APEC is responsible for major worldwide economic losses, including morbidity, mortality and the condemnation of poultry carcasses [21]. Because of the development of multidrug resistance among most APEC strains through alteration in the bacterial protein expression or mutations [22, 23], ZnO-NPs have attracted significant attention in medicine as an alternative or through regaining the activity of the available antimicrobials due to their small size, large surface area, low-cost, profuse nature, growth promotion, selective toxicity, antimicrobial and therapeutic activity, and safety as reported by the Food and Drug Administration [24, 25].
The XRD profile of sample 1 (bare ZnO-NPs) and sample 2 (the in situ prepared ZnO-NPs in the presence of PEG-6000 as a surfactant and absolute ethanol) showed the same distinct diffraction peaks typical for the wurtzite ZnO structure at 2θ = 31.70, 34.36, 36.21, 47.5, 56.57, 62.86, 67.92, and 69.1° with crystallite sizes of 25.8 nm and 28.7 nm, respectively, that came in accordance with the standard ZnO XRD pattern JCPDS No. 043–0002. The sharp characteristic peaks and absence of other peaks in the pattern of both samples reveal a desirable nano-crystalline structure, suggesting the high purity of the obtained ZnO. This result isn’t in congruence with [13], who concluded that using zinc sulfate as a precursor for bare ZnO-NPs yields particles with a size in the micrometer range.
The in situ addition of PEG-6000 (as a polymer) and ethanol during ZnO-NPs synthesis elicited a relatively larger crystallite size than the bare ZnO-NPs, which didn’t come in line with [13], who established that Polyvinyl Alcohol, as a polymer, addition during preparation of the ZnO-NPs decreases the particle size.
The XRD patterns of bare ZnO-NPs compared with that in situ prepared in the presence of PEG-6000 and ethanol indicates that the in situ addition of PEG-6000 during ZnO-NPs synthesis didn’t alter the XRD pattern of the ZnO-NPs and exhibited no additional reflections specific to PEG-6000.
Although being matched with the XRD pattern of the pristine wurtzite ZnO, the ex-situ coating of the bare ZnO-NPs with PEG-6000 (sample 3) exhibited additional features represented in peak broadening and the size dropped from 25.8 to 15.5 nm. While coating the in situ prepared ZnO-NPs (sample 4), the two additional reflection peaks at 2θ = 18.99 and 23.18°, which are distinctive ones for PEG-6000 with a dropped crystallite size of 28.7 to 18 nm. It is obvious that coating of either bare or in-situ synthesized ZnO-NPs using PEG-6000 significantly affects both the peak shape and peak position of the XRD pattern. The peaks get less intense, broader, and shift positions, indicating crystallite size reduction and structural changes. Conversely, to the authors who noticed that adsorption of PEG on the bare ZnO-NPs didn’t alter the crystalline construction of ZnO nanoparticles, and increasing the polymer molecular weight didn’t change the size of the coated NPs [14].
Regarding the Infrared (IR) spectra, the four samples presented absorption peaks characteristic to Zn-O stretching vibration (~ 432, 1635, and 3453 cm− 1) according to [20]. The FT-IR profile of sample 3 displayed lower intensity peaks corresponding to PEG-6000 compared to sample 2 (the in situ synthesized ZnO-NPs using PEG-6000 and ethanol) and sample 4 (the coated sample2). The more intense absorption peaks in sample 2 and their shift to low wave number reflect that the in situ use of PEG-6000 and ethanol during preparation of ZnO-NPs optimized the interaction and compatibility between the ZnO and PEG-6000. As well, the coating process was more efficient in case of sample 4 than sample 3 (ex situ coating of the bare ZnO-NPs).
The PEG-6000 coated in situ-prepared ZnO-NPs (Sample 4) represented nano-spherical morphology (∼19–67 nm) by TEM which indicate their capability to exhibit antibacterial activity. The smaller the particle size, the better biological activity through increasing interfacial area of reaction, the higher ROS release and the easier bacterial cell membrane penetration, hence inducing bacterial death more efficiently [24, 26]. In addition, ZnO-NPs release Zn2+ ions as a size-dependent phenomenon that may be responsible for the antimicrobial activity of ZnO-NPs causing bacterial cell wall rupture.
From the Minimum Inhibitory Concentration results in this study, the PEG-6000 coated in situ-prepared ZnO-NPs (Sample 4) elicited the best antibacterial effect (MIC = 0.1 mg/mL) followed by the (Sample 2) non-coated in situ-prepared ZnO-NPs (MIC = 0.2 mg/mL). The ex situ coated ZnO-NPs (Sample 3) mildly inhibited the bacterial growth, while the bare ZnO-NPs (Sample 1) completely failed to inhibit the bacterial growth in the broth media.
Failure of bare ZnO-NPs to induce antibacterial activity in the Minimum Inhibitory Concentration test may be due to their poor dispersibility and rapid precipitation in the broth (fluid) media, which accounts for the poor bioavailability, upon its application. Similarly, the scanning electron microscope (SEM) clarified that the bare ZnO-NPs are held together due to weak physical forces explaining particle agglomeration [13]. Moreover, suspending bare ZnO-NPs in the broth media, water molecules present, stimulated the formation of inter-particular Zn-O-Zn bonds and harsh particle agglomeration [10].
The in situ addition of PEG-6000 and ethanol as stabilizers (sample 2) lessened, somewhat, particles sticking together and allowed good dispersion in the broth media that displayed good antibacterial activity in the MIC. Likewise, the introduction of Poly Vinyl Alcohol as a surfactant during the synthesis of ZnO-NPs using zinc sulfate as a precursor sorted out the negative impact of particle agglomeration and particle separation [13].
Further coating of sample 2 with PEG-6000 (as a surface adsorbent) allowed better particle dispersion in the broth media and their protection against agglomeration or flocculation, making the NPs more attainable to the bacteria and improving the cellular incorporation of the nanoparticles and bacterial cell damage, consequently giving the maximal MIC value. This is attributed to the local solubilizing effect of PEG-6000 [27]. The polymer coating can effectively prevent water moiety in addition to modifying the particles’ surface through a combination of chemical and electrostatic interactions [14].
In this study, the antibacterial and synergistic activities of the optimized PEG-6000 coated in situ synthesized ZnO-NPs in the presence of PEG-6000 and ethanol (sample 4) were widely evaluated against 10 multidrug-resistant APEC strains. The minimum inhibitory concentration value of sample 4 was 0. 1 mg/mL.
A higher Minimum Inhibitory Concentration values of ZnO-NPs against E. coli were detected by [28] (0.15 mg/mL) and [29] who depended on ZnO nano-rods (0.250 mg/mL). It was noticed that the inhibition of E. coli requires high concentrations of non-coated ZnO NPs due to the cell wall lipopolysaccharides of APEC [6].
Based on agar well diffusion test, all E. coli strains were sensitive to PEG coated ZnO-NPs. The inhibition zone started to appear at a concentration of 0.1 mg/mL with a minimum zone (9 mm) while the best result was achieved with a concentration of 0.4 mg/mL of ZnO-NPs, where the inhibition zone reached its maximum level (20 mm).
At concentrations of 0.1 mg/mL and 0.2 mg/mL the percentage of sensitivity was 20 and 40%, respectively. Similar results were observed by [30], who reported the inhibition zone of Zn-ONPs against E.coli at 13 mm. Also [31], who stated that the best antimicrobial activity of ZnO-NPs against E. coli was at 1 mg/mL, and the maximum zone of inhibition was at 13 mm. Authors stated that coating ZnO nano-crystals with polymers have antimicrobial effect may reach 100% inhibition of bacteria at low concentrations [25].
The interaction between florfincol and streptomycin with PEG-6000 coated ZnO NPs was observed as follows: 30, 10% synergism, 30 and 20%partial synergism, 20 and 50% additive effect, respectively. Drugs exhibited a 20% indifferent effect and there was no antagonistic effect using FICI. The maximum zone of inhibition to florfincol (30 mm) and streptomycin (20 mm) was detected in combination with 0.4 mg/mL ZnO-NPs by a well diffusion test.
The additive effect of ZnO-NPs with the other antimicrobial agents may be attributed to the difference in mode of action as when ZnO-NPs become in contact with the plasma membrane of a bacterial cell, leading to change in the cell permeability and ZnO NPs move to the cytoplasm and affect the normal functioning of cell resulting in the formation of zone of inhibition against the microbes [32]. Moreover, florfincol is proposed to disrupt bacterial protein synthesis and is generally considered to be bacteriostatic, while streptomycin works by blocking the ability of 30S ribosomal subunits to make proteins, which results in bacterial death, respectively. Furthermore, combinations of drugs are much more effective synergistically by improving the uptake or decreasing the elimination or degradation of another drug (e.g., by drug efflux pump blockage).