponding author: Bruno A. M. Carciofi - E-mail: [email protected]

Figure 1
XRD patterns of present ZnO-NPs (top) and for standard ZnO (bottom) provided by Joint Committee on Powder Diffraction Standards (JCPDS), International Centre for Diffraction Data, card number

Figure 2
Characterization of the ZnO-NPs: (a) TEM images and frequency distribution histograms of the (b) length and (c) diameter.

Figure 3
S. aureus cell concentration [Log (N)] over time for (○) ZnO-NPs MIC ( mg mL-1), (▲) ZnO-NPs MBC ( mg mL-1), (♦) negative control (Milli-Q water) and (□) positive control (ciprofloxacin, mg mL-1). N is the cell concentration in CFU mL-1.

Figure 4
S. Typhimurium cell concentration [Log (N)] over time for (○) ZnO-NPs MIC ( mg mL-1), (▲) ZnO-NPs MBC ( mg mL-1), (♦) negative control (Milli-Q water) and (□) positive control (ciprofloxacin, mg mL-1). N is the cell concentration in CFU mL-1.

Figure 5
P. aeruginosa cell concentration [Log (N)] over time for ZnO-NPs at (▲) mg mL-1 (○) mg mL-1 and (■) mg mL-1, (♦) negative control (Milli-Q water) and (□) positive control (ciprofloxacin, mg mL-1). N is the cell concentration in CFU mL-1.

Table 1
Log (N)/Log (N0)* for broth dilution tests at different ZnO-NPs concentrations after 48h incubation at 35°C. Positive control and negative controls were ciprofloxacin ( mg mL-1) and Milli-Q water, respectively.

Table 2
Literature results for S. Typhimurium and S. aureus inhibition, and MIC and MBC values of aqueous ZnO-NPs suspensions.

ZnO-NPs (mgmL-1)Positive controlNegative control
S. aureus****
S. Typhimurium****
P. aeruginosa**
Method of synthesis (reference)SizeBacteria evaluated and observationsMICMBC
Solochemical (Present work)97 nmGreat inhibition effect against S. typhimurium and S. aureusmg/mL (mM)mg/mL (mM)
Commercial sample from Sigma-Aldrich, USA (Tayel et al., )50 nmInhibition effect against S. typhimurium and S. aureus22 and 10mM, respectively-
Commercial sample from Teconan, Spain. (Mirhosseini and Firouzabadi, )20 nm2mM do not inhibit the growth of S. aureus after 24h (5mM showed % of growth reduction)Not found10 mM
Commercial sample from Sigma-Aldrich, USA (Jones et al., )8nm and nm mM (8nm) do not inhibit the S. aureus growth. 5 mM ( nm) presented % of growth inhibition.1mM-
Commercial sample from Inframat Advanced Materials LLC, USA. (Xie et al., )30 nmInhibition effect against Salmonella enterica serovar Enteritidis - 10 mg/ml led to 1- or 2-log reduction in viable cells after an 8h exposure.mg/mL-
Nosaka method (Emami-Karvani and Chehrazi, Emami-Karvani, Z., Chehrazi, P., Antibacterial activity of ZnO nanoparticle on gram-positive and gram-negative bacteria. African Journal Microbiology Research, 5, (). https://doi.org//AJMR
https://doi.org//AJMR )
3 nmInhibition effect against S. aureusmg/mL8mg/mL
Kawano method (Ramani et al., Ramani, M., Ponnusamy, S., Muthamizhchelvan, C., Cullen, J., Morphology-directed synthesis of ZnO nanostructures and their antibacterial activity. Colloids and Surfaces B: Biointerfaces, , (). https://doi.org//j.colsurfb
https://doi.org//j.colsurfb )
78 nmmg/mL partial growth inhibition effect against S. aureus and Salmonella typhimurium.--
Solochemical (Sornalatha et al., Sornalatha, D. J., Bhuvaneswari, S., Murugesan, S., Murugakoothan, P., Solochemical synthesis and characterization of ZnO nanostructures with different morphologies and their antibacterial activity. Optik, , (). https://doi.org//j.ijleo
https://doi.org//j.ijleo )
37 nmPresented antimicrobial index of 40% to mg/mL, 50% to mg/mL and 66% to mg/mL against S. aureus.--
Commercial from Sigma, Australia. (Duffy et al., )< 50nmInhibition effect against Salmonella typhimuriummg/mL>5mg/mL
Solvothermal synthesis (Raghupathi, Koodali and Manna, )12 nmSalmonella typhimurium growth was inhibited by about 50% with 10 mM.--

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Rua Dr. Diogo de Faria, – 9º andar – Vila Clementino São Paulo/SP - Brasil
E-mail: [email protected]

Sours: https://www.scielo.br/j/bjce/a/XDsVVb4z8b5BwGPbtWPyWTR/?lang=en


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