Pseudomonas indica-Mediated Silver Nanoparticles: Antifungal and Antioxidant Biogenic Tool for Suppressing Mucormycosis Fungi

Mucormycosis is considered one of the most dangerous invasive fungal diseases. In this study, a facile, green and eco-friendly method was used to biosynthesize silver nanoparticles (AgNPs) using Pseudomonas indica S. Azhar, to combat fungi causing mucormycosis. The biosynthesis of AgNPs was validated by a progressive shift in the color of P. indica filtrate from colorless to brown, as well as the identification of a distinctive absorption peak at 420 nm using UV-vis spectroscopy. Fourier-transform infrared spectroscopy (FTIR) results indicated the existence of bioactive chemicals that are responsible for AgNP production. AgNPs with particle sizes ranging from 2.4 to 53.5 nm were discovered using transmission electron microscopy (TEM). Pattern peaks corresponding to the 111, 200, 220, 311, and 222 planes, which corresponded to face-centered cubic forms of metallic silver, were also discovered using X-ray diffraction (XRD). Moreover, antifungal activity measurements of biosynthesized AgNPs against Rhizopus Microsporus, Mucor racemosus, and Syncephalastrum racemosum were carried out. Results of antifungal activity analysis revealed that the biosynthesized AgNPs exhibited outstanding antifungal activity against all tested fungi at a concentration of 400 µg/mL, where minimum inhibitory concentrations (MIC) were 50, 50, and 100 µg/mL toward R. microsporus, S. racemosum, and M. racemosus respectively. In addition, the biosynthesized AgNPs revealed antioxidant activity, where IC50 was 31 µg/mL when compared to ascorbic acid (0.79 µg/mL). Furthermore, the biosynthesized AgNPs showed no cytotoxicity on the Vero normal cell line. In conclusion, the biosynthesized AgNPs in this study can be used as effective antifungals with safe use, particularly for fungi causing mucormycosis.


Introduction
More than 1.2 billion people are infected by pathogenic fungus each year, resulting in at least 1.7 million fatalities [1,2]. Fungal pathogens now outnumber drug-resistant Mycobacterium tuberculosis in terms of mortality, and they even outnumber malaria [3]. Mucormycosis is one of the most dangerous fungal diseases for humans. Mucormycosis is a disease caused by a group of fungi called mucormycetes, which includes various genera such as Rhizopus, Mucor, Synsephalastrum, Absidia, and Cunninghamella [4,5]. These fungi invade people with a history of diabetes, stem cell transplants, cancer, injection drug use,

Isolation and Identification of Strain S. Azhar
The bacterial strain P. indica S. Azhar was isolated from a soil sample collected from the garden of the Faculty of Science, Al-Azhar University, Cairo, Egypt. For the isolation of bacterial strains, nutrient agar medium was used. The 16S rRNA gene sequencing for bacteria was used to identify it genetically. The improved approach was used to extract bacterial genomic DNA. Single colonies were taken from agar plates and suspended in sterile deionized water in 50 µL. The cell suspension was then incubated in a water bath for 10 min at 97 • C before being centrifuged for 10 min at 15,000 rpm to separate the DNA-containing cell lysate. Using a genomic DNA template and two universal primers, PCR was utilized to amplify the 16S rRNA gene. The primers 27-f (5-AGAGTTTGATCCTGGCTCAG- 3) and 1492-r were utilized . Amounts of 1× PCR buffer, 0.5 mM MgCl 2 , 2.5 U Taq-polymerase (QIAGEN, Hilden, Germany), 0.25 mM dNTP, 0.5 M of each primer, and 1 µL of isolated genomic DNA were added into the PCR mixture (50 µL). Sigma Scientific Services Company (Cairo, Egypt) used a Thermal Cycler to execute the PCR, which included a 3 min hot start at 94 • C, 30 cycles at 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 1 min, and 10 min of gene extension at 72 • C. GATC Company used an ABI 3730x1 DNA sequencer to evaluate the sequencing (Ebersberg, Germany). Using the NCBI BLAST software, the acquired sequences were compared to those in the GenBank database. The bootstrap technique was used to create the phylogenetic tree.

Bacterial Filtrate Preparation
P. indica cells were suspended in nutrient and injected in broth media for fermentation at 36 ± 2 • C for 48 h in an orbital shaker (120 rpm). The biomass was collected using filter paper No. 1 and rinsed in sterilized distilled water to eliminate any medium components before being suspended in 100 mL distilled water. At 34 ± 2 • C, the mixture was stirred for 24 h. Finally, the biomass filtrate was produced by passing it through Whatman filter paper No. 1 and centrifuging it for 5 min at 2000 rpm to sediment any remaining cell debris. The supernatant was used for AgNPs biosynthesis.

Biosynthesis of AgNPs by Biomass Filtrate
For the production of AgNPs, the previously prepared bacterial biomass filtrate of P. indica was employed as follows: in a 250 mL flask, 1 mM silver nitrate was combined with 100 mL biomass filtrate and incubated at 36 ± 2 • C for 24 h, and agitated at 150 rpm, as shown in Figure 1. Negative controls (cell filtrate) were also run along with the experiment.

Characterization of AgNPs
The absorption characteristics of biogenically produced AgNPs were evaluated using UV-vis spectrophotometry (JENWAY 6305 Spectrophotometer, UK). Quartz cuvettes were used to investigate solutions containing AgNPs in the wavelength range 200-800 nm. Allocating the peak in the region of 400 to 500 nm indicated the existence of AgNPs. In a mortar, a known weight of AgNPs, 1 mg, was ground with dry 2.5 mg of KBr. The powder was then placed in a 2 mm micro-cup with a 2 mm internal diameter and loaded onto a FT-IR set at 26 • C ± 1 • C. The sample was scanned using infra-red in the range of 400-4000 cm −1 , utilizing Fourier transform-infrared spectrometty. To determine the size and form of nanoparticles, TEM (JEOL 1010, Tokyo, Japan) was used to characterize AgNPs. Drop-coating the AgNPs solution onto the carbon-coated copper grid and loading it onto a specimen holder were used to produce the sample. The sizes and shapes of AgNPs were validated using TEM micrographs. Stress analysis, residual-austenite quantitation, crystallite size/lattice, crystallite calculation, and materials analysis by overlaid X-ray diffraction patterns with a Shimadzu apparatus, using a nickel filter and Cu-Ka target (Shimadzu-Scientific Instruments (SSI), Kyoto, Japan), were obtained with the XRD 6000 series. The average crystallite size of AgNPs can also be measured utilizing the Debye-scherrer equation: D = kλ/β Cos θ.

Antioxidant Activity
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity of the biosynthesized AgNPs was determined according to the method used by Khalil et al. [64], with minor modifications. Different concentrations of biosynthesized AgNPs (400-0.78 µg/mL) were used to determine the antioxidant activity. The DPPH solution (800 µL) was combined with 200 µL of the sample concentration and maintained at 25 • C in the dark for 30 min. After that, the absorbance was measured at 517 nm against a blank after centrifugation for 5 min at 13,000 rpm. The standard for this experiment was ascorbic acid. The following formula was used to determine antioxidant activity: Antioxidant activity (%) = Control absorbance -Sample absorbance Control absorbance × 100

In-Vitro Cytotoxicity
The cytotoxicity of AgNPs, at concentrations ranging from 31.25 to 1000 µg/mL, was tested against normal Vero cell lines obtained from ATCC using the MTT procedure [65] with minor modifications. As mentioned in Equation (1), the viability percentages were computed as follows:

Bacterial Strain Identification
Isolate S. Azhar's 16S rRNA gene sequence was 1140 bp, and the sequence was uploaded to NCBI (accession number MW820210). Strain S. Azhar has the greatest sequence similarity to P. indica, based on the 16S rRNA gene sequencing analysis. A neighbor-joining tree phylogenetic study further revealed that strain S. Azhar belonged to the Pseudomonas genus ( Figure 2).

Synthesis and Characterization of AgNPs
In this study, biomass filtrate of P. indica was incubated with 1 mM AgNO 3 for 24 h in dark conditions. Appearance of brown color after contacting of the filtrate of P. indica S. Azhar with precursor (AgNO 3 ) at the reaction completion indicated AgNPs' formation; maximum absorbance peaks at 420 nm were seen in the UV-vis spectral analysis, which might correlate to spherical AgNPs, as seen in Figure 3A. Surface plasmon resonance (SPR) excitation might be responsible for the color alteration. After calcination, AgNPs were obtained as a black powder. The functional groups of those found in AgNPs were characterized using FT-IR analysis. As seen in Figure 3B. The occurrence of functional assemblies of biomolecules was discovered using FTIR wavelengths of 400 to 4000 cm −1 . Ten prominent peaks in the FTIR spectra of biosynthesized AgNPs were found at 474.4, 617.1, 1110.8, 1382.7, 1617.9, 1637.2, 2032.6, 2921.6, 3235.9 , and 3415.3 cm −1 ( Figure 3B). The peaks at 3235.9 and 3415.3 cm −1 correspond to the alcohol O-H stretching group or the secondary amine N-H stretching group. NH stretching of the protein's amide I band is shown by the bands at 1637.2and 1617.9 cm −1 . Alkyne stretch bands are represented by the peaks at 2032.6 and 2921.6 cm −1 . Furthermore, the bands seen at 1382.7 and 1110.8 cm −1 might be attributed to aromatic and aliphatic amine C-N stretching vibrations. Finally, the peaks at 617.1 and 474.4 cm −1 correspond to the bending of alkene (C=H) groups. As a result, the current work reveals that proteins or bacterial extracts attach quickly to AgNPs via the proteins' free amino or carboxyl groups; moreover, the acquired form of NPs changes as the protein binding with AgNPs varies.
The most effective approach for identifying morphological features, such as the size and form, of biosynthesized AgNPs is TEM examination. Figure 4 reveals the effective manufacture of spherical AgNPs using metabolites found in P. indica S. Azhar's filtrate, with typical sizes ranging from 2.4 to 53.5 nm. Furthermore, the biologically produced nanoparticles were evenly diffused, with no aggregation or morphological discrepancy. XRD Figure 5A. The average sizes of crystallite Ag-particles were calculated using Scherrer's equation. In this context, the size of Ag particles ranged between 8 to 80 nm, which were the outputs from the analysis of the equation. In line with our clarification of the results, we reported the successful fabrication of crystallite, monoclinic-phase AgNPs at the same XRD diffraction planes utilizing metabolites of P. indica S. Azhar.

Antifungal Activity
In this study, the antifungal activity of biosynthesized AgNPs against R. microsporus, M. racemosus and S. racemosum was carried out as shown in Figure 6. Results illustrated that the biosynthesized AgNPs exhibited outstanding antifungal activity against all tested fungal strains. Moreover, the antifungal activity was the highest toward R. microsporus, where the inhibition zone was 38 mm at a concentration of 400 µg/mL of AgNPs. Furthermore, AgNPs at concentration 400 µg/mL exhibited potential antifungal activity against S. racemosum, but this was lower than for R. microsporus, where the inhibition zone was 24 mm. On the other hand, the antifungal activity was the lowest toward M. racemosus where the inhibition zone was 19 mm, as shown in Figure 6A. Moreover, the MIC of AgNPs was assessed using the broth microdilution method against all tested fungal strains. Results revealed that the MIC of AgNPs was in the range of 50-100 µg/mL according to the fungal strain, where MIC was 50 µg/mL toward R. microspores and S. racemosum, while it was 100 µg/mL against M. racemosus, as shown in Figure 6B.

Antioxidant Activity
In this study, the antioxidant activity of AgNPs at different concentration (400-0.78 µg/mL) was evaluated using the DPPH method as shown in Figure 7. Results showed that AgNPs exhibited antioxidant activity, where the IC 50 was 31 µg/mL when compared to ascorbic acid (0.79 µg/mL). In addition, antioxidant activity at 400, 200, 100, and 50 µg/mL were 96%, 86.6%, 72%, and 59.6%, respectively, and antioxidant activity decreased with decreasing concentrations, where the activity was 1% at 0.78 µg/mL. This suggests the possible application of AgNPs as an alternative antioxidant in the treatment of diseases that are caused due to free radicals.

Cytotoxicity
Evaluation of the cytotoxicity of compounds in human normal cell lines is considered the first step to detect their safety [64]. Cytotoxicity of AgNPs at different concentrations was tested against the Vero cell line CCL-81, as illustrated in Figure 8. Results revealed that the IC 50 of the biosynthesized AgNPs was 132.2 µg/mL, and the cell viability of Vero cells at two concentrations, 31.25 and 62.6 µg/mL, was 99% and 98%, respectively, which confirmed no cytotoxic effect of AgNPs at these two concentrations. In general, if the IC 50 is ≥90 µg/mL, the material is classified as non-cytotoxic [66]. Therefore, this confirmed that the biosynthesized AgNPs are safe to use.

Discussion
Green biosynthesis of AgNPs has risen in prominence as a potential alternative to chemical and physical techniques. Metabolites secreted by P. indica S. Azhar are effective in the formation of AgNPs, besides stabilizing the formed NPs. P. indica S. Azhar filtrate was utilized as a bio-reactor for the formation of AgNPs through harnessing bioactive macromolecules that are secreted from it. P. indica nanoparticles are currently in the early phases of development. P. indica-derived biogenic AgNPs are more appealing and less hazardous to the environment than other approaches. The proteins or enzymes involved in cell-free filtrates of P. indica that change nitrate to nitrite, and then reduced silver ions to silver in the metallic form, may be responsible for the synthesis of AgNPs. The absorbance peak at 420 nm in the UV-visible spectrum corresponded to the typical band of AgNPs generated by the P. indica cell free filtrate. The absorption peak at around 400-450 nm is attributed to AgNPs surface plasmon resonance, confirming the production of AgNPs [67]. Similarly, Alsharif et al. [68] observed that AgNPs generated by Bacillus cereus presented a single symmetric maximum at wavelength 420 nm, which is linked to spherical structure. The capping of biosynthesized AgNPs was discovered using Fourier transform-infrared spectroscopy. The FT-IR spectral analysis of AgNPs revealed N-H aliphatic amino group peaks at 3415.3 and 3235.9 cm −1 , whereas the -CH 2 group is indicated by the peak at 2921.6 cm −1 [69]. The (OH) stretch of the carboxyl group is shown by the spectrum peak at 2032.6 cm −1 . The binding of the amide I band of protein with the (N-H) stretch is associated with the peaks at 1637.2 and 1617.9 cm −1 [22]. The C-H and C-O stretching bands may be recognized at 1382.9 and 1110.8 cm −1 , respectively [47]. The relationship between Ag and physiologically active chemicals that are responsible for the production and stability of AgNPs as capping agents was investigated using FTIR. The infrared area of the spectrum is designated by the carboxylic acid group (CHOO-), obtained with an amine (NH 2 ) in the amino acid of proteins [70]. The most efficient approach for identifying morphological features, such as the size and form of produced AgNPs, is TEM examination. According to TEM data, the biosynthesized AgNPs had a spherical form with a size range of 2.4 to 53.5 nm. According to a previous study, researchers successfully formed spherical AgNPs with a size range of 6-50 nm using TEM, as seen in Alsharif et al. [68]. Other reports showed a difference in the shape and size of AgNPs biologically formed by bacteria [71,72]. The size range of AgNPs generated by B. licheniformis filtrate during incubation with 1 and 3 mM AgNO 3 was (3-130 nm) and (45-170 nm), respectively, according to Sarangadharan and Nallusamy [73]. When the biomass of B. licheniformis was incubated with 1 mM AgNO 3 , the mean size of AgNPs reached around 50 nm in another investigation [74]. Specific peaks in the XRD spectra were used to illustrate the XRD pattern of the AgNPs biosynthesized by P. indica. The face center cubic (fcc) nano-structures of AgNPs (111) [75]. Previous research that demonstrated the creation of AgNPs utilizing microbes had comparable XRD results [69,76,77].
Mucormycosis disease is very dangerous to humans and is caused by members of the Mucorales genera, such as Rhizopus, Mucor, and Sycephalastrum. Therefore, the control of these fungi by biomaterials is required. The biosynthesized AgNPs in this study revealed promising antifungal activity toward Mucorales genera species such as R. microsporus, M. racemosus, and S. racemosum. Medda et al. [78] synthesized AgNPs using Aloe vera leaf extract, and found antifungal activity toward Rhizopus sp. and Aspergillus sp. Alananbeh et al. [79] revealed that promising antifungal activity of AgNPs toward M. hemalis and R. arrhizus. AgNPs might be critical in breaking down such resistance. AgNPs' efficacy can be attributed to a variety of mechanisms, including cell wall disintegration, surface protein degradation, nucleic acid damage caused by the formation and buildup of reactive oxygen and nitrogen species (ROS and free radicals), and proton pump blocking. AgNPs are thought to cause a buildup of silver ions, which obstructs respiration by causing intracellular ion efflux, causing harm to the electron transport system [80].
Oxidative stress is a condition in which the balance between a cell's antioxidative defense and oxidants is disturbed as a result of oxidant excess [78]. Furthermore, the presence of oxidants causes oxidative changes of biological systems at the molecular level (unsaturated bonds of lipids, proteins, DNA, and so on), resulting in damage and, ultimately, hastened cellular death [79]. Antioxidants are natural or manmade compounds that can help to prevent or postpone oxidative cell damage (ROS, RNS, free radicals, other unstable molecules) [79]. Therefore, antioxidant compounds are continuously required to resist oxidative stress. The biosynthesized AgNPs in the current study revealed antioxidant activity where the IC50 was 31 µg/mL. Previous studies have confirmed the antioxidant activity of AgNPs from different sources [81][82][83]. González-Ballesteros et al. [82] reported that AgNPs, silver nanoparticles, can be made from Lactobacillus brevis exopolysaccharides, and these were tested for antioxidant capabilities. At 100 g/mL, AgNPs had a moderate DPPH radical scavenging potential of 34.09% [83]. Evaluation of the cytotoxicity of compounds on human normal cell lines is considered the first step to detect their safety [64]. Therefore, the cytotoxicity of biosynthesized AgNPs was assessed, where results confirmed that AgNPs are safe in use.

Conclusions
In the current study, a fast, green, eco-friendly method was used to synthesize AgNPs by P. indica S. Azhar for controlling fungi that cause mucormycosis, as well as antioxidant activity. Results revealed that AgNPs were fabricated using metabolites of P. indica, then, the biosynthesized AgNPs were characterized using different modern techniques. Moreover, the biosynthesized AgNPs exhibited outstanding antifungal activity against R. microsporus, M. racemosus, and S. racemosum. Furthermore, results revealed antioxidant activity without any cytotoxicity on the Vero normal cell line. The results of this study warrant further in vivo experiments.

Data Availability Statement:
The data used to support the conclusions of this study are accessible upon request from the corresponding author.