Potentials of Iron Oxide Nanoparticles (Fe 3 O 4 ): As Antioxidant and Alternative Therapeutic Agent Against Common Multidrug-Resistant Microbial Species

For the treatment of pathogenic bacterial infections, multidrug resistance (MDR) has become a major issue. The use of nanoparticles is a promising strategy for combating medication resistance in a variety of pathogens that cause deadly diseases. The goal of our research was to extract multidrug-resistant bacteria from wound infections and then use iron oxide nanoparticles (Fe 3 O 4 ) as alternative therapeutic agents in vitro. Gram staining, morphological attributes evaluation, and biochemical testing were used to assess the microbes. The Kirby-Bauer disk diffusion method was used to test MDR-bacterial strains against several antibiotics; the majority of these isolates were resistant to ceftazidime, amoxicillin, Gentamicin, and tetracycline. the iron oxide nanoparticles were produced by the co-precipitation method and were confirmed by changing the color to dark black as well as the Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) analysis that shows the shape and average size between (29.03-56.54) nm. The highest effect of iron oxide nanoparticles (Fe 3 O 4 ) on the growth of Proteus.mirabilis ( P.mirabilis ) was as it was found that the average diameter of the inhibition zone was 22.66±1.15 mm, followed by Staphylococcus.epidermidis (S.epidermidis), Acinetobacter.baumannii ( A.baumannii ) with the average diameter of the inhibitory zone it was 21.66±1.52 mm, 20.33±1.53 mm respectively, and Candida.albicans ( C.albicans ) was 18.33±1.15 mm at 100 µgmL -1 (stock). The synthesized iron oxide nanoparticles (Fe 3 O 4 ) are used to capture rapidly microbes under the magnetic field effect. The antioxidant activity DPPH of the iron oxide nanoparticles (Fe 3 O 4 ) showed 29.3%, 42.2%, 58.6%, 67.4%, and 74 % at a concentration (6.25, 12.5, 25, 50, 100) µgmL -1 respectively, it demonstrated that the scavenging percentage increase with increasing the iron oxide nanoparticles (Fe 3 O 4 ) concentrations.


Introduction
Wound infections include surgical wound infections, acute soft tissue infections, bite wound infections, burn wound infections, and pyogenic wound infections. Wounds may take a long time to heal and may spread due to human habits. They are also aggravated in people with diabetes, obesity, or heart disease. Infectious wounds are dangerous, unsightly, odorous, and hypersensitive, giving patients pain and discomfort. S. epidermidis, P. mirabilis, A. baumannii and C. albicans. were found to be the most common isolates for wounds in a recent study [1].
Due to widespread antibiotic resistance and an increased incidence of infections caused by polymicrobial flora, wound infection control has become difficult [2]. Numerous antibiotics are becoming ineffective in managing bacterial illnesses as a result of multi-drug resistance in many bacteria. Because of overuse, self-medication, random prescribing of inappropriate medications, and prolonged antibiotic use, bacteria have developed drug resistance [3].
Furthermore, the conjugation of antibiotic resistance genes results in the emergence of resistant microorganisms [4]. Because of the rising number of antibiotic-resistant "superbugs," many diseases are becoming increasingly resistant to commonly used antibiotics. Drugresistant germs kill 25,000 people in Europe per year, whereas MDR-bacterial infections kill 23,000 people in the United States every year. Drug resistance in bacteria has been detected in all parts of the world, according to WHO studies. Approximately 50% of S. epidermidis, P. mirabilis, and A. baumannii infections were resistant to most effective antibiotics, such as Gentamicin, as a result, these drug-resistant bacterial isolates have a high fatality rate [5].
S. epidermidis is one of the wound infections, involving surgical wound infections, indwelling foreign devices, and bacteremia in immunocompromised patients, cardiac devices, and catheters [6], S.epidermidis, is a coagulase-negative gram-positive cocci bacteria, that is also a catalase-positive, non-motile and facultative anaerobe, it is the most common coagulasenegative Staphylococcus species that live on the human skin [7]. It is a very hardy microbe that forms white elevated cohesive colonies about 1-2 mm in diameter and is not hemolytic on blood agar, grouped in grape-like clusters, it is grow on mannitol salt agar medium but does not ferment mannitol it is an important test to distinguish it from another staphylococcus, it is spherical bacteria that grows easily on several growth media and varieties of varying medical importance, ability to cause disease and is present on the skin and mucous membranes in a large proportion and acts as opportunistic bacteria that occurs in damaged tissue [8].
P. mirabilis is most frequently a pathogen of Urinary tract infections (UTI), also one of the other bacteria that are present in wound infection. Gram-negative bacteria can cause bacteriuria and catheter obstruction [9], the swarming property of these bacteria appears, which helps them to spread widely in the area of injury, and the adhesion factor is one of the important virulence factors in it [10]. P.mirabilis strains are responsible for the majority of MDR ability to use antibiotics [11].
A. baumannii is a gram-negative bacteria, coccobacillus, non-motile, non-fermented lactose when grown on MacConkey agar and strict aerobic, catalase positive and oxidase negative [12]. Bacterial colonies developing on blood agar medium appear lead to light or white, it is not hemolytic, however, colonies on MacConkey agar appear smooth, small, and pale due to not fermenting lactose and size are 1-2 mm [13]. A. baumannii is an opportunistic pathogen and dangerous to humans because of the severe injuries it causes to patients who are hospitalized, including skin infections, soft tissue infections, Urinary tract infections, and Wound infections [14].
C. albicans this fungus is often grown on a sabouraud dextrose agar medium, is the only species that has the potential to form the germ tube in this test and in the presence of the stimulus that works to form it around the yeast cell, and the germ tube is crucial in the penetration process of the epithelial cell layer lining the body and tissues and access to the bloodstream [15], fungus infection as Candidiasis has been discovered in chronic and surgical wounds [16].
The routine use of antibiotics leads to the spread of antibiotic resistance, fighting against infections has become more difficult due to the emergence of different bacterial strains that are resistant to antibiotics (MDR bacteria). To overcome this, scientists are looking for new and more effective antibacterial agents. A good alternative for this purpose is nanotechnology [17]. We think that the antibacterial therapy can be improved considerably using iron oxide nanoparticles (Fe3O4) as Nano-vehicles for supporting the cellular growth and viability of pathogens, some anti-oxidants could be delivered externally through food and referred to as exogenous antioxidants. Lately, the development of biocompatible NPs having antioxidant properties has gained a great deal of attention [18]. Iron oxide nanoparticles (Fe3O4) ranging in size from 1-100 nm represent a new trend that is increasingly being developed and of interest for adoption in research related to medical applications [19]. Magnetic properties such as superparamagnetism, high magnetic susceptibility, high coercive, etc. [20,21]. The aim of this study is to diagnose MDR microbes, chemically synthesized iron oxide nanoparticles (Fe3O4), and study their antimicrobial and antioxidant activity against (S.epidermidis, P.mirabilis, A.baumannii, and C.albicans ).

Collection of microbial isolates
This study included the collection of isolates from wound infections in Hospitals. The diagnosis was made based on the morphological characteristics of the isolates by growing them on culture media, and they were diagnosed with biochemical tests and a final diagnosis with the Vitek2 System. The following microbes A.baumannii, P.mirabilis, S.epidermids and C.albicans were obtained.

Morphological and microscopic examination
Microbial isolates were cultured on the following culture media: MacConkey agar, this medium was used to investigate the ability of bacteria to ferment the sugar lactose, especially for gram-negative bacteria. Blood agar, this medium was used to detect the ability of bacteria to produce the hemolysin enzyme by observing the transparent areas surrounding the colonies. Mannitol salt agar, this medium was used for the ability of bacteria to ferment mannitol. Sabouraud dextrose agar, is used for the cultivation of fungi, then stained with Gram stain [22].

Preparation of McFarland solution
The McFarland standards (0.5) were prepared according to [23]. This solution was used as a reference to altering the turbidity of bacterial suspensions so that the number of bacteria stayed within a certain range, allowing microbiological testing to be more consistent [24].

Antibiotic Sensitivity test
In this study, 6 types of antibiotics were used based on the Clinical and Laboratory Standards Institute (CLSI 2021) [25]. The diffusion method was used, the colony was taken by a sterile loop from a culture grown for 24 hours and placed in a normal tube containing 5 ml of normal saline to make a bacterial suspension and compared with the turbidity of the preparation McFarland concentration of 0.5. On Muller Hinton agar medium, 100 μL of the bacterial suspension was taken and spread by sterile cotton swab then the dishes were left to dry and an antibiotic tablet was then placed in the dish, the plates were incubated for 24 h at 37°C, then the inhibition diameters were determined [26].

Synthesis and characterization of iron oxide nanoparticles (Fe3O4)
The iron oxide nanoparticles (Fe3O4) were prepared chemically using a co-precipitation method, which was done in the University of Technology/Applied science/Biotechnology laboratory. Initially, 4.8834g of iron (II) chloride di-hydrate (FeCl2·2H2O, 98%) and 7.2995g of iron (III) chloride anhydrous (FeCl3) were dissolved into 50 mL of De-ionized (D.I) water. Next, 25 mL of NH4OH was added under steady stirring. The solution was heated at 80 °C with constant stirring for one hour. After the reaction was completed, the black precipitated nanoparticles were washed several times with D.I. water to remove impurities and collected using an external magnet [27]. Scanning electron microscopy (SEM) analysis used to determine the size and shape of nanoparticles were taken on Scanning Electron Microscope (Zeiss, Jena, Germany). The morphological features and particle size distribution of the iron oxide nanoparticles (Fe3O4) were examined using a Transmission electron microscope (Zeiss, Jena, Germany) and Image J software [28] .

Detection of the antimicrobial activity of iron oxide nanoparticles (Fe3O4)
The test was carried out using the method of well diffusion, as Muller's medium was prepared according to the manufacturer's instructions, then left to solidify, then the microbial suspension was prepared and compared with a standard McFarland tube. Cotton swabs were used to disseminate the microorganisms, and wells were punched into the agar with a sterile well cutter. All types of microorganisms wells were poured with 100 μL of iron oxide nanoparticles (Fe3O4) at different concentrations (12.5, 25, 50, 100) µgmL -1 , then incubated in the incubator at a temperature of 37 ° C for 24 hours. The diameter of the inhibition resulting from each concentration was recorded [29,30].

Capturing of microbial isolates by iron oxide nanoparticles (Fe3O4)
S. epidermids, P. mirabilis, A. baumannii, and C. albicans were cultured in nutrient broth media for 24 hours to reach the density at 1.5x10 8 colony-forming units (CFU) ml in a typical experiment. After that, 1 mL of bacteria was collected by centrifugation. The bacteria were dispersed in 900 µL of PBS (phosphate buffer solution) and mixed with 100 μL of iron oxide nanoparticles (Fe3O4) solution and incubated in darkness at 37°C for 1 hour. For one minute, an external magnetic field was applied to the combination to see if the bacteria could be separated from the solution [31].

Antioxidant activity (DPPH assay)
The antioxidant activity of iron oxide nanoparticles (Fe3O4) with modest changes was evaluated using DPPH radicals. Iron oxide nanoparticles (Fe3O4) were used at concentrations (6.25, 12.5, 25, 50, and 100 µgmL -1 ) to look into the scavenging activity. The 750 µl of samples were blended with 750 µl of 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) solution and mixed well, then 750 µl of DPPH with 750 µl of methanol was used as a negative control, 750 µl of DPPH with 750 µl of ascorbic acid was used as a positive control. For 30 minutes, the samples and controls are kept at 37 °C temperature in the water bath [32]. At 517 nm, the optical density was read. Scavenging activity is calculated by the equation formula:

Microbial isolates and identification
To identify the microbial isolates of S.epidermidis, P.mirabilis, A.baumannii, and C.albicans the isolates were cultured on Maconkey agar, Gram-negative bacteria A.baumannii, P.mirabilis were cultured on this medium, the colonies appeared in pale color due to their inability to ferment lactose sugar, Mannitol-salt agar: Gram-positive bacteria S.epidermids were cultured on this medium to distinguish them from the other Staphylococci by their inability to ferment mannitol [33]. Sabouraud dextrose agar: C.albicans was grown on this medium, specific to the fungus [34]. Blood agar: was cultured on this medium, all these colonies of bacteria appeared gamma(ϒ) hemolysis this is evidence of the inability to produce hemolycin enzyme [35]. As shown in Figure (

Microscopic examination
Gram stain was used to distinguish the shapes, colors, and sizes of cells under the microscope. Gram-positive bacteria of S. epidermidis looked purple in color and formed like clusters under the microscope [6]. P. mirabilis bacteria had a pink color and were Gramnegative bacteria, but A. baumannii bacteria had a rod shape and were Gram-negative bacteria, as shown in Figure )2(. Gram-positive bacteria stained with the test dye crystal violet appear purple. This is due to the thick peptidoglycan coating of the bacterial cell wall that it retains. Gram-negative bacteria, are unable to maintain a violet dye. The anti-dye (safranin or fuchsine) is absorbed and turns red or pink due to the fact their peptidoglycan layer is drastically thinner and trapped among an internal membrane and a bacterial outer membrane [36].

Biochemical tests
Biochemical tests were carried out, such as S. epidermidis gram-positive bacteria, P. mirabilis, and A. baumannii, which appeared gram-negative bacteria. In terms of the ability of gram-negative bacteria P. mirabilis and A. baumannii to ferment lactose, they were not fermentable when grew on macConkey agar medium, and there was no hemolysis of these bacterial isolates when they grew on the medium of blood agar, and S .epidermidis bacteria it was not fermented mannitol when grown on mannitol salt agar medium, other tests showed negative results for both coagulase test and oxidase test, whereas positive result for Catalase test, growth test at 44 temperature was positive for A. baumannii bacteria and motility test was positive for P. mirabilis bacteria [37,38] as shown in Table (1).

Antibiotic sensitivity test of bacteria
Sensitivity was studied by the disk diffusion method to determine the extent of resistance of bacterial isolates to antibiotics and by comparing it with the standard tables mentioned in a previous study (CLSI,2021). The results showed that S. epidermidis was resistant to Ceftazidime, Amoxicillin and Gentamicin at a percentage of with an inhibition diameter of (11,12,13) mm. While it was sensitive to Imipenem, Ciprofloxacin, and Tetracycline with an inhibition diameter of (38, 35, and 20) mm respectively [39]. As for P.mirabilis bacteria, it showed resistance to Gentamicin and Tetracycline at a percentage of with an inhibition diameter of (10, 7)mm. While it was sensitive to Amoxicillin, Ceftazidime, Ciprofloxacin and Imipenem with an inhibition diameter of (26,24,38,22) mm respectively [40]. A. baumannii bacteria, resistant to Gentamicin with an inhibition diameter of (12) mm. While it was sensitive to Tetracycline, Ceftazidime, Amoxicillin, Ciprofloxacin and Imipenem and with an inhibition diameter of (32,24,20,32,38) mm. [41] A. baumannii has a high rate of resistance to most aminoglycoside antibiotics, and there is no correlation between biofilm development and antibiotic resistance [42]. As shown in the Figure )

Synthesis and characterization of iron oxide nanoparticles (Fe3O4)
Synthesis of iron oxide nanoparticles (Fe3O4): The results showed the iron oxide nanoparticles (Fe3O4) were successfully synthesized and had a dark black color as shown in Figure (4B) with ferromagnetic properties, which was in agreement with [43], who synthesized iron oxide nanoparticles (Fe3O4) by a chemical technique. The produced nanoparticles were strongly attracted by an external magnet as illustrated in Figure (

Characterization of iron oxide nanoparticles (Fe3O4)
Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) is used to analyze the morphology of the materials produced. Figure (5) shows SEM images of iron oxide nanoparticles (Fe3O4), which appear to be made up of small particles. Iron oxide nanoparticles (Fe3O4) synthesized by the co-precipitation method indicated the mean size of particles was (29.03-56.54) nm. Because the properties of Nano-crystal are significantly dependent on the dimension of nanoparticles, controlling the monodisperse size is critical [44]. Transmission electron microscopy (TEM) indicates that the iron oxide nanoparticles (Fe3O4) are spherical with an average mean size of 28.69 nm by using the Image J software, as shown in Figure (6) [45].

Antimicrobial activity of iron oxide nanoparticles (Fe3O4) against microbial isolates
Results of studying the effect of iron oxide nanoparticles (Fe3O4) by agar well diffusion method appeared that they have a significant inhibitory effect. The highest effect of iron oxide nanoparticles (Fe3O4) on the growth of P.mirabilis was as it was found that an average diameter of the inhibition zone was 22.6 mm, followed by S.epidermidis, A.baumannii with the average diameter of the inhibitory zone it was 21.6 mm, 20.3 mm respectively and C.albicans was 18.3 mm at 100 µgmL -1 (stock). A concentration of 12.5 µgmL -1 was the lowest inhibitory concentration, and an inhibitory diameter of at least 11.6 mm against P. mirabilis. As shown in Table (2) and Figure (7) [46,47]. It is obvious that as concentrations rise, the rate of inhibition rises as well. Iron oxide nanoparticles (Fe3O4) showed an antibacterial effect on Gram +ve and Gram -ve bacteria indicating that those nanoparticles are effective antibacterial agents. The presence of reactive oxygen species (ROS) produced by various nanoparticles is the primary cause of bactericidal activity. The chemical reaction among chemical material in the iron oxide nanoparticles (Fe3O4) and the outer bilayer of bacteria, as well as the reason for antibacterial of iron oxide nanoparticles (Fe3O4). The great efficiency of iron oxide nanoparticles can be attributed to their high biocompatibility and low toxicity to humans, in addition to their superparamagnetic properties. The iron oxide nanoparticles (Fe3O4) have a positive charge, while the microorganisms have a negative charge this is the mechanism of action, which forms electromagnetic gravitation between the iron oxide nanoparticles (Fe3O4) and the microorganisms, when the interaction takes place, the microbes are oxidized & depart this life immediately [48].

Capturing of microbial isolates by iron oxide nanoparticles (Fe3O4)
Magnetic response of iron oxide nanoparticles (Fe3O4) is confirmed via their migration in a matter of minutes toward a magnet placed near the solution, and the capture efficiency of microbial isolates before and after trapping was evaluated by iron oxide nanoparticles (Fe3O4). Figure (8) shows the capturing of S.epidermidis, P.mirabilis, A.baumannii, and C.albicans by magnetic IONPs dissolved in PBS solution. Iron oxide nanoparticles (Fe3O4) are very prominent in biomedicine; doesn't just for their inherent antimicrobial properties, but also for their super-paramagnetic nature [49]. Iron ions generate-oxygen radicals by converting hydrogen peroxide to the extra reactive hydroxyl-radical through Fenton re-action. Hydroxyl radicals generated via those iron ions can de-polymerize polysaccharides; due to DNA strand breakage, inactivated enzymes and initiate lipid-peroxidation. Moreover, magnetic nanoparticles bound to the cell membrane or cell membrane proteins; by electrostatic interactions is any other possible mechanism that could disrupt bacterial function and cause their death [50].

Antioxidant activity using DPPH radicals scavenging assay
When dissolved in organic solvents, DPPH is a free radical that is strong at room temperature and creates a darkish violet color. The UV saw absorption curve acquired for the synthesized samples as exemplified in Figure (9) it could be visible that the height depth of DPPH constantly dropped, and the lower in absorbance throughout 517 nm changed into used to calculate the unfastened radical scavenging percentage. Figure (9) shows the antioxidant activity of iron oxide nanoparticles (Fe3O4) exhibit potential free radical scavenging action. Ascorbic acid was used as a positive control in the same concentration range. The DPPH scavenging activity of iron oxide nanoparticles (Fe3O4) at five different concentrations (6.25, 12.5, 25, 50, 100 µg/mL) was 29.3%, 42.2%, 58.6%, 67.4%, 74 % respectively, it becomes referred to that this effect will increase with increase the concentration of iron oxide nanoparticles (Fe3O4) however it stays decrease as compared to ascorbic acid [51]. The presence of bioactive components in the extract with potent antioxidant activity can be attributed to the nanoparticles' radical scavenging capabilities, as a result, the surface functionalized iron oxide nanoparticles have considerable free radical scavenging activity. Because of their high cyto-compatibility and antioxidant activity, the produced nanoparticles could be a prospective candidate for a variety of biomedical applications. Surface functionalization with bio-entities can be employed to alter nanoparticle features as regards chemical functionality and applications [32].

Conclusions
The most commonly isolated microbes from septic wound infections were S. epidermidis, P. mirabilis, A. baumannii, and C. albicans. These isolates had a high resistance to ceftazidime, amoxicillin, Gentamicin, and tetracycline. Because physiological activities occur at the nanoscale, the association of iron oxide nanoparticles (Fe3O4) is expected to alleviate numerous biological and healthcare difficulties. The activity of the NPs is effective against common multidrug-resistant (MDR) species, and this is a pressing need as antibiotic abuse continues to promote the emergence of resistant strains around the world. Our research indicates the successful production of iron oxide nanoparticles (Fe3O4), as well as their use in antimicrobial efficacy against MDR-bacterial isolates from wound infections and antioxidants. We can conclude that the iron oxide nanoparticles (Fe3O4) have superparamagnetic and very effective antimicrobial agents.