1 Introduction

Disorders caused by viral infection [1, 2], mutations [3], and further environmental factors [1, 4], such as tumour [5], and diabetes [6] are performing a critical difficulty for people. Therefore, the increasing bacterial defense to practically all recognized types of traditional antibiotics and the slightly greater bacterial infection have achieved important notification across the past decade [7, 8]. Identifying different approaches for primary development and prevention of diseases is highly important [8,9,10]. Nanotechnology was adopted to use nanomaterials with small size and more significant surface areas concerning their bulk equivalents [11,12,13]. The pathogenic bacteria that quickly resist the related antibiotics are Escherichia coli and Staphylococcus aureus. They were unique species of bacteria that were obtained directly from the intestinal area of animals. E. coli and S. aureus contributes to an enteric infection, and various species produce other intestinal disorders [14]. Many nanoparticles (NPs) were examined for their antimicrobial potential by applying the agar-disc diffusion process [7, 15]. The benefits of inorganic NPs are the powerful surface-to-volume ratio, many unusual features, and their nano-scale, which promotes actions to connect over the actual organisms like pathogenic fungi, bacteria, and unicellular yeast [16,17,18].

Recently, the binary metal molybdates (RMoO4; R=Nickel, Cobalt, Zinc, etc.) possess outstanding features that make them excellent materials in critical applications such as energy storage [19], corrosion resistance [20], sensors [21], water remediation [22], antimicrobial agents [23], and water splitting applications [24]. Among them, NiMoO4 (NMO) NPs possess the intense of researchers attributable to their merits such as extraordinary electric conduction, exceptional chemical stability, cost-effective [25], improved electrochemical display [26], photo-luminescence [27], magnetic behavior [28], optical, and photodegradation characteristics [29].

Also, between the NPs that applied in broad areas, silver (Ag) NPs have been distinctly utilized in many applications attributable to their outstanding characteristics such as antibacterial [30], antiviral [31], and antifungal activities [32], anti-inflammatory [33], and anticancer [34, 35]. In addition, nanomaterials showed promising antimicrobial abilities against various pathogenic microorganisms-forming biofilms [16]. The formed biofilms are networks of multi-cellular threads protecting these microorganisms from applied drugs [36,37,38].

Radiation-induced synthetic techniques, especially those that employ gamma rays combined with solvents, may have favorable characteristics and exceptional choices that make them prevail over their competitors from conventional procedures [39, 40]. The preference for this method is due to its ease of preparation, uncomplicated controllable on size, limited procedures for obedience, environmentally friendly to precursors and solvents [41, 42]. Recently, Abdel Maksoud et al. [43], have studied the antibacterial and antibiofilm activities of Ag@ zinc ferrite NPs against some pathogenic bacteria.

Accordingly, for the first time, this paper listed the gamma irradiation supported the sol–gel process for the construction of Ag modified-NMO NPs (Ag@NMO). Then, the pure NMO NPs, and Ag modified-NMO NPs were examined toward pathogenic strains separated from surgical rooms. The integrated Ag modified-NMO NPs were used as antimicrobial and antibiofilm factors against pathogenic bacteria. So, we assume that the organized NMO NPs, and Ag modified-NMO NPs can be applied for various purposes for biomedical and industrial applications.

2 Materials and Methods

2.1 Materials

Nickel(II) nitrate hexahydrate (Ni(NO3)2·6H2O), ammonium molybdate ((NH4)2MoO4), and silver nitrate (AgNO3) were chosen as source material for Ni, Mo, and Ag, respectively. At the same time, citric acid monohydrate (C6H8O7, 99.57%), and ethylene glycol (C2H6O2, 99.8%) were used in the synthesis of NMO NPs via a sol–gel method [10, 44,45,46,47]. All chemicals were obtained from Sigma-Aldrich (UK) and used as received in the solid-state.

2.2 Preparation of NMO NPs

The NMO NPs were synthesized via the facile sol–gel technique as follows [10, 44,45,46,47,48]. 0.1 M of Ni(NO3)2·6H2O and 0.1 M of (NH4)2MoO4 were taken in different beakers containing 40 ml di-ionized water. These aqueous solutions were mixed dropwise with the aid of magnetic stirring for 30 min to form a greenish-yellow precipitate (solution A). Further, the stoichiometric ratio of citric acid (in a molar ratio of 1:1 to the precursor metal salts) was dissolved in 40 ml of ionized water under magnetic stirring for 30 min (solution B). Then, solution B was added dropwise to solution A and accompanied by the addition of ethylene glycol (10 ml) as a polymerization promoter to begin the gel formation [49]. The resulting solution ultrasonic irradiation for 45 min where the reaction temperature was reached 80 °C at the end of the sonochemical process [28]. Then, the produced solution was dried (at 400 °C) to initiate the gel formation. Finally, the dried gel was cooled to room temperature and ground to obtain NMO powder. The resulted NMO powder was washed by water and ethanol several times and collected by centrifugation (4000 rpm for 10 min). The NMO powder dried at 900 °C via utilizing a muffle furnace [10, 50].

2.3 Preparation of Ag Modified-NMO NPs

5 g of pure NMO NPs was mixed with 0.25 g of AgNO3 and dispersed in deionized water. Further, the resulting suspension solutions were mixed under ultrasonic irradiation for 45.0 min. Finally, the mixture solution was subjected to 50.0 kGy at a dose rate of 1.1 kGy/h at ambient conditions. The irradiation was conducted employing Co60 gamma-cell sources [43, 51, 52]. After filtration and washing by a mixture of water/ethanol several times, the final product was collected by centrifugation (4000 rpm for 10 min). Then, the powder was dried in a vacuum at 60 °C to give Ag modified-NMO (Ag@NMO) NPs.

2.4 Characterization of Ag@NMO NPs

The pure NMO NPs and Ag-coated NMO NPs were examined via employing the energy dispersive X-ray analysis spectra (EDAX), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction technique (XRD), scanning electron microscopy (SEM), and SEM-EDX mapping analysis [53, 54].

2.5 Antimicrobial Potential of Pure NMO NPs and Ag Modified-NMO NPs

The tested pathogenic bacteria and unicellular fungi were separated from medical places and identified in our published articles [44]. They were Staphylococcus sciuri, Staphylococcus vitulinus, Staphylococcus aureus, Enterococcus columbae, Staphylococcus lentus, Candida tropicalis, and Candida albicans, which were helpful taken from Microbiology Lab., NCRRT, Cairo, Egypt. All the examined bacteria and unicellular fungi were maintained on nutrient agar slants at 5 °C for another usage [55]. Well diffusion method was used to estimate the impacts of pure NMO NPs and Ag@NMO NPs on the examined bacteria and unicellular fungi [56]. To investigate the antimicrobial effect of pure NMO NPs and Ag@NMO NPs applying the well diffusion assay, bacterial and Candida suspension was developed and set to standard 0.5 McFarland’s concentration equal to (1–3) × 108 CFU/mL. The examined bacteria and unicellular fungi were swapped on a nutrient agar medium. Then 6.0 mm wells were cut on each nutrient agar plate outside and loaded by 50 μL of the examined treatments, and Amoxicillin (AX) and Nystatin (NS) as a positive control. All the tests were done in triplicates, and the tested plates were incubated overnight at 37 °C [57]. The microbial cloud lack of growth was described as a zone of inhibition (ZOI) in mm.

2.6 Minimum Inhibitory Concentration (MIC) Analysis of Pure NMO NPs and Ag@NMO NPs

The well distribution assay determined MIC of pure NMO NPs and Ag@NMO NPs upon the examined bacterial and Candida strains [58]. The overnight examined cultures were incubated for 3.0 h at 37 °C. Following that, the inoculum of the established bacteria and unicellular fungi was set to 0.5 McFarland. A complete volume of 50 µL of bacterial and Candida suspension was injected into nutrient agar medium plates. Pure NMO NPs and Ag@NMO NPs were serially diluted two-fold with sterile distilled water to varying concentrations (as µg/mL) then set separately in 6.0 mm wells formed on the agar plate's surface. The examined plates were incubated overnight at 37 °C, and the ZOI was assessed in mm. MIC was determined as the most negligible concentration of pure NMO NPs and Ag@NMO NPs that will hinder the possible extension of bacteria and Candida following 24 h incubation [59].

2.7 Antibiofilm Determination of Pure NMO NPs and Ag@NMO NPs

Pure NMO NPs and Ag@NMO NPs were examined for their antibiofilm potential toward the tested bacteria and Candida species by tubes method [60]. The examined test tubes, including 5.0 mL of the nutrient broth, were mixed with 10 µL of 0.5 McFarland (1–3 × 108 CFU/ml) of the tested bacteria and Candida species. Moreover, 0.25 mL of examined pure NMO NPs and Ag@NMO NPs was included in the tubes while the equivalent volume of water was combined with the control tubes. The prepared tubes were incubated overnight at 37 °C. Next to the incubation, the media in the tubes were excluded. The tubes were mixed with phosphate buffer saline (PBS; pH 7) and subsequently dehydrated [61]. About 5.0 mL of sodium acetate (3.0%) was applied for 10 min to maintain the adherent bacteria and Candida species, which were rinsed with deionized water [62]. The bacteria and Candida biofilms were dyed with crystal violet (CV; 0.1%) for 20 min and washed with deionized water. Subsequently, 3.0 mL ethanol was combined to terminate the CV. The biofilms were quantitatively determined by UV–Visible spectrophotometer (570 nm). The inhibition percentage was assessed by (Eq. 1) [63].

$${\text{Inhibition}}\,\% = \frac{{{\text{ODc}} - {\text{ODt}}}}{{{\text{ODc}}}} \times 100$$
(1)

where; ODc: The absorbance of the control sample (without treatment), ODt: The absorbance of the treated samples.

2.8 Reaction Mechanism Estimation by SEM

The tested bacterial and Candida cells were washed with PBS and flooded with 3.5% glutaraldehyde. The maintained bacterial and candida cells were rinsed regularly with PBS and cleaned with ethanol for 20.0 min at 24 °C before drying. Eventually, the bacterial and Candida cells were installed and set above the aluminium pieces to begin the SEM imaging [64]. The treated bacterial and Candida cells’ morphological and surface characteristics with the tested Ag@NMO NPs had identified by SEM (ZEISS), and comparing with control samples [65].

2.9 Statistical Analysis

The statistical estimate of our results was conducted using the ONE WAY ANOVA (at P < 0.05) and decided to be Duncan's multiple varieties studies and the least significant difference (LSD) report [57]. The results and data were analyzed and inquired by SPSS software version 15.

3 Results and Discussion

3.1 Structural Analyses

Figure 1 (A&B) exhibited the EDX spectra of NMO and Ag modified-NMO (Ag@NMO) NPs. The figure confirms the presence of all fundamental elements with a stoichiometric ratio. The EDX spectra are included several peaks such as Ni, Mo, O, and Ag, which affirm the existence of NMO and Ag@NMO NPs [66].

Fig. 1
figure 1

EDX spectra of A pure NMO NPs, and B Ag@NMO NPs

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XRD patterns of both the bare NMO NPs and Ag@NMO NPs are displayed in Fig. 2. The detected diffraction peaks of bare NMO NPs belonged with the (JCPDS cards No: 01–086-0361) and affirmed the successful synthesis of NMO NPs without any foreign phases [66,67,68]. While, for Ag@NMO sample, it is illustrated that the intensity of the detected NMO’s peaks was declined. This may be attributable to Ag coating over the NMO surface. Also, the figure confirms the presence of Ag NPs on the Ag@NMO sample. Three diffraction peaks were detected at 2 \(\theta\) = 38.44°, 44.51°, and 64.49° (denoted by *) that affirming the formation of Ag NPs (Fig. S1) [69, 70]. Further, the figure illustrated that the peaks were shifted to lower for Ag@NMO sample [71]. This means the increase in the lattice constant of Ag@NMO NPs attributable to the effect of gamma irradiation besides the decoration with Ag NPs [72,73,74]. The average crystallite size (D) for the bare NMO NPs and Ag@NMO NPs was estimated via Scherrer’ equation and is found to be 71.8 nm, and 48.28 nm respectively [75, 76].

Fig. 2
figure 2

XRD patterns of pure NMO NPs and Ag@NMO NPs

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Figure 3 shows the HRTEM images of NMO and Ag@NMO NPs. Figure 3A exhibits that the particles of NMO possess a hexagonal shape in the nanoscale regime. Also, Fig. 3B displayed that the particles of Ag modified (as bright particles) on the surface of the hexagonal NMO.

Fig. 3
figure 3

HRTEM images of A pure NMO NPs, and B Ag@NMO NPs

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Figure 4 presents the SEM images of pure NMO NPs and Ag@NMO NPs. Figure 4A confirmed that the pure NMO NPs have a nanoplate shape. At the same time, Fig. 4B illustrated the decoration of Ag nanorods on the NMO nanoplate.

Fig. 4
figure 4

SEM images of A pure NMO NPs and B Ag@NMO NPs

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Also, the mapping images confirm the presence of Ni, Mo, O, and Ag elements. Besides, the uniform distribution of these elements over the Ag@NMO sample was affirmed in Fig. 5 [77].

Fig. 5
figure 5

Represents the SEM and elemental mapping images of Ag@NMO NPs

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3.2 Antimicrobial Potential of Pure NMO NPs and Ag Modified-NMO NPs

The well diffusion experiment was performed to evaluate the antimicrobial performance of pure NMO NPs, and Ag@NMO NPs where Amoxicillin (AX) had been used as a positive antibacterial control against S. sciuri, S. aureus, E. columbae, S. lentus, and S. vitulinus and Nystatin (NS) had been used as a positive antifungal control upon C. albicans, and C. tropicalis as presented in Table 1 and Fig. 6.

Table 1 The antimicrobial activity (ZOI) and minimum inhibitory concentration (as µg/mL) of NMO NPs and Ag@NMO NPs
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Fig. 6
figure 6

Antimicrobial activity as ZOI for NMO NPs, and Ag@NMO NPs against different pathogenic bacteria and unicellular fungi

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The results revealed that both pure NMO NPs and Ag@NMO NPs express antimicrobial action toward the examined pathogenic bacteria and Candida species. Additionally, the potential antimicrobial progress with the treatments, including silver. Accordingly, Ag@NMO NPs reported the most significant ZOI against E. columbae (33.3 ± 0.115 mm), and C. albicans (30.8 ± 0.572 mm) which was mainly influenced by Ag@NMO NPs.

3.3 MIC Investigation

MIC testing of pure NMO NPs and Ag@NMO NPs were studied. This analysis was carried out on pure NMO NPs, and Ag@NMO NPs at various concentrations of (as µg/mL) performing well diffusion method [78], and the results have been included in Table 1. MIC results are diversely proportional with the potential of the tested treatment as antimicrobial action. The lowest MIC concentration of pure MNO NPs was recorded at 12.5 µg/mL upon S. lentus. As matched with well diffusion method results, Ag@NMO NPs were a further efficient antimicrobial factor than NMO NPs so, the MIC results in the presence of Ag were lower than pure NMO NPs. At the same time, Ag@NMO NPs showed the lowest MIC concentration. In Ag@NMO NPs, MIC concentration was low against E. columbae (0.048 µg/mL). Ag@NMO NPs have double function working as bacteriostatic in low concentration, on the other hand, acting as bactericidal at the high concentration [79].

3.4 Antibiofilm Activity of Pure NMO NPs and Ag Modified-NMO NPs

Pure NMO NPs and Ag@NMO NPs were adopted to assess their potency to restrain the examined bacterial strains' biofilm development and Candida species. About 0.5 mL of pure NMO NPs or Ag@NMO NPs were mixed into the tubes, including 5.0 mL of the nutrient broth injected with 10 µL of 0.5 McFarland (1–3 × 108 CFU/mL) of the tested bacteria and unicellular fungi. At the same time, the equivalent volume of sterile distilled water was attached to the negative control tubes. It was pointed in Fig. 7 that Ag@NMO NPs was more effective in inhibiting the biofilm of all the examined bacteria and unicellular fungi preferably than pure NMO NPs.

Fig. 7
figure 7

Anbiofilm activity as inhibition % for NMO NPs, and Ag@NMO NPs against different pathogenic bacteria and unicellular fungi

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The antibiofilm potential of Ag@NMO NPs was the most powerful, recording 94.32% for E. columbae, 91.99% for S. vitulinus, and 90.98% for C. albicans (Table 2). Since the antimicrobial potency of NPs effectively depends on the quantity of the particles, the small size of Ag@NMO NPs produces strong antibiofilm potential to the bacteria and unicellular fungi. It had known that biofilms are the effect of a general disease, and around 80% of bacterial infections in the world had combined with biofilms formation [80]. The ecological specifications and appearance of particular genes generated by adhesion may probably manage biofilms’ metabolic activity [81]. The environmental requirements and expression of particular genes produced by adhesion may theoretically control biofilms' metabolic action. [82]. The low cellular metabolism preserves the antimicrobial agents that improve within bacterial extension [83].

Table 2 Semi-quantitative inhibition% of the biofilm formation for non-treated and treated bacterial and yeast pathogens with NMO NPs, and Ag@NMO NPs
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3.5 Reaction Mechanism Determination by SEM Analysis

SEM examination was carried out to demonstrate the possible antimicrobial reaction mechanism against E. columbae, as observed in Fig. 8. The SEM examination of the control untreated E. columbae in the absence of Ag modified-NMO NPs showed bacterial accumulations that had normally growen with regular appearance. The usual bacterial surface and semi-developed biofilm had been observed (Fig. 8a). Simultaneous Ag@NMO NPs treatment, morphological alterations had been discovered in E. columbae (Fig. 8b), such as the complete lysis of the exterior surface accompanied by deformations and malformation of the E. columbae cells with the decrease in the total viable number, and the biofilm extension was restricted.

Fig. 8
figure 8

SEM imaging of E. columbae; where a regular bacterial cells without Ag@NMO NPs treatment, b abnormal, deformed and irregular bacterial cell with complete lysis following Ag@NMO NPs treatment, fully-irregular and deformed bacterial cell through Ag@NMO NPs treatment presenting the full lysis of E. columbae cell, and malformed bacterial cell after the treatment of Ag@NMO NPs

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The schematic design presented in Fig. 9 exhibits the possible antibacterial mechanism. It must be understood that Ag@NMO NPs start their action with adhesion at the outer surface of the bacterial cell, causing membrane damage and alter the transport flow. Then, the diffusion of Ag+ in the bacterial cell (at pH = 3) and interacting with all intracellular structures such as a plasmid, DNA, and other necessary organelles. Ultimately, cellular and genetic toxicity occurs because of the oxidative tension created by reactive oxygen species (ROS) production. Finally, NMO NPs have resisted the bacterial cells' acidic environment, and the transfer did not happen [64], but kept the antibacterial potential by altering the signal transduction mechanisms [84].

Fig. 9
figure 9

Schematic representation regarding the four prominent ways of antibacterial potential of Ag@NMO NPs, where (1) Ag@NMO NPs adhere to the bacterial cell surface and results in membrane damage and altered transport activity; (2) Ag@NMO NPs block the ions transport from and to the bacterial cell, (3) Ag@NMO NPs create and increase the ROS leading to bacterial cell wall damage, and (4) Ag@NMO NPs penetrate inside the bacterial cells and interact with cellular organelles and biomolecules, and thereby, affect respective cellular machinery, and modulate the cellular signal system and causing cell death. Ag@NMO NPs may serve as a vehicle to effectively-deliver Ag+ ions to the bacterial cytoplasm and membrane, where proton motive force would decrease the pH to be less than 3.0 and therefore improve the release of Ag+ ions

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4 Conclusion

For the first time, this work exhibits that gamma irradiation supported the sol–gel structure of Ag modified-NMO NPs. The XRD established the strong development of Ag@NMO NPs. Also, the EDX spectra confirmed that the fundamental elements were uniformly distributed within the Ag@NMO NPs without any foreign particles, which declared the purity of the Ag@NMO NPs. Also, the HR-TEM image shows that the particles of NMO possess a hexagonal shape in the nanoscale regime. Ag modified-NMO NPs was the well-known decisive antimicrobial activity towards all the tested pathogenic bacteria and unicellular fungi. The capability of Ag@NMO NPs decreases in the following form E. columbae (33.3 mm) ˃ S. vitulinus (32.0 mm) ˃ C. albicans (30.8 mm) ˃ S. aureus (25.5 mm) ˃ C. tropicalis (21.2 nm) ˃ S. sciuri (18.2 nm) ˃ S. lentus (16.0 nm). In Ag@NMO NPs, MIC concentration was low upon E. columbae (0.048 µg/ml). The antibiofilm potential of Ag@NMO NPs was the most powerful, recording 94.32% for E. columbae, 91.99% for S. vitulinus, and 90.98% for C. albicans. Since the antimicrobial strength of NPs dramatically depends on the particles size, the small size of Ag@NMO NPs provides effective antibiofilm behavior against the tested bacteria and unicellular fungi. It had recommended that Ag modified-NMO NPs replace some disinfectant liquids used for the surface disinfection in hospitals and some colors in the medical operating offices to protect the environment from entering the pathogenic bacteria. Furthermore, Ag modified-NMO NPs may have been acquired as a component in medical cosmetics and pharmaceuticals for biomedical approaches. Finally, Ag modified-NMO NPs may be employed in construction and environmental goals like rainwater processing from the contaminants and protect the atmosphere from deadly pathogens and dangerous diseases.