1 Introduction

Hemostasis, inflammation, proliferation, and maturation are interrelated biological systems contributing to wound healing, leading to minimal damage and eventual recovery [1, 2]. Wound healing aims to reduce patients’ pain and scarring while preventing severe infection and accelerating wound healing [3]. Nanotechnology is evolving as a rapidly expanding field with its use in science and technology to produce effective nanoscale materials [4]. It opens a new era in wound treatment by providing methods that accelerate wound healing and showcasing unique qualities as bactericidal agents [5]. Nanoparticles (NPs) are particles with one dimension ranging from 1 to 100 nm [6]. Due to nanoparticles’ tiny size, they exhibit unique properties that significantly differ from those displayed by their relative bulk materials [7]. These particles, such as zinc oxide nanoparticles (ZnO-NPs), have huge surface areas, which enhances their activities [8]. ZnO-NPs are less toxic and more affordable than other metal oxide nanoparticles. They have biological uses, including drug delivery, wound healing, and anti-inflammatory purposes [9,10,11,12]. Reactive oxygen species (ROS) produced by ZnO-NPs can prohibit cell adhesion, proliferation, and migration [13, 14]. When free Zn2+ ions interact with biomolecules like proteins, all of the bacteria’s essential processes fail [15]. Numerous physical, chemical, and biological processes can be used to synthesize ZnO-NPs. However, physical and chemical procedures have several drawbacks, including using poisonous and dangerous substances, high energy requirements, and expensive costs, which restrict their suitability for biomedicine purposes [16, 17]. Because it is more economical and environmentally friendly than physical and chemical processes, biological ways (green process) are favored for the preparation of nanoparticles [18,19,20]. Moreover, investigations have indicated that prokaryotic bacteria are used as a component in the synthesis of NPs due to their simpler culturing, short development times, and tolerant experimental conditions (pH, pressure, and temperature) [21]. Considering where nanoparticles are biosynthesized, two distinct production processes can be categorized as intracellular and extracellular [22]. Additional procedures, such as ultrasound or interaction with suitable detergents, are needed to release the synthesized NPs after intracellular production [23]. Because it is less expensive and does not require a complicated downstream stream technique, the extracellular approach to producing NPs is preferred over the intracellular method [24]. Microbial enzymes and proteins reduce metal ions to create NPs in the extracellular process [25].

ZnO-NPs have broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. The antibacterial mechanism of action is thought to involve the release of zinc ions and the generation of reactive oxygen species (ROS) on the surface of NPs. These zinc ions and ROS can cause oxidative damage to bacterial cells, leading to cell death. In comparison, some other metal and metal oxide nanoparticles, such as silver nanoparticles (AgNPs) [26] and copper oxide nanoparticles (CuO NPs) [27], also exhibit antibacterial activity by releasing metal ions and ROS [28]. Some other metal and metal oxide nanoparticles, such as titanium dioxide nanoparticles (TiO2-NPs) and gold nanoparticles (AuNPs), have also been shown to promote the wound healing [29, 30]. ZnO-NPs have relatively low toxicity compared to other metal and metal oxide nanoparticles. This is thought to be due to the formation of a protective protein corona around NPs, which can reduce their interaction with cells and tissues [31]. ZnO-NPs have been shown to promote wound healing by stimulating cell proliferation, migration, and differentiation. The mechanism of action is thought to involve the release of zinc ions, which can activate signaling pathways involved in wound healing. ZnO-NPs can also enhance angiogenesis and extracellular matrix synthesis, which are important for tissue regeneration. A significant advantage of biosynthesized ZnO-NPs is their eco-friendliness and sustainability. Biosynthesis method typically uses natural and renewable sources, such as plant extracts, and microorganisms (fungi, actinomyctes and bacteria). Another advantage of biosynthesized ZnO-NPs is their potential for enhanced biocompatibility and reduced toxicity level. Biosynthesis methods can produce nanoparticles with controlled sizes and shapes, improving their biocompatibility and reducing their potential toxicity. In addition, the presence of natural compounds in the synthesis process can provide additional benefits, such as antioxidant or anti-inflammatory properties, which can be beneficial for wound healing applications [32]. Biosynthesis method can be considered as an affordable strategy owing to the utilization of natural and green resources, reduced energy and equipment costs, and potential for large-scale production [33]. On contrary, the chemically synthesized nanoparticles often require toxic solvents and reducing agents, which can be harmful to the environment and human public health. The microbiota of the skin plays a significant role in the development and homeostasis of cutaneous immunity. The skin is home to a diverse community of microorganisms, including bacteria, fungi, and viruses, collectively known as the skin microbiota [34]. The skin microbiota interacts with the host immune system and helps to maintain skin health and protect against pathogens. The skin microbiota can influence the development of the immune system in early life. Studies have shown that the skin microbiota composition in infants is associated with the development of the immune system and the risk of allergic diseases later in life. For example, infants with a higher abundance of Staphylococcus aureus on their skin have been found to have a higher risk of developing atopic dermatitis [35]. The skin microbiota can also modulate the immune response in the skin. The skin microbiota can interact with immune cells in the skin, such as Langerhans cells and T cells, and can influence the production of cytokines and other immune molecules. This interaction can help to maintain immune homeostasis in the skin and protect against infection and inflammation [36]. Other prevalent bacterial microflora species are found on the epidermis include Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes species.

This study aimed to investigate the wound-healing properties of green synthesized ZnO-NPs with three different concentrations using E. coli metabolic filtrated extract. The biofabricated ZnO-NPs were characterized to ensure their synthesis in nanoform. The antimicrobial performance of prepared biogenic synthesized ZnO-NPs was explored against some skin-causing bacterial infection.

2 Materials and methods

2.1 Materials

Zinc acetate (Zn(CH3COO)2) and sodium hydroxide (NaOH) were obtained from Sigma Aldrich Co. (Germany). Deionized water was used for dilution, analysis, and application.

2.2 Production of biomass

The bacterial strain E. coli ATCC 25922 was cultured in LB medium to produce the biomass for biosynthesis. The culture flask was incubated on an orbital shaker at 37 °C and agitated at 200 rpm. The biomass was harvested after 24 h of growth and centrifuged at 10000 rpm for 10 min. The supernatant was collected for further reaction [37].

2.3 Biological synthesis of ZnO-NPs

Three different concentrations of ZnO-NPs were synthesized by adding 12.5 mL of zinc acetate Zn(CH3COO)2 [0.11,0.23,0.35 M] to 12.5 mL of E. coli metabolic filtrated extract. After that, 1.25 mL of NaOH (10 g NaOH in 50 mL of distilled water) was slowly added drop by drop into the beaker containing Zn(CH3COO)2 and E. coli metabolic filtrated extract solution under stirring conditions for 30 min. Then, the mixture was centrifuged at 10000 rpm for 40 min; the white precipitate was washed with distilled water H2O and dried in an oven at 150 °C for 24 h. The obtained three concentrations were coded as ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3. ZnO-NPs-1 refers to the low concentration of ZnO-NPs prepared using 0.11M of zinc acetate. Meanwhile, the sample ZnO-NPs-1 was defined for the nanoparticles prepared using zinc acetate (0.23 M). Finally, ZnO-NPs-3 is the code for ZnO-NPs synthesized using a high concentration (0.35 M) of zinc acetate.

2.4 Physical characterization of ZnO-NPs

The optical characteristics of the biosynthesized ZnO-NPs were evaluated using UV-visible spectroscopy (UV-vis) in the wavelength range of 250–500 nm. The particle shape of ZnO-NPs was examined using TEM analysis. A small amount of ZnO-NPs was deposited on a coated copper grid to generate thin films, and any extra solution was blotted off using paper towels. Then, the grids were left for drying in the air before TEM investigation. Additionally, using dynamic light scattering (DLS), the particle size, polydispersity index, and zeta potential were determined via Malvern Instruments (UK). Scanning electron microscopy (SEM) was used to examine the ZnO-NPs’ surface morphology. With the application of energy-dispersive X-ray analysis (EDX), The elemental analysis of the scanned samples can be detected.

2.5 Microorganisms used

Six different types of skin infectious pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii as a model for Gram-negative (G-ve), Staphylococcus aureus and Staphylococcus epidermidis as a model for Gram-positive (G+ve) and were donated by the Rhizopus oryzae and Aspergillus niger as a model for fungal species were selected and inoculated aerobically in nutrient agar for bacteria and Sabouraud dextrose agar (SDA) for fungal species. The stock solution of each NPs was made by dissolving the appropriate mass of the tested ZnO-NPs (50 mg) into a tube having 5 mL of DMSO/deionized water and placed for sonication for 30 min.

2.6 Evaluation of antimicrobial behavior of ZnO-NPs

2.6.1 Inhibition zone assay

The antibacterial effects of biologically synthesized ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 were evaluated using the agar well diffusion experiment. The turbidity of the bacterial culture was modified to correspond with the 0.5 McFarland standards solutions after 24 h incubation in a nutrient broth medium. The microbes were then dispersed using streaking techniques over the upper layer of the Mueller-Hinton agar (MHA) and sabouraud dextrose agar (SDA) plate to induce sporulation. An aseptic cork borer was utilized to punch wells of 6 mm diameter on the MHA and SDA agar plates. The wells already inoculated with the tested bacterial/fungal cultures were then treated with different concentrations of the treatment solution (5, 15, 25, and 50 mg/L). Positive control substances were augmentin and voriconazole (500 mg/mL) for bacteria and fungi. While sterile distilled water was applied as a negative control. For an additional hour, the culture plates were held at 4 °C to give the NPs an adequate period to disseminate. The plates were after that maintained in the incubator for growth at the standard conditions of each type of microbe. Following the growth condition, the inhibition zones were measured to determine the antibacterial performance [38].

2.6.2 Estimation of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC)

The macrodilution (broth) technique used for the MIC assay was approved by the Clinical and Laboratory Standards Institute (CLSI) [39]. For the bacteria and fungi, the constant volume (5 mL) each of sterile Mueller-Hinton broth (MHB) and Sabouraud dextrose broth (SDB) were poured separately into the sterile test tubes. The evaluated ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 stock solutions served as the basis for making different concentrations (0–65 ppm). Following that, a standardized microbial inoculum resulted in the final approximation of log 6.2 CFU/mL microbial cell densities in a separate tube. After that, 5 mL of the broth medium containing the respective concentration of each NPs was applied successively along with 0.1 mL of the inoculum (3.6 × 106 CFU/mL). As positive control samples, broth media (MHB and SDB) having bacterial or fungal organisms were introduced into a tube. The fungal cultures were incubated for 5–7 days at 28°C, whereas the bacterial cultures were incubated for a day at 37 °C [40]. After the suitable incubation span of all tubes, each tube received 100 μL of a resazurin solution (0.1% w/v), which was allowed to incubate at 37 °C for approximately 2 h.

The term “MBC” or “MFC” means the smallest dosage of the antimicrobial agent, which can eradicate at least 99.9% of the microbes. In order to calculate the MBC/MFC, 100 mL of treated samples from MIC tubes that were withdrawn but had no observable microbial growth on them were streaked onto sterile plates of MHA for bacteria and SDA for fungi. The streaked plates were then incubated. The MBC/MFC levels were those at which no discernible proliferation was witnessed [41].

2.6.3 Time-kill kinetics assays

The time-kill assay was employed to determine the sufficient killing duration of NPs (ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3) against explored microbes. In essence, the cell suspension was modified to 1×106 CFU/mL. The estimated concentration levels that are MIC-equivalent, twice-MIC-equivalent, and four-MIC-equivalent of each NPs were utilized by inoculation in the 5 mL of PBS with 10 μL of each explored microbes. All mixtures were set in a frequent shaking incubator at 200 rpm. At frequencies of 0, 5, 15, 30, 45, 60, 75, 80, 105, and 120 min, aliquots (1.0 mL) of the medium mixture were withdrawn and aseptically seeded into MHA and SDA agar in sterile Petri dishes. The inoculated Petri dishes were incubated at optimal conditions for each type of bacteria and fungi. In parallel, another trial was run as a control using only the organisms. The test organisms’ colony-forming unit (CFU) was calculated, and the procedure was carried out three times. Log CFU/mL was displayed against time on a graph. Furthermore, the nonlinear dose-response modeling method was used in GraphPad Prism to obtain the IC50 values and log the IC record for the MIC values of each NPs [42].

3 Results and discussion

3.1 Biological synthesis of ZnO-NPs and their physical characterizations

Because of their straightforwardness, affordable prices, and advantages of preservation of the environment when compared to traditional chemical and physical paths, green synthesis approaches of metal and metal oxide nanoparticles have recently been brought to the interest of investigators [43, 44]. In the current investigation, ZnO-NPs were observed firstly by the naked eye via the appearance of a white precipitate that formed after 20 min of mixing the E. coli metabolic filtrated extract with zinc acetate solution. The appearance of this white precipitate is owing to the formation of zinc hydroxide coated by the metabolic filtrate of E. coli. In the second step, zinc hydroxide was converted to ZnO-NPs via a drying process. Figure 1 shows ZnO-NPs with absorbance peaks at 352 nm. As demonstrated in Fig. 1, the absorption of ZnO-NPs increased with the concentration of the utilized zinc acetate. The obtained data are in accordance with another study that affirmed that the absorption peaks of ZnO-NPs were around 340–385 nm [45].

Fig. 1
figure 1

Uv-vis spectra of ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3

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The morphology of the biosynthesized ZnO-NPs was examined with transmission electron microscopy (TEM). Figure 2a–c represent the particle shape of ZnO-NPs-1, ZnO-NPs-2, and, ZnO-NPs-3. It was observed that ZnO-NPs were prepared with an irregular shape. The aggregation and particle size were increased while the concentration of ZnONPs increased, as shown in Fig. 2c for the sample (ZnO-NPs-3).

Fig. 2
figure 2

TEM photographs of the biogenic ZnO-NPs synthesized with different concentrations: (a) ZnO-NPs-1, (b) ZnO-NPs-2, and (c) ZnO-NPs-3

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The appearance of aggregation particles is mainly attributed to nanoparticles’ reactivity which tends to combine with each other’s to form cluster particles. Thus, the formed nanoparticles need to be stabilized using the external stabilizing agent. In this current study, the metabolic filtrate of E. coli is able to prevent the aggregation of NPs. However, the metabolic filtrate cannot avoid the agglomeration of ZnO-NPs prepared using a high zinc acetate concentration (as shown for the sample ZnO-NPs-3).

The average particle size (nm), PDI, and zeta potential (mv) of the biosynthesized ZnO-NPs were calculated using dynamic light scattering (DLS) and zeta potential analyses and set in Table 1. The average particle size of the biological prepared ZnO-NPs-1, ZnO-NPs-2 and ZnO-NPs-3 recorded 79.19, 79.83 and 91.57 nm, respectively as seen in Table 1. As observed, the size of ZnO-NPs was marginally increased as zinc acetate concentration was increased.

Table 1 Average particle size (nm), polydispersity index (PDI), and zeta potential value of the biogenic ZnO-NPs with different concentrations
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Table 1 presents the PDI values for ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 as 0.265, 0.274 and 0.385, respectively. The homogeneity of ZnO-NPs is indicated by the illustration of nanoparticles with PDI values between 0 and 0.5. Therefore, all biosynthetized ZnO-NPs are assumed to contain homogenous particles. The negative protein groups covering the produced nanoparticles are responsible for any negative charges on their surface. The negative protein groups covering the generated nanoparticles are responsible for any negative charges on their surface [46]. In previous study, ZnO-NPs’ charge at the surface was assessed via zeta potential analysis, which revealed a mean zeta potential of -20.9 mV and suggested they had moderate stability, while the stability is considered as a good when the value is above -30 mV or +30 mV [47]. The zeta potential values of ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 are as follows: −34.3, −33.7, −33.4 mV (Table 1), respectively, indicating the stability of the biosynthesized ZnO-NPs.

3.1.1 Scanning electron microscopy (SEM) analysis

Morphological features of the bio-synthesized ZnO-NPs were examined via SEM. Figure 3 shows SEM of the three prepared samples: ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3. Each sample was scanned at two different magnifications. From the scanned samples, it was observed that the as-prepared ZnO-NPs exhibit rough surfaces with definite structures. Thus, SEM for analyzed samples (ZnO-NPs-1 and ZnO-NPs-2) shows the uniform shape of nanoparticles. Regarding the sample (ZnO-NPs-3), the shape was changed, and there were more aggregated particles. Overall, E. coli metabolic filtrated extract succeeded in forming ZnO-NPs with small size.

Fig. 3
figure 3

SEM images of the biogenic ZnO-NPs: (a, b) ZnO-NPs-1; (c, d) ZnO-NPs-2; (e, f) ZnO-NPs-3. Each sample has been scanned for two different magnifications

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The elemental analysis of the bio-synthesized ZnO-NPs (ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3) was determined using energy dispersive X-ray analysis (EDX). In all EDX graphs (Fig. 4), the existence of zinc and oxygen proved the preparation of ZnO-NPs. Meanwhile, the other elements such as nitrogen, carbon, phosphorus, and sulfur are mainly attributed to the E. coli metabolic filtrated extract. As the weight and atomic percentages of each component were recorded in the onset table, the variety of the percentage could be attributed to the scanned sample spot. As known, the surface elements are randomly deposited, so it is normal to find the percentage difference of each component in the scanned three samples. The percentage of zinc and oxygen elements of ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 was 41.52/40.16%, 45.97/34.62%, and 45/36.57%, respectively, affirming the successful biosynthesis of ZnO-NPs.

Fig. 4
figure 4

EDX spectra for ZnO-NPs: (a) ZnO-NPs-1, (b) ZnO-NPs-2, and (c) ZnO-NPs-3

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3.1.2 Antimicrobial properties of ZnO NPs

The Zone of inhibition measurements

ZnO-NPs have been shown to have antibacterial properties, and their use in wound healing applications has been explored due to their ability to promote wound healing and reduce inflammation. Bacterial cell-free metabolites, on the other hand, are compounds produced by bacteria that can have various biological activities, including antimicrobial activity. Six skin-infectious pathogenic microorganisms, namely, P. aeruginosa, A. baumannii, S. aureus, S. epidermidis, R. oryzae, and A. niger, were explored using the agar well diffusion technique to look into the effect of three biogenic synthesized ZnO-NPs with varying concentrations of zinc acetate on the proliferation of the selected potential pathogens (Fig. 5). From the stock solution of each separate ZnO-NPs, six various concentrations (5, 15, 25, 50, 75, and 100 mg/L) were prepared for antimicrobial trials. The highest measured width of ZOI of ZnO-NPs-3 indicated that vigorous antimicrobial action was observed against all selected pathogens, where the diameters of ZOI ranged between 16 and 36 mm for P. aeruginosa, 18 and 39 mm for A. baumannii, 13 and 31 mm for S. aureus, 14 and 33 mm for S. epidermidis, 12 and 28 mm for R. oryzae, and 11 and 25 mm for A. niger (Figs. 5 and 6). Results depicted that the antimicrobial potential of ZnO-NPs against G-ve bacteria was higher than other selected G+ve and fungal species. This phenomenon is attributed to the rigid structure and thick peptidoglycan in the cell wall of G+ve bacteria compared with the cell wall structure of G-ve bacterial which has a thin layer of peptidoglycan [48]. To assess their antibacterial effectiveness, the well-diffusion test was used with biologically produced ZnO-NPs against several kinds of microorganisms. According to the data, P. aeruginosa and A. flavus both exhibited a greatest inhibiting zone, measuring 22 ± 1.8 mm and 19 ± 1.0 mm, respectively [49].

Fig. 5
figure 5

Zones of inhibition of biogenic synthesized (A) ZnO-NPs-1, (B) ZnO-NPs-2, and (C) ZnO-NPs-3 (5 and 100 mg/L) against skin infectious pathogens using agar well diffusion assay

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Fig. 6
figure 6

Image of agar diffusion assay of different concentrations of ZnO-NPs-3 against tested bacterial species

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Furthermore, results have shown that augmentin has only antibacterial action with ZOI of 17 and 18 mm against P. aeruginosa and A. baumannii and 14 and 16 mm against S. aureus and S. epidermidis. On another side, voriconazole has a potent antifungal activity with no significant effect against bacterial species, where the ZOI for R. oryzae and A. niger was 14 and 12 mm, respectively.

3.1.3 MIC and MBC values

The MICs and MBCs/MFCs of three distinct NPs, including ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3, are presented in Table 2. The fact that the estimated MICs and MBCs of the studied ZnO-NPs-3 were much smaller than those of the ZnO-NPs-1 and ZnO-NPs-2 displays that nanoparticles have superior antimicrobial properties. Compared to other G+ve and fungal species, all antimicrobial NPs are considerably diminished in the MICs and MBCs/MFCs in G-ve bacterial species. ZnO-NPs-3 was found to have the most antibacterial capability for all of the chosen microbial species, followed by ZnO-NPs-2 and ZnO-NPs-1. This is likely because the ZnO-NPs-3 was biologically generated with increasing zinc acetate concentrations. The results summarized in Table 2 indicated that the measured MIC values of ZnO-NPs-3 against P. aeruginosa, A. baumannii, S. aureus, S. epidermidis, R. oryzae, and A. niger were 10 ± 0.14, 15 ± 0.06, 30 ± 0.21, 20 ± 0.43, 30 ± 0.12, and 35 ± 0.38 mg/L, respectively, while MBC/MFC values were 15 ± 0.54, 20 ± 0.24, 35 ± 0.18, 25 ± 0.37, 35 ± 0.82, and 40 ± 0.60 mg/L, respectively. Notably, the ZnO-NPs were highly efficient against P. aeruginosa and A. baumannii, compared to the others. The interaction effects of the respective microbes and ZnO-NPs provide a clearer picture of how the ZnO-NPs could interact with selected microbes. The diameter, structure, durability, and quantity of ZnO-NPs all affect their bactericidal properties. Rectangle ZnO-NPs produced using a green-mediated synthesis have efficient antimicrobial action toward S. aureus and E. coli. Findings indicate that the MIC value was 20.0 mg/L and that ZnO-NPs with a rectangular shape had a significant antimicrobial property at lower doses [50]. The results suggested that the size of NPs exhibits more vigorous antibacterial activity than the size of their counterparts [51].

Table 2 MIC and MBC values of the investigated nanomaterials against some human pathogenic microbes
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3.1.4 Time-kill kinetics assay

Figure 7 depicts the time-kill kinetics patterns of the MIC dose of biologically synthesized ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 against a variety of microbial pathogens. Findings demonstrate that after treatment with the tested NPs, there was a noticeable decrease in cell viability, followed by a rise for up to 120 min when compared to the control (microbial evolution without NPs). Concerning G-ve bacteria like P. aeruginosa and A. baumannii, time-kill kinetics profiles of investigated ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 at the MIC dosage employed demonstrated a corresponding decrease in the amount of cell viability after time points of 60, 30, and 15 min and 45, 15, and 10 min, respectively, followed by a steady rise up to the 24 h and a consequent decrease against its own control (Fig. 7a, b). Results of the time-kill kinetics profile of investigated ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 at the MIC dosage for G+ve bacteria, including S. aureus and S. epidermidis, revealed a decrease in the amount of cell viability for S. aureus at 60, 45, and 30 min and 60, 30, and 15 min for S. epidermidis, endured constant until 24 h, in comparison to its control (Fig. 7c, d). For R. oryzae and A. niger, the time-kill kinetics profile of investigated ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 against fungal species revealed a decrease in the amount of cell viability between 90, 60, and 45 min (Fig. 7e, f).

Fig. 7
figure 7

Time-kill kinetics of ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 against (a) P. aeruginosa, (b) A. baumannii, (c) S. aureus, (d) S. epidermidis, (e) R. oryzae, and (f) A. niger

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To explain the biosynthesized ZnO-NPs, the calculated values of IC50, Log IC50, and R2 were accomplished for three explored NPs (ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3) toward P. aeruginosa, A. baumannii, S. aureus, S. epidermidis, R. oryzae, and A. niger and are represented in Fig. 8 and Table 3. The estimated values of IC50, Log IC50, and R2 fluctuated according to the as-prepared and concentrated zinc acetate.

Fig. 8
figure 8

Normalized absorbance (%) of ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 against P. aeruginosa, A. baumannii, S. aureus, S. epidermidis, R. oryzae, and A. niger

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Table 3 The calculated values of IC50, Log IC50, and R2 for explored ZnO-NPs-1, ZnO-NPs-2, and ZnO-NPs-3 against P. aeruginosa, A. baumannii, S. aureus, S. epidermidis, R. oryzae, and A. niger
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Our findings (Table 3) show that the examined bacteria and fungus interacted with produced ZnO nanoparticles as antimicrobial agents in considerably different ways, with varying ZI marginal means. Compared to S. aureus and the other examined fungi, P. aeruginosa was the microbe that was more affected by the ZnO-NPs, which shows that it tended to be resistant to the particles. ZnO-NPs can inhibit the evolution of G+ve bacteria, G-ve bacteria, and fungi. The potency of the antibacterial effect depends on how sensitive the bacteria are [52]. The cell wall of G+ bacteria comprises a thick layer of peptidoglycan with a diameter of 20–80 nm that functions as a defensive barrier to safeguard the cell’s surroundings.

In contrast to G-ve bacteria possess a thin layer of cell walls that make these types of bacteria more sensitive to antimicrobial agents [53]. According to Baek and An [54], reported that G-ve bacteria are less resistant to ZnO-NPs than G+ve bacteria. Where it has been shown that the antibacterial capabilities of ZnO NPs directly correlate with their dosage and particle surface area. It has been demonstrated that zinc oxide nanoparticles’ concentration and particle surface area directly connect with their antibacterial properties [55]. Compared to ROS, ZnO-NPs could affect the media surrounding bacterial cells by forming them or altering the microbe’s cell wall due to their electrostatic adhesion to the cell surface, which can cause cellular damage [56]. Many infections, such as E. coli, S. aureus, P. aeruginosa, Bacillus subtilis, and others, can be suppressed by ZnO-NPs because of their exceptional features, including their crystallinity, permeability, particle size, and form [57]. Moreover, under UV and visible light irradiation, ZnO-NPs’ intra and interparticle pores enhance photocatalytic antimicrobial reactions based on ROS [58].

The possible mechanisms of antimicrobial wound healing formulations can depend on the specific ingredients and formulation [59]. Some possible mechanisms include the following: Direct killing of bacteria: Antimicrobial agents in the formulation can directly kill or inhibit the growth of bacteria at the wound site. For example, ZnO-NPs or iodine-containing compounds commonly use antimicrobial agents in wound healing formulations [60]. Modulation of the immune response: Antimicrobial agents can also modulate the immune response to enhance the body’s natural defense mechanisms against infections. For example, some antimicrobial agents can stimulate the production of antimicrobial peptides or enhance the activity of immune cells, such as neutrophils and macrophages, which can help to eliminate bacteria at the wound site. In summary, the hypothesis behind antimicrobial wound healing formulations is that they can expedite wound healing by preventing or treating bacterial infections. The possible mechanisms of these formulations can vary depending on the specific ingredients and formulation but may include the direct killing of bacteria, modulation of the immune response, promotion of tissue regeneration, and reduction of inflammation [61].

4 Conclusion

With the global need not to use chemicals harmful to humans and the surrounding environment, the focus of the current research was on the use of green chemistry in the preparation of zinc oxide nanoparticles (ZnO-NPs) with different concentrations to know the ability of extracellular metabolites of E. coli to stabilize zinc oxide in nanoform. Thus, ZnO-NPs are biologically synthesized from extracellular metabolites of E. coli, which is an inexpensive and environmentally friendly strategy for synthesizing ZnO-NPs with antimicrobial properties for potential use during wound repair and healing. The particles’ enhanced morphologies and extremely small sizes enable them to infiltrate cell membranes readily. Biogenic ZnO-NPs have powerful microbicidal effects against some skin-infectious pathogens when reduced by microbes, and toxicological studies have shown they have biocompatibility features. Therefore, the biogenic ZnO-NPs produced through biosynthesis could be utilized for biological purposes such as wound curing.