Ultrasound assisted-phytofabricated Fe3O4 NPs with antioxidant properties and antibacterial effects on growth, biofilm formation, and spreading ability of multidrug resistant bacteria

Abstract Complicated issue in infectious illnesses therapy is increasing of multidrug resistant (MDR) bacteria and biofilms in bacterial infections. In this way, emerging of nanotechnology as a new weapon specifically in the cases of metal nanoparticle (MNPs) synthesis and MNPs surface modification has obtained more attention. In this study, ultrasound-assisted green synthesis method was utilized for the preparation of Fe3O4 NPs with novel shape (dendrimer) through leaf aqueous extract of Artemisia haussknechtii Boiss. Ultraviolet–visible spectroscopy, energy dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), atomic force microscopic (AFM), X-ray diffraction (XRD) techniques were applied for MNPs physicochemical characterization. Also, disc diffusion assay, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), planktonic and biofilm morphology of three pathogenic bacteria involving Serratia marcescens ATCC 13880, Escherichia coli ATCC 25922, and methicillin-resistant Staphylococcus aureus (MRSA) were evaluated upon treatment of Fe3O4 NPs as antiplanktonic and antibiofilm analysis. Results showed efficient antiplanktonic and antibiofilm activities of biosynthesized Fe3O4 NPs with average diameter size of 83.4 nm. Reduction in biofilm formation of S. aureus ATCC under Fe3O4 NPs stress was significant (66%) in higher MNPs concentration (100 μg/mL). In addition, as first report, spreading ability of S. aureus as important factor in colony expansion on culture medium was reduced by increasing of Fe3O4 NPs. Present study demonstrates striking antiplanktonic, antibiofilm, antispreading mobility and antioxidant aspects of one-pot biosynthesized Fe3O4 NPs with novel shape.


Introduction
Every year, approximately 100,000 tons of antibiotics against infectious diseases are produced worldwide [1]. Misusage of this amount of antibiotics has lead to multidrug resistance emerging among pathogenic strains, specifically in bacteria [2]. In addition to high costs, MDR has resulted in high-mortality rates through the inefficiency of conventional antimicrobial agents [3]. According to natural selection phenomenon, spreading of resistant pathogens such as bacteria, fungi, and viruses are caused extremely by using new mechanisms of MDR in these microorganisms [4]. For the case of bacteria species, these mechanisms include mainly the application of multidrug efflux pumps and resistance plasmids [5]. Another problem associated with resistance to antibiotic chemotherapy is the formation of chronic biofilm [6]. As a definition, bacterial biofilms are a slimy layer of bacteria that adhere to biotic and abiotic surfaces [7]. With regard to the medicinal aspect, biofilm formation can be contributed to chronic infections such as cystic fibrosis and periodontitis [8]. In order to remove biofilm, using an efficient strategy to disrupt the multicellular structure of the biofilm is necessary [9]. Recently, nanotechnology was used as a novel and powerful tool in medicinal offers [10]. In this technology, NPs application, specifically metal NPs (Ag, Cu, TiO 2 , ZnO, MgO, and Fe 3 O 4 NPs) illustrated antimicrobial and antibiofilm activities against MDR pathogens [11,12]. Among these effects, antibacterial activities of magnetic iron oxide (Fe 3 O 4 ) NPs have been reported by many investigators [13,14].
Based on bottom-up and top-down approaches, there are many ways for preparation of Fe 3 O 4 NPs including hydrothermal synthesis, thermal decomposition, ultrasound-assisted reduction, co-precipitation, electrochemical synthesis, and laser pyrolysis techniques as chemical and physical methods [15]. These methods have an advantage by the uniformity of MNPs distribution and disadvantages by consumption of toxic and expensive materials in MNPs preparation [16]. In recent years, green synthesis was introduced and applied by many scientists as a novel and effective process [17,18]. Several types of living organisms such as microorganisms (specifically magnetotactic bacteria), plants, and fungi were used for MNPs synthesis [19]. Among these organisms, plants have the advantages of more biocompatibility and availability than microbes and fungi [20]. These advantages are caused by this fact that plants have various secondary metabolites like flavonoids, flavonols, and terpenoids which they can contribute in the reduction and stabilizing of metal ions and MNPs structure, respectively [21]. Major disadvantages of green synthesis method are agglomeration and ununiform size and shape of NPs. For reducing these unsuitable results, we used ultrasonic wave as ultrasound-assisted reduction method. Advantages of this strategy were reported for the biosynthesis of Pd/Fe 3 O 4 nanocatalyst by green tea leaves [22].
Physicochemical properties of MNPs such as surface plasmon resonance (SPR) and local field enhancement (LFE) can be changed by the alteration of diameter size and surface composition. For instance, LFE of MNPs with bipyramids shape is higher than other shapes by the sharp tips [23]. In this way, there are spherical, semi-spherical, cubic, triangular, rod, wire, flower-like, and dendrimer shapes. It is worth to note that biological activities including antimicrobial, anticancer, cytotoxic, antifungal, biocompatibility, and bioavailability of MNPs are also affected by the size and morphology alterations [24]. These properties of MNPs can be tunable by the selection of synthesis methods and reducing and stabilizing agents [25]. In this regard, the present study illustrated novel shape (dendrimer) of green synthesized Fe 3 O 4 NPs with enhanced antibacterial and antibiofilm abilities.
Different medicinal aspects of Artemisia L. genus were approved by many investigators [26,27]. Among this genus, Artemisia haussknechtii Boiss. is one of the local species in Iran [28]. Therefore, based on the above information, in this study, we utilized A. haussknechtii aqueous extract to synthesize Fe 3 O 4 NPs with a new shape of dendrimer. Assays of disc diffusion, MIC/MBC, and growth kinetics were used to measure antibacterial effects of green synthesized Fe 3 O 4 NPs on three sensitive and MDR bacteria species of S. aureus ATCC 43300, E. coli ATCC 25922, and S. marcescens ATCC13880. In addition, antibacterial mechanisms of these MNPs were determined through analyzing changing in biofilm and bacterial morphology properties.

Leaves extract preparation and biosynthesis of Fe 3 O 4 NPs
Plant species of A. haussknechtii were sampled based on the previous study [29]. Aqueous leaf extract of A. haussknechtii was prepared by 20 g of freshly amassed leaves. The leaves surface were cleaned with running tap water, followed by washing with distilled water and boiling in 250 ml volume of distilled water at 90 C temperature for half hour. Suspensions were filtered two times with Whatman No. 40 filter papers. The filtered sample was collected and stored at 4 C for next stage. This extract was utilized as reducing as well as stabilization/capping agents.
Duing the preparation Fe 3 O 4 NPs, the conical flask containing 50 ml of ferric chloride hexahydrate (0.2 M) was added to 0.001, 0.01, and 0.1 M concentrations of FeCl 2 .4H 2 O (50 ml) under stirring on a magnetic stirrer for 2 h. Afterward, 10 mL of the aqueous extract of A. haussknechtii leaves was mixed with 90 ml of resulted solution and pH is adjusted to 8, by the addition of 0.1 M NaOH solution. The above reaction was under a stirring condition at room temperature followed by the application of ultrasonic irradiation for 2 h with a frequency of 40 kHz and the total acoustic power of 50 W [30]. In comparison analysis, the effect of different temperatures (4,25,35,45,55, and 65 C) on MNPs synthesis was measured in the range of 1-7 h. In order to purify MNPs, the resulting solution was centrifuged at 4000 rpm for 5 Â 10 min and washed several times with 1:1 mixture of absolute methanol and distilled water. Powder MNPs were prepared by incubation of solution at 50 C for 48 h and then, they were stored in an airtight condition for further characterization by XRD, FTIR, and SEM analysis. The chemical reaction of Fe 3 O 4 precipitation is given in the below Equations 1 and 2 Total amounts of phenol, flavonoid, flavonol, and tannin Folin-Ciocalteu assay was applied to assess the total amount of phenolic compounds as one of the important group of secondary metabolites [31]. To prepare 3 ml volume of solution sample, distilled water was added to leaf extract and MNPs samples until we get an amount of 200 lL (1 mg/mL), blended completely by 0.5 ml of Folin-Ciocalteu reagent for 3 min followed by the addition of 2 ml of 20% (w/v) sodium carbonate (Na 2 CO 3 ). The mixture was heated at 45 C for 15 min and then absorbance was indicated at OD 765nm . The total phenolic amount was calculated via calibration curve. Measurements were carried out as triplicate repeat for each sample and demonstrated as mg of gallic acid equivalent (GAE) per g dry weight (/gDW). Flavones, flavonols, flavanones, isoflavonoides, neoflavonoides, flavanols, flavan-3-ols, anthocyanins, and chalcones are important subgroups of flavonoides secondary metabolites in the plant kingdom [32]. For indication of the total flavonoids amount of each sample, AlCl 3 colorimetric assay was used with slight modification [33]. Standard and treatment solutions (0.5 ml) were separately mixed by distilled water (2 ml) and 5% sodium nitrite (150 lL). Mixtures were combined with aluminum chloride 10% solution (150 lL) and 4% sodium hydroxide (2 ml) followed by standing for a period of 6 min. Then, distilled water was added to make a volume of 5 ml in a 5 ml volumetric flask. The mixture was allowed again to stand for 15 min and absorbance of the solutions was evaluated at OD 510 nm against blank and the total flavonoid content was also expressed as rutin equivalents in mg per g of dried extract (mg/g DW).
In order to determine total flavonol content, 250 ml of 2% AlCl 3 and 250 ml of 5% CH 3 COONa solution were added to 200 ml of each sample (1 mg/mL) [34]. Samples were sealed and incubated for 2-3 h at 25 C. The absorbance was indicated at OD 440 nm and results were expressed as mean ± standard deviation by mg of (þ)-catechin equivalents per g of dried extract (mg catechin/g DW).
Moreover, measurement of total tannin content was performed based on the method of Sun et al [35]. About 1.5 ml of concentrated hydrochloric acid and 3 mL methanol solution of 4% vanillin were added to 50 lL of diluted samples. After 15 min incubation of mixture in room temperature, absorption was determined at OD 500nm against methanol as a blank. Total tannin contents were presented as mg of (þ)-catechin equivalent (CE)/g DW and all samples were also analyzed in triplicate repeat.

Total antioxidant capacity (TAC) and DPPH assays
Total antioxidant capacity (TAC) of the biosynthesized Fe 3 O 4 NPs was measured in accordance with phosphomolybdenum assay [36]. About 100 mg of dried leaf extract and the biosynthesized NPs were separately taken into a reaction vial and mixed with 0.05% dimethyl sulfoxide (DMSO). About 0.1 ml aliquot solution of the samples was mixed with 1 ml of the reagent solution (0.6 M H 2 SO 4 , 0.028 M Na 3 PO 4 and 0.004 M [(NH 4 ) 6 Mo 7 O 24 4 H 2 O]). Samples were incubated at 95 C for 90 min followed by cooling at 25 C. Then, absorbance of the resulted samples were indicated at OD 695 nm against a reagent solution (without the annealed samples) as blank. Ascorbic acid was utilized as a positive control. The absorbance of the samples were reported as the total antioxidant activity which the higher antioxidant activity was illustrated by the higher absorbance.
DPPH scavenging activity of MNPs and leaf extract were determined by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging assay in 96 well microtiter plate [36]. Briefly, 100 lL of each concentration (100-500 lg/mL in methanol) of the green synthesized Fe 3 O 4 NPs and aqueous leaf extract was mixed with 100 lL of DDPH (100 lM) solution and incubated in the dark condition and at room temperature for 1 h. After color change from violet to pale yellow, the absorbance of the mixtures was indicated at OD 517 nm . Ascorbic acid was used for comparison assay. The capacity of the samples to scavenge DPPH radical was determined by: Percentage of DPPH scavenging activity

MDR and sensitive bacteria
Reprehensive MDR and sensitive bacteria of gram negative (E. coli ATCC 25922 and S. marcescens ATCC13880) and grampositive (S. aureus ATCC 43300) were used to determine the antimicrobial activity of Fe 3 O 4 NPs. These strains were obtained from bacterial archive of microbiology laboratory, Razi University of Kermanshah. Following evaluation, bacterial strains were maintained on nutrient agar slants at 4 C.

Disc diffusion assay
Antibacterial activity was determined by using disc diffusion assay [37]. Overnight MHB cultures of pathogenic bacteria of E. coli ATCC 25922, S. marcescens ATCC13880, and S. aureus ATCC 43300 were prepared freshly for each assay. These cultures were mixed with sterile physiological saline and turbidity was indicated by adding physiological saline until obtaining 0.5 McFarland turbidity standard (1.5 Â 10 8 CFU/ mL). Petri plates were prepared by 20 ml of sterile MHA and prepared bacterial inoculations were swabbed on the surface of the solidified media. After drying of media for 10 min, biosynthesized Fe 3 O 4 NPs were impregnated on discs at different concentrations of Fe 3 O 4 NPs (0.1, 0.01, and 0.001 M of FeCl 2 .4H 2 O) and were compared to plant leaf extract [38].

Determination of MIC/MBC
The bacteriostatic and bactericidal activities of Fe 3 O 4 NPs were measured by MIC/MBC assays [39]. An appropriate volume of bacteria (2 lL) in MHB was added to suspensions of Fe 3 O 4 NPs whose concentration varied using serial two-fold dilution from 100, 50, 25, 12.5, 6.25, and 3.12 lg/mL, respectively. These concentrations were taken from 0.1 M concentration of MNPs which had a higher antibacterial effect in agar diffusion assay. After incubation of medium for 24 h at 37 C, the tubes monitored for turbidity as growth and non-turbidity as no growth. The MIC values were interpreted as the lowest concentration of the sample, which illustrated clear fluid with no development of turbidity. Ten lL of the samples from each tube with no growth of bacteria were subcultured onto an MHA. The minimum bactericidal concentration (MBC) was determined as the highest dilution of the Fe 3 O 4 NPs that did not produce a single bacterial colony on the MHA after a 24 h of incubation period [40].

Effect of Fe 3 O 4 NPs on bacterial growth kinetic
Bacterial growth kinetics of E. coli ATCC 25922, S. marcescens ATCC13880, and S. aureus ATCC 43300 were evaluated under Fe 2 O 3 NPs effect at different concentrations (1000, 500, 250, 100, 50, 25, 12.5, 6.25, and 0 lg/mL as control). These bacteria were grown in liquid LB medium until they reached the log phase [41]. In order to obtain the first point of optical density (OD 600nm ), two different concentrations of 0.1 and 0.2 were determined by dilution of cell culture medium with fresh LB liquid medium. Different concentrations of Fe 3 O 4 NPs were added into the cell culture medium. Then, the culture medium was incubated at 37 C and 250 rpm. Bacterial growth kinetics was evaluated by measuring OD at 600 nm (bacterial concentration) at interval each hour.

Bacterial morphology analysis upon Fe 3 O 4 NPs treatment
Morphology of bacteria upon Fe 3 O 4 NPs treatment was visualized firstly by phase contrast microscopy (OLYMPUS-BX51, Shinjuko, Tokyo, Japan) using OLYMPUS-DP12 digital live camera and Q-capture pro7 software, Shinjuko, Tokyo, Japan, taking samples directly upon the cover-slide from stationary phase of growth kinetics.  (20,40,60,80, and 100%). These samples were coated with gold or platinum for FE-SEM scanning analysis [42].

Biofilm formation measurement
Ninety-six-well polystyrene plates was for evaluating biofilm formation. Initially, overnight cultures of bacteria were adjusted to an OD 600 nm of 0.5 in LB medium and co-cultured by different concentrations (100, 50, 25, 12.5, 6.25, 3.12, and 0 lg/mL as control) of green synthesized Fe 3 O 4 NPs as treatments and without MNPs as control for 24 h at 37 C without shaking. Bacterial growth was determined by the assessment of absorbance at OD 600 nm by UV-Vis spectroscopy. In order to remove planktonic bacteria and, plates were rinsed with water several times. Biofilms were stained with 350 lL of crystal violet (0.1%, v/v) for 30 min at 25 C. Then, plates were emptied, washed with water, blotted onto tissue paper towels. Dried crystal violet was extracted with ethanol (95%, v/v), and total biofilm formation was then assayed at OD 570 nm [43]. All experiments were carried out as three replicate tests independently. In addition, results were presented as the averages plus standard deviations (SD) of three replicate cultures. The meaningful inhibition of biofilm was indicated by Tukey's test (p .05).

Morphology analyses of biofilm by light microscopic and AFM
Light microscopic and AFM were utilized to observe changes of biofilm morphology of S. aureus ATCC 43300 under treatment by Fe 3 O 4 NPs. In the case of observation by light microscopic; MRSA strains were cultured on small glass slides in 24-well microtitre plates with different concentrations of MNPs (100, 50, 25, 12.5, 6.25, 3.12, and 0 lg/ mL as control) for 48 h. After this time, planktonic cells were eliminated and the biofilm was stained with crystal violet dye for 10 min followed by washing gently and then drying for 10 min. Afterward, the biofilm morphology was observed under light microscopy (40Â) (OLYMPUS CX31 with camera model KECAM CMOS 10000 KPA) [44]. After approval of morphology changes of biofilm under various amounts of biosynthesized NPs stress via light microscopic, AFM analysis was applied to assess obviously these changes. In this way, the topography of biofilm structure was evaluated at higher concentration of Fe 3 O 4 NPs (lg/mL) compared with control sample. Preparation of biofilm by S. aureus was carried out on silicone slides for 24 h incubation at 37 C followed by washing with PBS and air drying. Images were obtained at a resolution of 4.42 Â 4.42 lm by non-contact AFM (Nanosurf Mobile S).

FTIR analysis of biofilm
Biological macromolecules including polysaccharides, proteins, and DNA are framework of biofilm structure which can be affected by MNPs stress [45]. Biofilm surfaces on the glass slides were analyzed from the point of macromolecular composition in two conditions of high concentration (100 lg/mL) of Fe 3 O 4 NPs stress and without MNPs treatment as control for a period of 24 h. For this analysis, FTIR spectrometer was used with the reflectance mode of wavenumber over the range of 400-4000 cm À1 (Bruker, Germany, model: ALPHA).

Spreading assay of S. aureus
This assay was carried out via the method of Kaito et al. with slight modification [46]. In this case, 3 g of Muller Hinton agar was used and 0.8 g of nutrient broth in 100 ml distilled water followed by autoclaving of solution. Afterward, filter sterilized 10% (w/v) D-glucose in distilled water was added to the final solution. Different concentrations of MNPs (3.12, 6.25, 12.5, 25, 50, and 100 lg/mL as treatments) were coincubated with 5 lL of S. aureus ATCC 43300 sample onto the center of plates for 48 h at 37 C. Finally, the spreading of bacteria was indicated on the culture medium.

Statistical analysis
SPSS version 16 software (SPSS Inc., Chicago, IL, USA) was utilized to obtain one-way ANOVA (Tukey's test) results of experiments. Furthermore, all tests were carried out in triplicates, means with standard deviation were measured and p .05 was determined statistically as the meaningful difference between samples and control. In order to determine the dose-response relation between the plant extracts and Fe 3 O 4 NPs, regression analysis was applied. Furthermore, linear regression analysis was used to measure the correlation coefficient.

Physicochemical properties of NPs
Ultraviolet-Visible (UV-Vis) spectroscopy UV-visible absorbance spectroscopy is used to survey the NPs characterization such as concentration, size, aggregation, and bioconjugation [47]. haussknechtii leaf aqueous extract medium was indicated by color change from pale green to blackish (Figure 1(a)). Phytochemicals such as flavonoids and polyphenols of A. haussknechtii leaf extract can be firmed by color change. This concentration (0.1 M) was selected to evaluate explicitly the effect of temperature parameter (4,25,35,45,55, and 65 C) on the rate of MNPs growth in a period of 7 h ( Figure  1(b)). Similar to the previous study about Ag and Au NPs synthesis, there was a direct relationship between temperature and absorbance values [49].  XRD patterns of leaf extract were reported by previous investigations [48,50]. Figure 2( [51]. Debye-Scherrer equation is the relationship between crystallite size and X-ray diffraction peak broadening. This equation is shown as follows:

XRD analysis
where d is the crystallite size of green synthesized Fe 3 O 4 NPs for (hkl) phase, k is Scherrer constant (0.9), k is the X-ray wavelength of radiation for Cu Ka (0.154 nm), b hkl is the fullwidth at half maximum at (hkl) peak in radian, and h hkl is the diffraction angle for (hkl) phase. Dependent upon atom density, each crystallographic facet contains energetically distinct sites. High atom density at (311) may be associated with high reactivity of these crystalline facets [52]. By the equation, the estimated crystallite size of synthesized Fe 3 O 4 NPs was 83.4 nm, which figured out to be high purity crystalline [53].

FT-IR analysis
FT-IR analysis was used due to the indication of possible molecules for capping and reducing of MNPs [54]. As seen in Figure 3, the absorbance peaks at 604.46 cm À1 and 1043.86 cm À1 are attributed to C-Cl and C-F stretching strong bands for alkyl halide compounds [55]. The peak at 1427.06 cm À1 corresponds to C¼C stretching multiple bands of aromatic groups [56]. The intensity of the absorption bands at 1607.39 cm À1 and 3411.81 cm À1 correspond respectively to C¼O (carbonyl) and N-H (amine) stretching bands [57]. Results of FT-IR analysis illustrated the attribution of different functional groups in the leaf extract including amine, carbonyl, polyphenols, and alkyl halide. Therefore, some polyphenols may be stabilizing of Fe 3 O 4 NPs by attaching to the MNPs surfaces through interacting with free amine or carbonyl groups. Also, the absorption band at 1607. 39

SEM images, EDAX, and AFM analysis
As shown in Figure 4(a), the results of SEM images demonstrated the denderimer shape of Fe 3 O 4 NPs with branched surfaces at nanometer magnifications. This structure may have resulted from chemical interactions such as hydrogen and electrostatic bonds between the organic capping agents of plant secondary metabolites and core of Fe 3 O 4 NPs [59]. Diameter sizes of NPs were in the range of 1-150 nm by the maximum size between 120 and 130 nm (Figure 4(b)). Compared to similar reports, green synthesized Fe 3 O 4 NPs by Solanum trilobatum and Kappaphycus alvarezii extract had 18 and 14.7 nm size with spherical shape [48,60].
Elemental composition analysis of biosynthesized Fe 3 O 4 NPs was performed by energy-disperse X-ray spectroscopy (EDAX) method (Figure 4(c)). EDAX graph indicated elemental signal for iron with an intensity of 111.8, which is specific for the absorption of metallic iron nanocrystallites resulted from surface plasmon resonance. Also, signals were observed for Cl, O, Ca, K, and S elements respectively with intensity of 138.4, 53.1, 12.6, 6.5, and 3.2 with also elemental distribution (Figure 4(d)) which may be related to protein/enzymes presence in A. haussknechtii leaf extract [61].
Surface topography, size, structure, agglomeration, and height of Fe 3 O 4 NPs were surveyed by AFM analyses ( Figure  5). Various dimensions of images ( Figure 5(a-f)) were utilized to detect clearly NPs. As illustrated in Figure 5(d), lower height of 2.73 nm and uniformity of size distribution were observed for Fe 3 O 4 NPs. In this case, the previous study showed size distribution range 7-77 nm for green synthesized magnetic Fe 3 O 4 NPs by fruit extract of Couroupita guianensis Aubl [62]. Therefore, as a comparative approach, our results showed reduction and uniformity of Fe 3 O 4 NPs size.

Antioxidant activities
Total contents of phenolic, flavonoid, flavonol, and tannin Standard curve of gallic acid was used for quantitative evaluation of total phenols. As shown in Figure 6(a), there is linearity curve of the calibration from 20 to 100 lg/mL concentration for gallic acid (R 2 ¼ 0.9987). Table 1 illustrates the total phenolic content of the methanolic leaf extract and Fe 3 O 4 NPs with 15.98 ± 1.30 and 3.22 ± 0.77 mg gallic acid equivalent (GAE)/g DW, respectively. In accordance with standard curve of rutin, measurement of total flavonoids was quantitatively performed and linearity of the calibration curve was obtained from 20 to 100 lg/mL amount for rutin (R 2 ¼ 0.9652; Figure 6(b)). The leaf extract and Fe 3 O 4, respectively showed total flavonoid content  (Table 1). There was a significant difference (p .05) between total phenol and flavonoid content of leaf extract and Fe 3 O 4 NPs. Total flavonol content (TFC) around Fe 3 O 4 NPs with a value of 0.76 ± 0.27 was less than leaf extract (p .05). Also, as shown in Figure 6(c), total tannin amount was assessed as a standard curve of (þ)-catechin and linearity of the calibration curve (R 2 ¼ 0.967) was gained from 0 to 120 lg/mL concentration for (þ)-catechin. Fe 3 O 4 NPs and leaf extract, respectively showed total tannin content with 0.29 ± 0.19 and 2.36 ± 0.47 mg (þ)-catechin equivalent (CE)/g DW (Table 1). Also, as observed in this table,  flavonoid/phenol ratio did not demonstrate a meaningful difference between Fe 3 O 4 NPs and leaf extract.

Total antioxidant capacity (TAC) and DPPH assays
Total antioxidant capacity (TAC) of plant leaf extract, Fe 3 O 4 NPs, and ascorbic acid (control) was compared together by phosphomolybdenum method. Antioxidant activity of Fe 3 O 4 NPs was increased by the augmentation of samples concentration. As presented in Figure 7(a), amounts of absorbance were 0.788 ± 0.033, 0.599 ± 0.041, and 0.382 ± 0.055, respectively for ascorbic acid, plant leaf extract, and Fe 3 O 4 NPs at higher concentrations (500 lg/mL). Therefore, the total antioxidant ability of Fe 3 O 4 NPs was lower than ascorbic acid and plant leaf extract. In the case of DPPH assay, there were similar results (Figure 7(b)

Disk diffusion assay
The antibacterial capacity of Fe3O4 NPs was evaluated firstly against three bacteria E. coli ATCC 25922, S. aureus ATCC 43300, and S. marcescens ATCC13880 by disk diffusion assay. Inhibition zone diameter (IZD) was observed as confirmation of antibacterial ability after incubation of the plates (Figure 8). This assay illustrated that higher concentration (      of green synthesized Fe 3 O 4 NPs [64]. In this regard, the effective amount of previous antibacterial test (0.1 M of FeCl 2 .4H 2 O) was used as a basic concentration to measure MIC assay. As shown in Figure 9, the MIC values of Fe 3 O 4 NPs against MDR and sensitive bacteria were in the range of 12.5-50 lg/mL. The highest value of this assay was for E. coli and S. marcescens with 50 lg/mL. In contrast, S. aureus had the lowest amount of 12.5 lg/mL. In case of MBC, values ranges 50-100 lg/mL. MBC result for E. coli and S. marcescens was 100 lg/mL. Staphylococcus. aureus showed lower concentration of MBC (50 lg/mL) than E. coli and S. marcescens. Therefore, it can be concluded based on these assays that E. coli and S. marcescens as gram-negative bacteria had more resistant than S. aureus as gram-positive bacteria [65]. In gram-negative bacteria, multidrug efflux pumps as membrane-located transporters make a major contribution to this intrinsic resistance [66]. Multidrug transporters in gram-negative bacteria protect bacterial cells from the function of antibiotic agents on both sides of the cytoplasmic and outer membranes with the broad specific substrate [67]. NPs amount [70]. Higher reduction in growth kinetics of E. coli ATCC 25922 and S. marcescens ATCC13880 was observed at Fe 3 O 4 NPs concentrations with !50 lg/mL. Comparatively, in the case of S. aureus ATCC 43300, lower amounts of MNPs (!12.5) had a striking effect on bacterial growth reduction. Also, there was an obvious relationship between initial concentrations of bacteria (0.1 and 0.2 OD) with the antibacterial activity of MNPs. At higher initial OD (0.2), needed amounts of the Fe 3 O 4 NPs to inhibit bacterial growth were more than lower initial OD (0.1) [41]. As demonstrated in Figure 11, specific growth rate, l, (OD 600 nm /h) of three sensitive and MDR bacteria: (a) E. coli ATCC 25922, (b) S. marcescens ATCC13880, and (c) and S. aureus ATCC 43300S in the presence of MIC concentrations (50 lg/mL for E. coli and S. marcescens and 12.5 lg/mL for S. aureus) had negative rate after 3, 3, and 1 h, respectively. In contrast, there was no negative growth rate in control samples. Escherichia coli and S. marcescens had more resistance to Fe 3 O 4 NPs than S. aureus. In this way, response difference of these bacteria can be caused bythe difference in cell wall stability and growth rate of gram-negative (-) and grampositive (þ) bacteria under Fe 3 O 4 NPs stress [71].

Bacterial morphology analysis upon Fe 3 O 4 NPs treatment
Morphology changes of the sensitive strain of S. aureus ATCC 43300 (MRSA) in the presence of 12.5 lg/mL concentration of Fe 3 O 4 NPs was observed by SEM images (Figure 12). As illustrated in Figure 12  illustration, Figure 10(d) shows a schematic image of several antibacterial mechanisms of Fe 3 O 4 NPs against S. aureus.

Biofilm formation assay
As shown in Figure 13

AFM and morphology analyses of biofilm
Biofilm morphology changes of S. aureus stain under Fe 3 O 4 NPs stress was evaluated by a light microscope at a magnification of 40Â ( Figure 14). As illustrated in these images, the reduction of biofilm structure was enhanced by the rising concentration of Fe 3 O 4 NPs. Similar to biofilm formation assay, higher antibiofilm activity (66% reduction) was observed at 100 lg/mL of Fe 3 O 4 NPs. Therefore, the results of these two methods showed obviously antibiofilm effects of Fe 3 O 4 NPs at the higher concentrations. Changes in biofilm architecture were illustrated at 10 lL phosphatidylcholinedecorated Au NPs at 0.116 mg/mL concentration for 24 h incubation against Pseudomonas aeruginosa (PAO1) [72]. Also, biofilm formation of E. coli and S. aureus was assessed at the presence of biosynthesized silver NPs in 5, 10, and 15 lg/mL concentrations for incubation of 48 h. In this regard, more reduction was viewed at 15 lg/mL amount of Ag NPs [73]. AFM images of biofilm formation by S. aureus under high concentrations of biological synthesized Fe 3 O 4 NPs (as treatment) and free-NPs (as control) are presented in Figure 15. As obvious from Figure 15(a-c), biofilm roughness is lower in treatment (10.666 nm) compared to control (Figure 15(d-f) by a value of 45.955 nm). In addition, there were pores in treatment biofilm resulted from biofilm damage by Fe 3 O 4 NPs stress. The inhibition and disturbing of biofilm structure of S. aureus under Fe 3 O 4 NPs treatments were approximately similar to the results of light microscopic and AFM analysis. Similarly, reduction in roughness values as 12-36% and 40-60% has been reported, respectively for S. aureus and E. coli in presence of biosynthesized Ag and Au NPs [49].

FT-IR analysis of biofilm
Polymers involving polysaccharides, protein, and nucleic acids are essential macromolecules in biofilm formation. FT-IR spectra of biofilm formation by S. aureus on a glass slide in two conditions of Fe 3 O 4 NPs stress and NPs-free as control were compared due to analyzing chemical composition changes of biofilms after 24 h period ( Figure 16). Results showed peaks of 1114. 46 cm À1 and 1112.57 cm À1 , respectively for control and treatment which can be the presence of sign of nucleic acids and polysaccharides macromolecules. Peaks at 1655.16 cm À1 and 1655.36 cm À1 illustrate the presence of proteins [74]. In comparison, the treatment showed reduction and increasing of peak intensity at 621.14 cm À1 (C-Cl stretching bond) and 2363.91 cm À1 (CC bond), respectively.

Spreading assay of Staphylococcus aureus
Colony expansion of S. aureus on soft agar was investigated previously in particular conditions as finger-like dendrites [46]. As illustrated in Figure 17, motility ability of S. aureus was determined by special assay of spreading upon Fe 3 O 4 NPs stress in various concentrations (3.12, 6.25, 12.5, 25, 50, and 100 lg/mL). Compared to a clear pattern of bacterial colonies as finger-like dendrites in control sample and colonies under lower concentrations of Fe 3 O 4 NPs (Figure 17(a-g)), there was a decreasing pattern of colonies expansion by increasing MNPs amounts. These results indicate the sensitivity of S. aureus colony under NPs stress, which is the   antibacterial advantage of these MNPs. In fact, due tothe dependence of virulence, tissue colonization, and biofilm formation of S. aureus on colony spreading in initial stages of bacteria growth, infections related to this strain can be blocked by Fe 3 O 4 NPs [75].
Surface topography, size, structure, agglomeration, and height of Fe 3 O 4 NPs were surveyed by AFM analyses ( Figure  5). Various dimensions of images ( Figure 5(a-f)) were used to detect clearly NPs. As illustrated in Figure 5( [76]. Phenolic compounds act as antioxidants via their redox properties [77]. The total phenolic concentration could be applied as rapid screening of antioxidant activities. Plant secondary metabolites such as flavonoids, including flavones, flavanols, and condensed tannins have the antioxidant abilities on the basis of the presence of free hydroxyl functional groups, which they can reduce metal ions to NPs [78]. In this way, the metal ions including iron and copper ions bind to the various reducing/stabilizing flavonoids [79]. Several studies have reported antibacterial effects of MNPs and metal oxide NPs on gram-positive and gram-negative bacteria [80,81]. The efficiency of these antibacterial agents may be related to the cell wall and membrane difference of bacteria. Gram-positive bacteria have a thick cell wall (about 20-80 nm) compared to gram-negative with a thin layer of peptidoglycan (about 7-8 nm) and two cell membranes (outer and plasma membrane) [82]. It is worth mentioning that MNPs with a size range of 8-80 nm can penetrate the cell wall [83]. Antibacterial activities of Fe 3 O 4 NPs were demonstrated by several studies [68,84]. It was reported that these MNPs can cause damaging of E. coli membranes by diffusion of the tiny particles ranging from 10 to 80 nm [85]. Zero-valent iron NPs interact with intracellular oxygen and cause disturbing the cell membrane bythe production of oxidative stress [86]. Green synthesized Fe 3 O 4 NPs by fruit extract of Couroupita guianensis Aubl. had more antibacterial activity on gram-negative bacteria K. penumoniae MTCC 530, E. coli MTCC 2939, and S. typhi MTCC 3917 than gram-positive bacterium S. aureus MTCC 96 [62]. In this case, the generation of reactive oxygen species (ROS) as an antibacterial factor may result from MNPs unique properties. Also, CuO and Ag NPs have demonstrated that antibacterial activities can be raised by diameter size reduction of MNPs [80,87]. Based on cell wall characteristics including the thickness of peptidoglycan wall and number of the membrane, grampositive and gram-negative bacteria are different. In this case, gram-negative bacteria with the cell wall including a thin layer of peptidoglycan, an outer bilayer membrane (lipopolysaccharides and proteins) and inner membrane are more complex than gram-positive bacteria by having only a thick layer of peptidoglycan cell wall and plasma membrane [88]. In this regard, growth kinetic comparison of E. coli and B. subtilis (gram-positive) under different concentrations of iron oxide NPs showed higher growth inhibition for B. subtilis than E. coli [68]. Also, the core-shell Fe 3 O 4 @C-PVPS:PEDOT NPs (iron oxide NPs coated by catechol-conjugated poly (vinylpyrrolidone) sulfobetaines and encapsulated with poly (3,4-ethylenedioxythiophene)) illustrated a high antibacterial impact on S. aureus and E. coli [89].
Changes of bacterial morphology were proved by the impact of MgO NPs on S. enteritidis, E. coli O157:H7, and C. jejuni bacteria in the late-log phase of growth [81]. These alterations in morphology were involved in changing shape from spiral to coccoid and producing deep craters in the bacterial membrane. Cell wall clumping, membrane blebs, and rupture were observed in E. coli MTCC 443 at the stationary phase of growth kinetics upon treatment by ZnO NPs with a diameter range of 25-45 nm [90]. Toxicity of MNPs against bacteria can be related to several parameters involving the type of bacteria, physicochemical properties of MNPs such as the large surface area to volume (SA:V ratio), and chemical and biological functionalization of MNPs surface [91]. In this study, secondary  metabolites of A. haussknechtii leaf aqueous extract such as phenol and flavonoids can influence on this property [92].
Antibiofilm properties of green synthesized MNPs were reported by other studies [93,94]. Synergism effect of chemical synthesized Fe 3 O 4 NPs with antibiotics of streptomycin, vancomycin, and penicillin was estimated against biofilm formation of Enterococcus faecalis pathogen. Result of this study did not approve great antibiofilm activity of magnetic NPs with high concentration (1688-16988 lg/mL) [95]. Pseudomonas aureuginosa and S. aureus exhibited biofilm reduction from 125 and 250 lg/mL to 1000 lg/mL amounts of Fe 3 O 4 NPs, respectively [96]. Incubation of E. coli ATCC 15224 and S. aureus ATCC 25923 for 72 h at 37 C on glass and silicon surface with Fe 3 O 4 NPs coating showed a meaningful reduction in these bacteria [97]. Unique properties of MNPs including large surface to volume ratio augment reactivity of MNPs with surrounding materials. In this way, the generation of reactive oxygen species (O 2 , OH , and H 2 O 2 ) resulted from MNPs which can disrupt the biofilm structure of bacteria [98]. Also, the shape of MNPs can be an efficient factor in biofilms and bacteria damage [72].

Conclusions
In summary, antioxidant, antiplanktonic, antibiofilm, and antimotility capacities of biosynthesized Fe 3 O 4 NPs by leaf aqueous extract of medicinal plant A. haussknechtii on three MSR bacteria E. coli ATCC 25922, S. marcescens ATCC13880 and S. aureus ATCC 43300 were surveyed as ecofriendly and efficient approach (Figure 18). There are several reports in the case of antibacterial activities of MNPs, but it has been paid less attention about antibiofilm and antimotility aspects of MNPs, specifically biosynthesized Fe 3 O 4 NPs. This study introduced a novel shape of