A Comparative Study of Antibacterial Activity of CuO/Ag and ZnO/Ag Nanocomposites

The synergistic eﬀects of transition metal based nanocomposites are known to possess enhanced antibacterial activities. However, in-depth analysis of the relative antibacterial performance of some of the prominent nanocomposites remains unavailable. This study compares the antibacterial activity of two separate nanocomposites, which are copper oxide with silver (CuO/Ag) and zinc oxide with silver (ZnO/Ag). The individual CuO/Ag and ZnO/Ag nanocomposites were synthesised by a mixed wet-chemical method. The resulting particles were analysed by XRD, XRF, TEM, UV-Vis spectrophotometer, BET, and FTIR. The antibacterial activity of the nanoparticles were tested on Gram-negative and Gram-positive bacteria, Escherichia coli (ATCC25922) and Staphylococcus aureus (ATCC25923), respectively, using the Kirby–Bauer disc diﬀusion and the microdilution methods. The Kirby–Bauer disc diﬀusion test results had the same minimum inhibition concentration (MIC) value for both CuO/Ag and ZnO/ Ag against E. coli and S. aureus , which was 0.25mg/ml. The applied nanocomposites using microdilution showed that CuO/Ag had approximately 98.8% and 98.7% eﬃciency on the respective Gram-positive and Gram-negative bacterial species, while ZnO/ Ag achieved 91.7% and 89.3% eﬃciency, respectively, against the Gram-positive and Gram-negative bacterial species. This study presents a novel approach for relative analysis of the performance eﬃciency of transition metal based nanocomposites.


Introduction
Inorganic nanoparticles have demonstrated strong activity against broad-spectrum Gram-positive and Gram-negative bacterial species [1]. e toxicity of nanoparticles to bacteria depends on the kind and composition of the nanoparticles as well as the type of bacteria involved [2]. Silver has shown superior antibacterial activity among the family of inorganic nanoparticles [3]. Silver nanoparticles are effective against a wide span of microbes including Gram-positive and Gramnegative bacteria and viruses [4]. e antibacterial activity of silver is dependent on the antibacterial mechanistic routes of nanoparticles against bacterial species [5]. ese routes are (1) the mediating of reactive oxygen species (ROS), which may destabilize cell functionality or integrity, and (2) the direct penetration of nanoparticles, which may cause functional and structural changes to organelles [6]. Several researchers have proposed the strong interaction of ionic silver with thiol groups of important microbial enzymes, which disrupts their structure and causes them to be inactive as one of the main reasons for silver's high antibacterial activity [7]. It has also been proposed that once bacteria are treated with silver, their DNA replication function is lost. Moreover, there is experimental evidence showing profound structural changes in the cell membrane of bacteria once treated with silver [8].
However, in comparison with other inorganic nanoparticles, silver is very expensive [9]. Moreover, a major drawback of using silver nanoparticles is due to their associated small size, which enables them to cross biological membranes with ease of posing risks to humans and the environment. Inorganic nanoparticles such as zinc oxide (ZnO) and copper oxide (CuO) are cheaper, widely available, less toxic to mammalian cells, stable, and more environmentally friendly [10][11][12]. erefore, in order to ensure a fair balance between a potent antibacterial inorganic material and cost effectiveness of the material, a nanocomposite consisting of silver and other tested inorganic antibacterial material is proposed [13]. Nanometal oxides including zinc oxide and copper oxide have demonstrated antibacterial activity [14]. It is envisaged that nanocomposites of silver with each of these metal oxides will create a potent and yet more cost-effective inorganic antibacterial material [15]. e nanocomposites to be created would therefore be a separate silver with copper oxide (CuO/Ag) and silver with zinc oxide (ZnO/Ag) nanocomposites. e synergistic combination of these nanocomposites would enhance their antibacterial potential. In this case, each elemental composition of the nanocomposite will harness its own antibacterial activity through the production of its associated ions, acting as antibacterial agents [16].
is study, therefore, presents nanocomposites of copper oxide with silver (CuO/Ag) and zinc oxide with silver (ZnO/Ag). e investigation undertakes the task of comparing their bioactivities by representative Gram-positive and Gram-negative bacterial species, which are, respectively, the E. coli strain ATCC25922 and S. aureus strain ATCC25923 obtained from the Noguchi Memorial Institute for Medical Research, University of Ghana, Legon.

Synthesis of Copper Oxide/Silver Nanocomposite.
Copper oxide/silver (CuO/Ag) nanocomposites were synthesised by the wet chemical precipitation synthesis method. By this approach, 0.1 M copper acetate was dissolved in a 100 ml of deionised water. e solution was continuously stirred at 700 rpm on a magnetic stirrer hot plate at 80°C. A homogeneous solution was shortly formed. 2 M NaOH was added dropwise to reach a pH of 11. e synthesis was continuously stirred at the same speed and temperature for a further sixty minutes. A black suspension was formed, which is indicative of the formation of copper oxide. To form the CuO/Ag nanocomposite, 0.001 M of silver nitrate was added to the 100 ml of CuO aqueous precipitate at the same temperature and stirring speed. 5 ml of 0.004 M of sodium borohydride was added dropwise to the reacting solution for the reduction of silver ions. e temperature was gradually reduced to room temperature at the same stirring speed. e synthesised nanocomposites were separated by centrifugation and washed several times with distilled water to remove excess ions. e particles obtained were dried in an oven at 80°C overnight. en they were calcined at 500°C to remove impurities that may be present on particles surfaces. e particles were subsequently characterised and applied for antibacterial activities.

Synthesis of Zinc Oxide/Silver Nanocomposite.
Similarly, surfactant-free zinc oxide/silver nanocomposites were synthesised by the wet chemical precipitation method. 0.1 M zinc acetate was dissolved in a 100 ml of deionised water. A homogeneous solution was obtained after keeping the solution at 700 rpm on a hot magnetic stirrer plate at 80°C. e temperature and stirring speed were kept throughout the synthesis period. 2 M NaOH was added to the homogeneous solution to increase its pH to 11. A white precipitate was formed, which was stirred at the same speed and temperature for another sixty minutes. e synthesis process continued by the rapid addition of 0.001 M silver nitrate to the aqueous zinc oxide precipitate. Subsequently, 5 ml of 0.004 M sodium borohydride was then added dropwise to form reduced silver nanoparticles in the composite. e synthesis was allowed to continue for another sixty minutes, during which the temperature was allowed to gradually fall to ambient conditions. e fabricated nanocomposites were separated by centrifugation at 6000 rpm. e particles were severally washed to remove unreacted ions. ey were dried at 80°C overnight and further calcined at 500°C for 3 hours at a heating rate of 1°C/ S. e synthesised particles were successively analysed through a series of characterizations and utilized for antibacterial studies.
Equations (4) to (6) are a sequence of steps for the formation of zinc oxide/silver (ZnO/Ag) nanocomposite.

Antibacterial Testing.
e as-prepared nanocomposites (CuO/Ag and ZnO/Ag) were tested on E. coli and S. aureus.
e Kirby-Bauer disk diffusion and the microdilution using optical density methods were employed for antibacterial testing. All materials used for the antibacterial testing were sterilized at 121°C for 15 minutes. All the microbiology applications were done in a sterile laminar flow cabinet.

Disk Diffusion Assay.
e bacteria were separately cultured overnight in a Mueller-Hinton broth. 100 ul of separate bacteria cultures containing approximately 6 × 10 7 CFU was uniformly streaked on a solidified Mueller-Hinton agar in a glass Petri dish. Paper disks measuring about 6 mm were individually impregnated with CuO/Ag or ZnO/Ag at varying concentrations of 5 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, and 0 mg/ml. e concentration of 0 mg/ml was adopted as the control for the experiment. e impregnated paper disks were distinctly placed on the agar plates streaked with distinct bacteria. e agar plates were incubated overnight at 37°C. Visible zones of inhibition observed around the perimeter of the nanocomposite impregnated paper disks were measured and recorded for analysis of antibacterial activity.

Bacteria Growth Kinetic Assay.
e disk diffusion antibiotic test presented a method for exploring the efficacy of CuO/Ag and ZnO/Ag on bacteria strains. However, the growth kinetics of bacteria are highly dependent on their optical density. Spectrophotometric technique was used to study the optical density of bacteria cell cultures to determine their growth kinetics. e Varioskan LUX multimode microplate reader ( ermo Fisher Scientific, USA) was employed for reading the optical densities of bacteria in a 96well microplate. Separate bacteria cultures of E .coli and S. aureus were grown in a Mueller-Hinton broth to obtain an optical density of 0.2 corresponding to a bacteria density of 6 × 10 7 CFU. e bacteria inoculum was transferred into a 96-well microplate. CuO/Ag and ZnO/Ag were separately dispersed in an aqueous suspension by an ultrasonicator (Bransonic Ultrasonic Bath, omas Scientific, USA) operating at 60 s at 75 % amplitude at a pulse configuration of 0.5/0.5 s. e homogeneously dispersed nanocomposites were added to the respective bacteria inoculum. e bacteria were incubated for 24 hours at 37°C. e optical density of the inoculum was measured periodically during the culturing period to observe the bacteria growth state pattern by changes in the optical density. e data obtained from the spectrophotometric fluorescent reading were analysed to understand the kinetics of bacteria growth. Such information is fundamental to gaining in-depth knowledge on the antibacterial efficiency of silver nanocomposites.

X-Ray Diffraction (XRD) Analysis of Metal Oxide/Silver
Nanocomposite. All nanocomposites including copper oxide/silver (CuO/Ag) and zinc oxide/silver (ZnO/Ag) were studied with the X-ray diffractometer. e analysis was important to observe the prevalent crystal structure in the individual nanocomposites.
XRD was used to analyse the crystal structures of the synthesised copper oxide/silver (CuO/Ag) nanocomposites. e sharpness of the spectra is an indication of the crystalline nature of the nanocomposites. e XRD spectrum of synthesised CuO/Ag nanocomposite is presented in Figure 1.  48. e spectra peaks of copper oxide correlated with the spectra peaks of the CuO/Ag nanocomposite. e crystal structure analysis showed that the monoclinic phase of copper oxide was the dominant phase in the nanocomposite. e spectra peak of silver is weak due to the low concentration of silver in the nanocomposite. Silver has a face centered cubic (FCC) crystal structure.
e unit cell has perpendicular faces with equal lengths along all sides. Silver atoms are located at each corner of the unit cell and at the middle of each face. e absence of unwanted peaks indicates the purity of the nanocomposite. e purity of the samples that were further analysed by X-ray fluorescence (XRF) is critical to determine the true antibacterial effect of the nanocomposite antibacterial agents. Using the Scherer equation, the crystallite size of CuO/Ag was calculated to be 34.2 nm.
e ZnO/Ag nanocomposite spectra shown in Figure  . e spectra peaks corresponded with the exact patterns of zinc oxide, which again signifies the purity of the nanocomposite. e spectra peak at 2θ of 38°is the (111) plane of silver. Silver had weak peaks due to the low concentration of silver in the nanocomposite. Similar observation about the weak spectra peaks of silver due to low concentration in a nanocomposite has been previously observed [17]. e crystallite size of ZnO/Ag nanocomposite according to the Scherer equation was found to be 36.17 nm.

X-Ray Fluorescence (XRF) Analysis of Metal Oxide/ Silver Nanocomposite.
e elemental analysis of metal oxide/silver nanocomposites including separate CuO/Ag and ZnO/Ag showed the quantity of constituents contained in each individually analysed sample of CuO/Ag and ZnO/Ag. e constituent elements detected in the whole sample were expressed as oxides in relative percentage of all the elements present in the sample. e results for the XRF spectroscopy analysis of CuO/Ag are shown in Table 1. e XRF analysis of CuO/Ag shows that the sample had a high content of copper oxide (CuO) followed by silver (Ag). All elements were expressed in oxides; hence the silver was communicated as Ag 2 O. e weak peaks of silver recorded by the XRD spectrum are confirmed by its low content verified by the XRF elemental analysis.
Advances in Materials Science and Engineering e table also shows the XRF elemental analysis of ZnO/ Ag. e highest constituent of the sample is zinc oxide followed by a low content of silver. e results agree with the weak peaks of silver in the XRD spectra pattern.
e XRF results revealed that the individual nanocomposites had similar respective compositions of metal oxides and silver. e respective length and width of the CuO/Ag nanorods were measured and plotted against their frequency. e silver component of the nanocomposite appears spherical, being supported by the copper oxides that are rather rodshaped. A similar shape of nanorods has been observed among CuO nanoparticles [18]. As shown in Figure 3, CuO/ Ag nanocomposites have an average length of 400 nm. e CuO/Ag nanocomposites have an average width of 20 nm. However, the size of the particles recorded by the X-ray diffraction was 34.2 nm. e dry CuO/Ag powder might have undergone agglomeration, which eventually influenced the sizes of its particles. CuO nanorods of similar size dimensions have demonstrated activity against a range of microbes [19]. e as-synthesized ZnO/Ag nanocomposites assumed a plate-like shape. is morphology is similar to that of other ZnO nanoparticles by Jang [20]. e size distribution of ZnO/Ag ranges from 15 nm to 60 nm with an average size of 35 nm. e size was similar to that computed by Scherer's equation, which was 36.17 nm. Such range of particle size is known to possess commended toxicity to bacteria species [21]. According to Scherer's equation performed on the highest intensity peak of the XRD spectra to compute the particle size, CuO/Ag and ZnO/Ag had similar sizes, which were 34.2 nm and 36.17 nm, respectively. e antibacterial activity of metal based nanoparticles among other factors is influenced by the size of the particle. Particle size influences the reactivity of the nanoparticle through the number of atoms available on the surface for reactivity. e number of atoms occupying the surface of a nanoparticle increases with decreasing size. e particle size affects other factors such as dissolution to produce associated ions mediating cell death. e cellular internalization of nanoparticles through cellular membranous pores leading to cell death is also highly dependent on particle size. e similar range of sizes of the particles recorded by the XRD suggests that the separate particles had similar antibacterial effects with respect to their sizes.

UV-Vis Spectroscopy Analysis of Metal Oxide/Silver
Nanocomposites.
e UV-Vis (GENESYS 150 UV-Vis, ermo Fisher Scientific, USA) spectrophotometric absorption spectra of CuO/Ag nanocomposites are found in Figure 5. CuO/Ag nanocomposite had highest wavelength peak at 542 nm. e excitation of electrons from the valence band to the conduction band of CuO/Ag is predominant at Ag (FCC phase) ref. Ag (FCC phase) ref.
ZnO/Ag nanocomposite the peak wavelength [22]. e energy band gap of CuO/Ag nanocomposite is 1.2 eV. However, the energy band gap of CuO has previously been reported to be 1.54 eV [23]. e presence of silver enhances the band gap. Silver electrons act as dopants of CuO, which improves its energy band gap. Band gap enhancement affects the electrical conductivity of the resulting material. CuO/Ag with an energy band gap of 1.2 eV can cause the release of electrons at a lower energy wavelength than CuO, which had a band gap of 1.54 eV. e release of electrons by the nanoparticle is important for initiating cell death. e release electrons interact with bacteria cells or the fluid medium to produce reactive oxygen species (ROS), which become injurious to bacterial cells.
It is realised that UV-Vis of copper oxide/silver nanocomposite had a spectra peak extending from the UV range to the visible range. e copper oxide/silver particles could therefore extract energy from the UV or visible range of the spectrum. e acquisition of energy promotes electron transition preceding essential antibacterial activities of metal based nanomaterials such as copper oxide/silver Advances in Materials Science and Engineering nanocomposites. In the presence of nanomaterials, the right incidence of the required energy causes the generation of reactive oxygen species.
ZnO/Ag nanocomposites are demonstrated in Figure 6. Absorption wavelength peak of 372 nm was recorded as the wavelength of energy for electron excitation in ZnO/Ag. Similar wavelength has been reported for ZnO [24]. is agrees with the predominance of the ZnO in the nanocomposite as revealed by the XRD and XRF results. e corresponding energy band gap was 2.88 eV. e energy band gap is lower than the traditionally reported band gap of zinc oxide, which is known to be 3-4 eV but mostly found to be 3.2 eV by a majority of researchers [23]. e enhanced band gap of ZnO/Ag is attributed to the presence of silver, which dopes the zinc oxide semiconductor, thereby enhancing its optical properties. e silver acted as a dopant to enhance the band gap of the ZnO particles. Band gap enhancement modifies the electrical conductivity of the ZnO particles.
e band gap of ZnO/Ag recorded as 2.88 eV generates electrons at lower heat of light photon energy as compared to the band gap of the traditional band gap of ZnO, which is larger.
us, it required lesser energy for electron transition, which accounts for cell death through the initiation of reactive oxygen species. e phenomena might account for the high antibacterial activity of doped zinc oxide and that of other doped nanomaterials. Kumar and others demonstrated that doped zinc oxide exhibited higher antibacterial activity [25]. e lower energy band gap of CuO/Ag compared to ZnO/Ag means that the excitation of electrons in CuO/Ag would require a lower energy than ZnO/Ag. Also, according to the UV spectra, unlike CuO/Ag, ZnO/Ag did not have peak spectra within the visible range of the spectrum but rather within the UV range. us, ZnO/Ag would require higher energy for electron transition between energy states as compared to CuO/Ag, which recorded a broad spectra peak extending into the visible range of the spectrum. Electron transfer between energy states which principally occurs at the wavelength of the spectra peak creates electron excitation generating hole and electron pairs, resulting in reactive oxygen species that interact with bacteria species that are toxic to bacteria cells. CuO/Ag from the UV spectra analysis could be expected to cause cell death at a lower energy than ZnO/Ag, since it had spectra peak extending into the lower energy of the visible range.

Brunauer-Emmett-Teller (BET) Analysis of Clay-Nanocomposite Pellets.
BET analysis was carried out to provide information on the surface area of the respective nanocomposites.
e BET provided information on the surface area by the adsorption of nitrogen gas molecule as a function of relative adsorption pressure (P/P 0 ), which spans from 0.05 to 1. At (P/P 0 � 1), the gas molecules condense within the pores. As the pressure is decreased from its highest value of 1, gas desorption takes place. e process provides information on the adsorption-desorption of the gas molecules from the nanocomposite surfaces. e information is used to obtain properties about the material surface such as surface area, pore volume, and pore size distribution by the implementation of the BET equation. e adsorption-desorption curves are termed isotherms, which are classified into six different types depending on the IUPAC 1985 classification [26]. e analysis of surface area is crucial among the characteristics of nanomaterial based antibacterial agents. e larger the surface area of the nanomaterial, the greater its adsorption onto bacteria surfaces, thereby causing subsequent cell death. Surface area also increases with decreasing particle size. e smaller the particle size, the larger the surface area. Smaller particle sizes have demonstrated higher antibacterial activity [19]. e argument may be attributed to the fact that smaller particles by virtue of their size can easily penetrate into the cytoplasm through cellular pores. Particle size again influences reactivity. Decreasing the particle increases the reactivity because more atoms possessing free bonds become available on the surface for reactivity. Figure 7 contains adsorption-desorption isotherm and the corresponding BJH (Barrett-Joyner-Halenda) plot describing its pore size distribution of CuO/Ag. e adsorption-desorption curve depicts a Type IV isotherm. Type IV isotherm is characteristic of pore sizes within the range of 1.5-100 nm. In Type IV isotherm, the micropores are filled at extremely low relative pressure. At the knee of the curve, a monolayer is formed, which is followed by a multilayer formation at a moderate pressure. As the pressure increases further, there is a condensation of gas molecules within pores. Gas adsorption continues at high relative pressure, which is an evidence of agglomeration confirming the aggregation already reported due to difference in particle size reported by XRD and TEM results. e BJH plot confirms the Type IV isotherm of the CuO/ Ag nanocomposite. e nanocomposite had pores ranging from micropores to mesopores, which is typical of Type IV isotherm. According to the BET equation, the surface area (S BET ) was calculated to be 18 m 2 g −1 . Again, by applying the same BET equation, the pore volume of CuO/Ag was found to be 4.2 cm 3 g −1 . Figure 8 is the adsorption-desorption isotherms and the BJH plot of ZnO/Ag. e nitrogen adsorption and desorption at 77 K of ZnO/Ag nanocomposites behaved similarly to CuO/Ag. ZnO/Ag demonstrated a Type IV isotherm, in which the micropores were filled at extremely low relative pressure. At a low relative pressure at the knee of the isotherm curve monolayer of nitrogen gas was adsorbed on the ZnO/Ag surface. As the pressure increased to a moderate level, multilayer of adsorbed gas was formed, which condensed within pore structures at high pressures. At high pressure, gas adsorption continued.
e BJH plot of ZnO/Ag revealed that the nanocomposite possessed a range of pore sizes. e pores spanned from micropores to mesopores, which is characteristic of Type IV isotherms. e surface area (S BET ) of ZnO/Ag was found to be 18 m 2 g −1 ; the pore volume was found to be 4.0cm 3 g −1 .

Advances in Materials Science and Engineering
Comparatively, ZnO/Ag and CuO/Ag possessed equivalent BET characteristic features. eir similarity in surface area suggests they had comparable particle size, which was also consistent with the XRD results of their particle size which suggested the same. us, ZnO/Ag and CuO/Ag have similar antibacterial activities with respect to their respective surface areas.

FTIR Analysis of Metal Oxide/Silver Nanocomposite.
Fourier transform infrared (FTIR) spectroscopic analysis was carried out on the wet-chemical synthesised metal oxide/silver nanocomposites to ensure that surfactant free particles were used for the antibacterial studies. Surfactants are reported to influence the antibacterial activity. e FTIR analysis provided information on the surface properties on the respective nanocomposites. In this study, the FTIR spectra of CuO and ZnO are, respectively, consistent with those of CuO/Ag and ZnO/Ag, which is attributed to their high relative quantities as already demonstrated by the XRD and XRF analyses. Slight shifting in the wavenumber (cm −1 ) of the bonds is attributed to the interactions between metal oxide (CuO or ZnO) and silver (Ag). Figure 9 shows the FTIR spectra analysis of CuO/Ag nanocomposite. Band at 572 cm −1 is attributed to Cu-O bond. e bands observed between 3600 and 3500 cm −1 indicate the presence of the O-H associated with water. Peaks corresponding to organic surfactants are absent because surfactant free nanocomposites were synthesised for testing on bacteria species to nullify any possible effects of surfactants in the antibacterial process. Figure 10 illustrates the FTIR spectra of ZnO/Ag nanocomposite. e O-H bond that occurs with the spectra band of 3600 to 3500 cm −1 was very weak, which means that the moisture content was almost absent in this sample. e bond confirming the presence of zinc oxide was present at 694 cm −1 , which is the band for Zn-O bond. e weak bands occurring between 2920 and 1381 cm −1 are because of interaction with silver. Significant absorption peaks corresponding to organic molecules are not observed because the nanocomposites surfaces were void of surfactants. is was important to observe the true antibacterial activity of nanocomposites alone without any surfactant influences. e surfactant free nanocomposites observed by FTIR analysis are in agreement with the high purity of the samples, which were also demonstrated by XRD and XRF analyses.  Advances in Materials Science and Engineering enhanced antibacterial activity of the nanocomposites was attributed to silver. Silver, apart from other reasons, is reported as having comparatively high antibacterial activity [3]. e reason for the high antibacterial activity of silver has been reported by many researchers. ey have suggested that the high affinity of silver for the sulphur in the thiol protein of bacteria is largely responsible for this [27]. e affinity leads to the breakage of disulphide bonds in thiols. e protein tertiary structure is eventually disrupted [28], resulting in cell death.     Figure 11 shows the antibiotic effect of CuO/Ag nanocomposite against E. coli. e zones of inhibition shows that CuO/Ag was effective against E. coli from a decreasing concentration of 5 mg/ml to 0.05 mg/ml. e MIC value of CuO/Ag against E. coli was 0.25 mg/ml. In previous work, the susceptibility of E. coli to Ag was high, while the same E. coli was resistive towards copper [29]. e literature report agrees with the finding in this study, in which the presence of silver in the nanocomposite demonstrates effectiveness against E. coli.

Antibacterial Study of CuO/Ag and ZnO/Ag against
Similarly, in Figure 12, the activity of CuO/Ag against Gram-positive bacteria (S. Aureus) is demonstrated. e MIC was 0.25 mg/ml. Figure 13 is a histogram of the summary of the activity of CuO/Ag on the Gram-negative and Gram-positive bacteria. e graph reveals that CuO/Ag nanocomposite was more effective against S. aureus than against E. coli. e observation can be made from the sizes of the inhibition zones of the respective strains of the bacteria species. e activity of CuO/Ag against S. aureus had relatively bigger zone of inhibition as compared to the activity of CuO/Ag against E. coli. e size of the inhibition zone depicts the sensitivity of the bacteria. A similar observation has already been reported by other researchers. It has been observed that Gram-positive bacteria such as S. aureus were more susceptible to antibiotics than the Gram-negative bacteria of E. coli [30]. Although E. coli had the smaller size of zone of inhibition, it had the same MIC as S. aureus, which was 0.25 mg/ml. is means that the same amount of CuO/Ag could result in a similar inhibition in both the E. coli and S. aureus strains. Strain specificity has been observed for E. coli among researchers [29]. While it was sensitive towards silver, it remained insensitive towards copper. erefore, the effectiveness of the nanocomposite against E. coli was attributed to the presence of the composition of silver. e nanocomposite created a synergy which broadened its range of antibacterial spectrum. erefore, it was effective against both Gram-negative and Gram-positive strains of the bacteria species.
ZnO/Ag nanocomposite was also tested on the two strains of bacteria species. First, ZnO/Ag was tested on E. coli. e results revealed that the ZnO/Ag nanocomposite was effective against E. coli. e activity of ZnO/Ag on E. coli can be observed in Figure 14. ZnO/Ag had an MIC value of 0.25 mg/ml. Figure 15 shows the activity of ZnO/Ag against S. aureus. e zones of inhibition in this case are larger than those observed in ZnO/Ag against E. coli in Figure 14. is shows the higher efficiency of ZnO/Ag against S. aureus than against E. coli. e antibacterial activity of ZnO/Ag on both E. coli and S. aureus is shown in Figure 16. Similar to the activities of CuO/Ag against E. coli and S. aureus strains, S. aureus was more susceptible than E. coli. e zones of inhibition of ZnO/Ag against S. aureus were comparatively bigger than those of ZnO/Ag against E. coli, which explains the higher activity of ZnO/Ag on S. aureus than on E. coli. e antibacterial activities of CuO/Ag and ZnO/Ag against E. coli and S. aureus are compared in Figure 17. CuO/ Ag concentrations of 5 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, and 0 mg/ml against E. coli produced respective inhibition zones of 9 mm, 8 mm, 8 mm, 7 mm, 6 mm, 0 mm, and 0 mm. Its activity against S. aureus was 14 mm, 13 mm, 13 mm, 10 mm, 7 mm, 0 mm, and 0 mm. ZnO/Ag concentrations of 5 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, and 0 mg/ml against E. coli produced respective inhibition zones of 8 mm, 7 mm, 7 mm, 7 mm, 7 mm, 0 mm, and 0 mm. Its activity against S. aureus was 12 mm, 12 mm, 10 mm, 9 mm, 8 mm, 0 mm, and 0 mm. e higher sensitivity of S. aureus was significant from its bigger zones of inhibition among the activities of each nanocomposite. Comparatively, the zones of CuO/Ag and ZnO/Ag had close values due to the influencing contribution of silver (Ag) in both nanocomposites. e Ag contribution is responsible for the common MIC of both nanocomposites for both species, which are indicated in Table 2. CuO/Ag had bigger zones in most instances, demonstrating its higher activity over ZnO/Ag.

Bacteria Growth Kinetic Study.
e Kirby-Bauer disc diffusion bacteria susceptibility test was used to study the antibiotic efficiency of engineered silver/metal oxide (CuO/ Ag and ZnO/Ag) nanocomposite. A major challenge with this technique is that it is difficult to determine the rate of antibacterial activity with respect to time [31]. In the disk diffusion technique, filter paper disk containing antibacterial agents is placed on an agar plate with bacteria spread on it. e antibacterial agents diffuse through the agar containing the bacteria [32]. e antibacterial potency is determined by the size of a zone of inhibition surrounding the filter paper disk after overnight incubation or approximately twelve hours of incubation. e size of the zone of inhibition produced in disk diffusion bacteria susceptibility technique is dependent on the amount of ions impregnated on the paper disc. e toxic ions interact with cells to cause cell death. e whole procedure takes approximately twelve hours to observe the resulting zone of inhibition. us, the disc diffusion method is not convenient for observing the time-kinetic effects of the antibacterial agents [33].
Antibacterial activity of nanoparticles involves a number of routes. Prominent among these routes are dissolution of nanoparticles to produce respective ions in solution, which are toxic to bacteria cells, nanoparticle interaction with bacteria cell walls, which disrupts the cell wall, and also the penetration of nanoparticles through cellular pores that bind to organelles, proteins, and genetic structures, which leads to cell death [6]. In the Kirby-Bauer disk diffusion method, it is difficult to differentiate between the ionic mediated cell death through the generation of reactive oxygen species and the nanoparticle-bacterial cell interaction. Diffusion typically involves the movement of ions rather than nanoparticles. In the disc diffusion approach, ionic mediated mechanistic pathway is popularly reported by a majority of researchers [34]. Other methods of antibacterial testing such as the broth microdilution present a modality of antibacterial testing which incorporates all possible routes of nanoparticle antibacterial mechanisms [35]. e broth microdilution technique is a liquid broth including a concentration of antibiotics and the bacteria to be tested [36]. In most cases, depending on the desired outcome, the antibiotics and the bacteria may be subject to varying concentrations. Broth microdilution allows the measurement of the optical density of the inoculum. e optical density value correlates with the quantity of bacteria present in the inoculum [21]. Hence, this technique, unlike disc diffusion, presents a quantitative method of measuring the bacteria growth with respect to time. e bacteria timekinetics are important for analysing the time-dependency effect of antibacterial agents [37]. e bacteria growth kinetic studies of the antibacterial activity of CuO/Ag and ZnO/Ag nanocomposites were performed by distinctly subjecting E. Coli and S. Aureus bacteria cells to the inhibiting activities of the respective nanocomposites. e growth inhibition assay included a broth containing approximately 6 * 10 6 CFU of respective bacteria cells with varying concentrations (1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, 0.05mg/ml, and 0 mg/ml) of the respective nanocomposite. A control experiment included that of a broth media with respective bacteria cells and no (0 mg/ml) antibacterial agent (zero amount of either of the individual nanocomposites). e optical density of the assay was initially and periodically measured. e optical density of broth media with the respective individual nanocomposite was used as a blank hence deducted from the optical density of the corresponding inoculum to obtain the actual optical density of only the respective bacteria cell. erefore, the optical density obtained for analysis was the exact representative of bacteria growth time-kinetics. Figure 11: Antibacterial activity of CuO/Ag nanocomposite against E. coli. e paper disks were impregnated with the following suspension of copper oxide nanoparticles: A � 5 mg/ml, B � 2 mg/ml, C � 1 mg/ml, D � 0.5 mg/ml, E � 0.25 mg/ml, F � 0.1 mg/ml, and G � 0 mg/ml.   Figure 18. e E. coli kinetic growth curve shows that the control (without antibacterial agent) sample had relatively high bacteria, which is shown by the exponential rise in its optical density measured at 600 nm. e samples with antibacterial agent show comparably low optical densities compared to their respective control samples as indication of their inhibited growth. e growth kinetic study curves of CuO/Ag nanocomposite recorded lower optical densities than those of ZnO/Ag. CuO/Ag nanocomposite had higher antibacterial activity than ZnO/ Ag nanocomposite, which confirms the observation made in the disk diffusion test. e curves of E. coli showed higher optical densities than those of S. aureus, which are representatives of the sensitivity of S. aureus to the antibacterial agents in comparison to E. coli. Analytically, both E. coli and S. aureus strains were more susceptible to CuO/Ag than to ZnO/Ag nanocomposite. Similar observations of the antibacterial performances of CuO/Ag and ZnO/Ag were made in the Kirby-Bauer disk diffusion method in the previous sections of this study. Figure 19 presents the percentage of bacteria cells that are alive in the inoculum with respect to time. From the kinetic growth study curves, the antibacterial activities of Figure 14: Antibacterial activity of ZnO/Ag nanocomposite against E. coli. e paper disks were impregnated with the following suspension of copper oxide nanoparticles: A � 5 mg/ml, B � 2 mg/ml, C � 1 mg/ml, D � 0.5 mg/ml, E � 0.25 mg/ml, F � 0.1 mg/ml, and G � 0 mg/ml.  us, the bacteria cells were completely inhibited without any growth potential. On the other hand, S. aureus also showed drastic sensitivity by a decrease in cell population after the 4 th hour of antibacterial activity against by ZnO/Ag. However, in the presence of lower concentrations (0.25 mg/ml, 0.1 mg/ml, and 0.05 mg/ml) of ZnO/Ag, there was an intermittent slight increase in cell population after  Advances in Materials Science and Engineering 13 the 4 th hour indicated by the gradual rise in the curves. is infers that the S. aureus bacteria cells became resistive towards ZnO/Ag antibacterial agents. is rise of the curve again assumed a downward slope, denoting that the cell population began to decrease again. e explanation to this is that, in aqueous medium, zinc oxide dissolves faster than silver, which is partially soluble [38]. Meanwhile, zinc oxide is a micronutrient for cellular metabolism and can be controlled, especially at lower concentrations [39]. However, as the quantity of the partially oxidative silver increases, the cells are again inhibited and assume a downward trend showing a decrease in cell population again. According to Figure 20, S. aureus showed more susceptibility against both antibacterial agents as compared to E. coli. After 4 hours of antibacterial activity, S. aureus showed significant activity against both antibacterial agents.
Meanwhile, E. coli recorded major activity against antibacterial agents after 18 hours of antibacterial activity. After 24 hours of antibacterial activity, the efficiencies of the respective nanocomposites were similar for the respective bacteria species. e respective antibacterial efficiency of the microdilution after 24 hours of activity is shown in Table 3. After the 24-hour antibacterial activity, both CuO/Ag and ZnO/Ag had significant activity on both E. coli and S. Aureus. However, the activity of CuO/Ag was observed to be higher than that of ZnO/Ag among the strains of both species. Figure 21 is a boxplot demonstrating the antibacterial activities of CuO/Ag and ZnO/Ag against the respective E. coli and S. Aureus strains after 24 hours of microdilution test. e strains of both species were sensitive to both CuO/Ag and ZnO/Ag. However, CuO/Ag recorded higher activity  us, the nanocomposite antibacterial agents exhibited no strain specificity as they are effective against both the Gramnegative and Gram-positive strains. Strain specificity has been reported by other authors; while B. subtilis was susceptible to copper oxide, it was insensitive to silver and also E. coli was more susceptible to silver than copper oxide [28]. e enhanced antibacterial activities of the nanocomposites (CuO/Ag and ZnO/Ag) against all the tested bacteria strains show that the synergy created by the nanocomposites produced a wider spectrum of antibacterial activity by being effective against both Gram-negative and Gram-positive bacteria species. e respective nanocomposites had similar effects on both E. coli and S. aureus. us, both CuO/Ag and ZnO/Ag had similar activity on E. coli and S. aureus. e activity of the different transition metals in the nanocomposites creates a wide spectrum of  relatively expensive than metal oxide nanoparticles. us, the creation of the nanocomposite provided a cost-effective highly efficient antibacterial agents.

Conclusion
In this investigation, the comparative assessment of the antibacterial activity of silver nanocomposites was carried out through a stepwise procedural approach of synthesis, characterization, and application. e antibacterial activities of CuO/Ag and ZnO/Ag were relatively compared by their activity on Gram-positive and Gram-negative bacteria, which were, respectively, S. aureus and E. coli. e silver nanocomposites of CuO/Ag and ZnO/Ag were prepared by the simple one pot mixed wet chemical synthesis method. e synthesised nanocomposites were characterised by XRD, XRF, TEM, UV-Vis spectrophotometer, BET, and FTIR. XRD results supported by evidence of the XRF revealed that the synthesised particles were of high purity and the calculation of crystallite size from the highest intensity XRD peak suggested that the particles had identical sizes. e spectrophotometric analysis shows that the prepared CuO/Ag and ZnO/Ag had an enhanced energy band gap different from known CuO and ZnO nanoparticle counterparts, respectively, due to silver doping influences. e TEM analysis of the nanocomposites showed that silver nanoparticles were interspersed within the respective metal oxide nanoparticles. According to the TEM, each nanocomposite had at least single size dimension within 1-100 nm, which defines their nanosize. e BET results further confirmed that the nanocomposites had equivalent surface area and hence particle size as predicted by the XRD spectra analysis of particle size. According to the FTIR spectra, both nanocomposites were devoid of organic surfactants, which could impede antibacterial activities. e nanocomposites were tested by two main methods of applications. e Kirby-Bauer disc diffusion and the microdilution optical density tests were used to access the antibacterial performance of each nanocomposite by testing them on Grampositive and Gram-negative bacteria strains including S. aureus and E. coli. e results showed that CuO/Ag was more effective against both E. coli and S. aureus as compared to ZnO/Ag. e results further revealed that S. aureus was more susceptible to the individual actions of CuO/Ag and ZnO/Ag nanocomposites as compared to E. coli.
e results prove the novelty that CuO/Ag nanocomposite has better antibacterial activity against both Gram-positive and Gram-negative bacteria species compared to ZnO/Ag. However, this study can be further extended to study a broader range of bacteria species to further confirm the relative high antibacterial activity of CuO/Ag in comparison to ZnO/Ag.

Data Availability
All data used in this manuscript have been presented within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.