Antibacterial, antibiofilm, antiquorum sensing, antimotility, and

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Characterization, Synthesis, and Modifications

Antibacterial, antibiofilm, antiquorum sensing, antimotility, and antioxidant activities of green fabricated Ag, Cu, TiO2, ZnO, and Fe3O4 NPs via Protoparmeliopsis muralis Lichen aqueous extract against multi drug resistant bacteria Mehran Alavi, Naser Karimi, and Tahereh Valadbaeigi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00274 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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ACS Biomaterials Science & Engineering

Antibacterial, antibiofilm, antiquorum sensing, antimotility, and antioxidant activities of green fabricated Ag, Cu, TiO2, ZnO, and Fe3O4 NPs via Protoparmeliopsis muralis lichen aqueous extract against multi drug resistant bacteria

Mehran Alavia,b, Naser Karimia,b* and Tahereh Valadbeigic aDepartment

of Nanobiotechnology, Faculty of Science, Razi University, Kermanshah, Iran.

bDepartment

of Biology, Faculty of Science, Razi University, Kermanshah, Iran.

cDepartment

of Biology, Faculty of Sciences, Ilam University, Ilam, IR Iran.

Email addresses: Mehran Alavi ([email protected]), Naser Karimi ([email protected]; [email protected]), Tahereh Valadbeigi ([email protected]) Tell/Fax: 0098-833-4274545.

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Consideration of lichen organisms as the ecofriendly source of metal nanoparticles (MNPs) and metal oxide NPs (MONPs) synthesis is seldom. In this study, Ag and Cu MNPs as well as TiO2, ZnO, and Fe3O4 MONPs were green synthesized by Protoparmeliopsis muralis lichen aqueous extract. Firstly, physicochemical characterization by UV-Vis spectroscopy, XRD, FT-IR, FESEM, and TEM techniques demonstrated the presence possibility of secondary metabolites around formed MNPs/MONPs with different diameters and shapes (spherical, triangular, polyhedral, and cubic). Then, antibacterial, antibiofilm, antiquorum sensing, and antioxidant abilities of these MNPs/MONPs against multi drug resistant (MDR) bacterium (Staphylococcus aureus ATCC 43300) and reference bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) were evaluated by in vitro tests. Results of disc diffusion and MIC/MBC assays of Ag NPs as an effective antibacterial agent illustrated higher sensitivity of P. aeruginosa pathogen than E. coli and S. aureus. In next steps, significant reduction was observed in biofilm formation of each bacterium and pyocyanin synthesis by P. aeruginosa under Ag NPs. Totally, this investigation presented novel clean production of five MNPs/MONPs with prominent advantages of ecofriendly, cost-effective, and antipathogen properties. Keywords: metal nanoparticles (MNPs), green synthesis, Protoparmeliopsis muralis, multi drug resistant (MDR) bacteria.


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1. Introduction Nanomedicine is medicinal application of nanostructures, especially nanoparticles (NPs), with having at least one dimension in the range of 1 to 100 nm. Based on synthesis method, these NPs can have different shapes such as spherical, triangular, rod, cubic, and polyhedral1-2. Also, various usages of NPs are resulted from excellent optical, electrical, thermal and magnetic properties derived from high surface area to volume (SA:V) ratio and high surface energy3-4. NPs may be organic, semi-organic, and inorganic nano-sized particles. In the case of inorganic NPs, MNPs/MONPs such as Ag Cu, TiO2, Fe3O4, ZnO, and MgO are more important due to their various applications in medical and non-medical technology

5-6.

For medical purposes,

MNPs/MONPs can be utilized in detection, diagnosis, and therapy of dangerous diseases7. Nanomedicine can resolve obstacles related to medicine challenges by using pure or modified NPs. Among these challenges, infectious diseases caused by resistant microbe specifically bacteria are quickly augmenting and expanding. In this regard, major problem is arising from multi drug resistant (MDR) bacteria 8. Fighting against these types of bacteria is expensive and time consuming that leads to patient dissatisfaction. For example, in U.S. approximately $20 billion and $35 billion have been spent respectively in health care and lost productivity for one year9. Meanwhile, there is a necessity of effective agents without disadvantages of previous antibiotics. In this way, the methicillin-resistant Staphylococcus aureus (MRSA) is one of the important infectious agents in hospital which we used in this study as suitable model of MDR bacteria10. Changes in environmental conditions can lead to biofilm formation from planktonic form of bacteria growth. Bacteria in biofilm produce extracellular matrix biomass including extracellular



polymeric substance (EPS), extracellular DNA (eDNA), flagella, pili, proteins and carbohydrates11. Biofilm is protected from disrupting agents such as irradiation, desiccation, and wide range of antibiotics via providing of hydrophilic extracellular matrix as protecting medium12. Inactivity of some bacteria in biofilm followed by regrowth after antibiotic treatment is major escaping mechanisms of biofilm13. Bacteria can cause many biofilm-related infections in human mainly including urinary tract, gingivitis, dental plaque, bacterial vaginosis, contact lenses, and middle-ear infections14. Therefore, discovery of new strategies for inhibition or reduction of biofilm formation is needed. In this way, NPs and MNPs/MONPs applications have obtained more attention because of their unique properties than to bulk type in disrupting and penetration into biofilm structure15-17. Development of technology and industry was simultaneous with environmental problems involving air, water, and soli pollutions18-20. Introducing of toxic materials into environment is one result from NPs synthesis. One way to remove these pollutions is application of natural sources such as plant and bacterial materials as precursors 21. In the case of MNPs/MONPs synthesis, there are several methods based on chemical, physical, and green approaches. Each of these methods has own advantages and disadvantages. Green synthesis method is preferred than to physical or chemical methods because important advantages of using eco-friendly (non-toxic) substances and low cost in synthesis22. In green synthesis, it can be used constitutes of plants, lichens, bacteria, fungi, and algae organisms as reducing or stabilizing agents for MNPs/MONPs synthesis23. In contrast to plant, bacteria, algae, and fungi, lichen extracts applications in MNPs/MONPs synthesis are largely unknown. Aqueous extract of Parmotrema praesorediosum lichen is used for silver nanoparticles with an average particle size of 19 nm and antibacterial against Salmonella typhi pathogen24. Lichens are composed of two parts of algal and fungi organisms as respectively


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mycobions and photobionts parts25. These organisms have several secondary metabolites such as usnic acids, phenolic, depsidones (lecanoric acid), depsones, and depsides (atranorin) compounds with antioxidant, antimicrobial, and anticarcinogenic activities26. In this study, we used P. muralis lichen aqueous extract as novel lichen for biosynthesis of Ag, Cu MNPs as well as TiO2, ZnO, and Fe3O4 MONPs with antibacterial and antibiofilm activities against MDR and sensitive bacteria. For this purpose, one-pot methods were utilized to prepare each MNP and MONP. As comparative study, growth rate related to each MNP/MONP was obtained in period of 96 hrs by UV-Visible spectroscopy. Physicochemical characterizations of MNPs/MONPs were analyzed by techniques of UV-Visible spectroscopy, Fourier transform infrared spectroscopy (FT-IR) analysis,

X-ray diffraction (XRD), energy dispersive X-ray

spectroscopy (EDX), scanning electron microscope (SEM), and transition electron microscope (TEM). Assays of total antioxidant activity, scavenging properties of DPPH, total phenolic, flavonoid, flavonol, and tannin contents were used due to assessment of biological characterizations of lichen aqueous extract and MNPs/MONPs. In addition, E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 43300 were applied as sensitive and MDR models of bacteria for evaluating of antibacterial and antibiofilm properties of lichen extract and MNPs/MONPs via disc diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), bacterial growth kinetic, bacterial morphology, and biofilm formation analyses. Mobility of each bacterium as important pathogenic property was surveyed upon Ag NPs effect. Furthermore, pyocyanin concentrations in P. aeruginosa bacteria were measured through antiquarum sensing assay, under different levels of biofabricated Ag NPs treatment. 2. Experimental



2.1.

Materials

AgNO3, CuSO4, TiO(OH)2, Iron(II) chloride tetrahydrate (FeCl2.4H2O≥98%), Iron(III) chloride hexahydrate (FeCl3.6H2O ≥98%), Zinc nitrate hexahydrate (Zn(NO3)2.6H2O ≥99%), FolinCiocalteu reagent (FCR), gallic acid, vanillin, (+)-catechin, rutin, sodium carbonate (Na2CO3), sodium nitrite (NaNO2), aluminum chloride (AlCl3), sodium hydroxide (NaOH), hydrochloric acid (HCl), dimethyl sulfoxide (DMSO), sulphuric acid (H2SO4), methanol, ethanol, sodium phosphate (Na3PO4), ammonium molybdate [(NH4)6Mo7O24•4H2O], sodium acetate (CH3COONa), 1,1diphenyl-2-picryl-hydrazyl (DPPH), 1X PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4), glutaraldehyde, tannic acid (C76H52O46), Mueller-Hinton broth, MuellerHinton agar, crystal violet, and chloroform (CHCl3) were purchased form Sigma-Aldrich (St. Louis, MO). 2.2.

Lichen aqueous extract preparation and green synthesis of five MNPs/MONPs

Healthy samples of P. muralis were collected from Kane Gonabad Mountains in Ilam province of Iran during May 2017. The lichen species were identified and authenticated by an expert of plant systematic center of Ilam University. In order to having future reference, voucher specimens were submitted at the herbarium. Aqueous extract of P. muralis were prepared by freshly amassed lichen samples (20 g). The samples were cleaned with running tap water, followed using distilled water, and air dried on a paper towel for 6 days. Dry samples were ground in a tissue grinder to fine powder, and boiled with 250 mL of double distilled water at 90 ˚C for 30 min. Suspensions were filtered with Whatman No. 40 filter paper. The ﬁltered suspension was collected and stored at 4 ˚C till further use. This extract was used as reducer and stabilizer/capping agent27. For synthesis of five MNPs/MONPs, the Erlenmeyer flask containing 100 mL of AgNO3, CuSO4, TiO(OH)2,


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and Zn(NO3)2.6H2O by 0.1, 0.01 and 0.001 M concentrations were stirred for 2 hrs. Ten mL of the aqueous of P. muralis was added in 50 mL of each metal salt at room temperatures under stirred condition for 24 hrs. In the case of Fe3O4 NPs synthesis, FeCl3.6H2O (0.2M) was added to 0.001, 0.01, and 0.1M of FeCl2.4H2O and then, pH is adjusted to 8, by 0.1 M NaOH solution. In this way, sodium hydroxide was utilized to prepare alkaline medium due to coprecipitate Fe3O4 NPs28. In addition, effect of different times (24, 48, 72, and 96 hrs) on growth of biosynthesized five MNPs/MONPs was surveyed at 65 °C. The resulted solution was stirred vigorously at 65 °C for 1 h, subsequently followed by cooling at room temperature and the supernatant was discarded. Afterward, NPs solutions were centrifuged at 4000 rpm for 30min after thorough washing and drying at 70 °C for 8 hrs. Crude pellets were then re-suspended in sterile double distilled water, filtered through Whatman No. 40 filter and stored at 4 °C in the dark condition before utilizing in following analyses. 2.3.

Physicochemical properties of NPs

The surface plasmon resonance (SPR) property, size/shape of liquid, and solid samples were obtained by UV-Vis spectroscopy and transmission electron microscopy (TEM) techniques respectively. The intensity of absorption peaks by MNPs/MONPs was examined by UV-Vis spectrophotometer (Tomas, UV 331) from 200 to 800 nm. X-ray diffraction (XRD) analysis and Fourier transform infrared spectroscopy (FT-IR) were applied for determination of crystalline size, functional groups, morphology, and elemental composition of dried samples. XRD analysis was performed via EQUNIOX 3000, diffractometer in the scanning range of 10˚- 80˚ (2θ) using Cu Ka radiations of wavelength 1.5406 Å for identification of the crystal phases and determination of the average crystal size of five NPs. Model XL30, Philips, Eindhoven, FE-SEM was used to study the



morphology of MNPs/MONPs and the elemental analysis. Spectrophotometer (Bruker, Germany, Model: ALPHA) was used to obtain FT-IR spectra of each sample. 2.4.

Phytochemical analysis: Total phenolic, flavonoid, flavonol, and tannin contents

Folin-Ciocaltue assay was applied for measurement of total phenolic content (TPC) in the case of lichen extract, Ag, Cu, TiO2, ZnO, and Fe3O4 NPs. This assay is important because of scavenging properties of phenol metabolites for free radicals including reactive nitrogen species (RNS) and reactive oxygen species (ROS)29. 0.5 mL of Folin-Ciocalteu reagent was added to 3 mL aqueous solution of each sample with 1 mg/mL concentration and maintained for 3 min. 2 mL of sodium carbonate (Na2CO3; 20%) was added to prepared solution. Heating at 45 °C for 15-20 min of solution was followed by indication of absorbance at 765 nm using UV spectrophotometer. Calibration curve was utilized based on the results of three replicates for each sample for evaluating TPC as mg of gallic acid equivalent (GAE) per g dry weight (/gDW). It was used colorimetric method of the aluminum chloride (AlCl3) based on previous study for assessment of the total flavonoid content (TFC) of each sample30. In this way, after preparation of samples, absorbance of the solutions was determined at OD510nm against blank and TFC was expressed as mean ± standard deviation of rutin equivalents in mg per g of dried extract (mg/gDW). Moreover, total flavonol content (TFLC) and total tannin content (TTC) were performed founded on previous study30. 2.5.

Antioxidant activity: Total antioxidant capacity (TAC) and free radical DPPH scavenging assay

It was used phosphomolybdenum assay to evaluate total antioxidant capacity (TAC) of the biosynthesized MNPs/MONPs and aqueous lichen extract at OD695 nm 30. All steps were carried


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out based on previous investigation and ascorbic acid was utilized for comparison assay. The 1,1diphenyl-2-picryl-hydrazyl (DPPH) assay was applied to determine the antioxidant ability of the lichen extract and MNPs/MONPs spectrophotometrically at OD517nm. Process of assay was similar to former study and the ability of the samples to scavenge DPPH free radical was assessed by below formula:

1. Percentage of DPPH scavenging activity =

2.6.

Control OD ― Sample OD × 100 Control OD

Microorganisms

Reprehensive bacteria of gram positive (S. aureus ATCC 43300) and gram negative (E. coli ATCC 25922 and P. aeruginosa ATCC 27853) were applied to indicate the antimicrobial properties of lichen extract and NPs of Ag, Cu, Fe3O4, TiO2, and ZnO. All of these strains were prepared from bacterial archive of microbiology laboratory, Razi University of Kermanshah. In order to following tests, bacterial strains were maintained on nutrient agar slants at 4 ̊C. 2.7.

Antiplanktonic activities

Disc diffusion assay was carried out on the basis of stages of previous study. For measurement of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and bacterial growth under NPs stress, various NPs concentrations (0.5, 1, 2, 4, 10, 20, 40, 60, 80, and 100 μg/mL) were applied. Following steps of these three methods were alike to prior investigation30. 2.8. Bacterial morphology analysis upon Ag NPs stress



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P. aeruginosa morphology at stationary phase of growth kinetic was visualized under Ag NPs treatment firstly by phase contrast microscopy (OLYMPUS-BX51) by OLYMPUS-DP12 digital live camera and Q-capture pro7 software, taking samples directly upon the cover-slide. Afterwards, impact of Ag NPs on bacterial morphology was observed completely via FE-SEM method in reference to former study31. 2.9. Antibiofilm activity: Biofilm formation assay Crystal violet (CV) assay was utilized in order to assessment of biofilm formation

32.

At first,

overnight cultures of bacteria in microtiter plates (96-well polystyrene plates) were adjusted to an OD600 of 0.5 in LB medium and co-cultured by different concentrations (3.12, 6.25, 12.5, 25, 50, and 100 μg/mL) of biosynthesized MNPs/MONPs as treatments for 24 hrs at 37°C without shaking. Absorbance at OD600 was determined by visible spectroscopy for indication of bacteria growth. In order to remove planktonic bacteria, plates were rinsed by water for three times. Biofilms in each well were stained by 100 μL of crystal violet (0.1%, w/v) for 30 min at 25°C. Then, plates were emptied, washed by water, blotted onto tissue paper towels. Ethanol (95%, v/v) was applied for extraction of dried crystal violet followed by evaluating of biofilm formation at OD570. Tests were carried out as three replicates tests independently. Also, results were presented as the means ± standard deviations (SD) of three replicates cultures. The meaningful inhibition of biofilm was determined by the Tukey’s test (p≤0.05). 2.10. Motility assay and pyocyanin measurement Motility activities of E. coli, S. aureus, and P. aeruginosa were evaluated at different concentrations of Ag NPs (0, 3.12, 6.25, 12.5, 25, 50, and 100μg/mL) in according with original protocol with slight modifications 33. Nutrient broth (0.8g) and Muller Hinton agar (3g) were added


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to 100mL distilled water for preparing of soft agar. Filtered sterilized D-glucose (10% w/v) was combined with autoclaved sample. 5 μL of each bacterium was co-incubated by various amounts of Ag NPs on center of prepared soft agar for 48 hrs at 37 °C. Then, motility of bacteria was measured on the culture surface. Based on our previous investigation, concentrations of pyocyanin pigment were evaluated for P. aeruginosa under stress of Ag NPs (0, 3.12, 6.25, 12.5, 25, 50, and 100μg/mL)34. 2.11. Statistical analysis SPSS version16 software (SPSS Inc., Chicago, IL) and one way ANOVA (Tukey’s test) were applied respectively to perform statistical analysis and evaluate significance of data (p≤0.05). Results of secondary metabolites content, antioxidant activities, disc diffusion, biofilm formation, and pyocyanin production assays were presented as the means ± standard deviations (SD) of three replicates samples. 3. Results and Discussions 3.1.

UV-Vis spectroscopy analysis

For the purpose of primary detection from MNPs/MONPs presence in medium, it was used UVVis spectrum. In this analysis, control solution was lichen aqueous extract which does not show any prominent absorbance in UV spectrum. Impact of three concentrations of metals salts (0.1, 0.01, and 0.001M) was considered as effective factor in MNPs/MONPs formation and growth in colloidal solution. Solution of metals salts containing AgNO3, CuSO4, (FeCl2.4H2O, FeCl3.6H2O), ZnO(NO3)2.6H2O, and TiO(OH)2 with lichen extract illustrated respectively absorbance peaks at 378, 576, 216, 328, and 283 nm (Figure 1a-e). Preparation of Ag NPs by artiﬁcial sweeteners showed maximum absorbance at 380–450 nm35. For phytofabricated Cu NPs via the fruit extract



of Duranta erecta, absorption peak at range of 570 to 600 nm was observed36. Also, phytosynthesized ZnO and Fe3O4 NPs by respectively extracts of Tecoma castanifolia leaf and Couroupita guianensis fruit demonstrated the highest absorbance peaks at the ranges of 370-400 and 250–350 nm respectively37-38. These absorbance peaks with color changes indicated MNPs/MONPs formation. In the case of Ag NPs, it was observed 0.846 a.u for 0.001M and 1.719 a.u for 0.1M concentrations of silver nitrate salt. In addition, for Cu, Fe3O4, ZnO, and TiO2 NPs, these different intensities were (0.810 - 1.110 a.u), (0.650 - 1.064 a.u), (0.642 - 1.057 a.u), and (0.750 - 0.931 a.u) respectively. Also, discrepancy percentages between highest and lowest concentrations of metals salts were presented in Figure 1 caption. Similar results were reported in the case of biological synthesis of TiO2 and ZnO NPs39. Concentrations of each MNP/MONP were detected within 96 hrs by absorbance in their peaks Figure 1f. It is worth emphasizing that green synthesis of MNPs/MONPs by lichen aqueous extract had slow rate than plant aqueous extract which can be resulted from lower reducing capacity of lichen extract30.


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1.2

(f)

1 Absorbance (a.u)

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0.8 24hrs

0.6

48hrs

0.4

72hrs

0.2

96hrs

0 Ag

Cu Fe3O4 ZnO TiO2 NPs

Fig. 1. Impact of various concentrations of metal salts (0.1, 0.01, and 0.001M) on absorbance peaks of MNPs/MONPs. Difference percentages between lowest and highest metals salt amounts for Ag (a), Cu (b), Fe3O4 (c), ZnO, (d) and TiO2NPs (e) were respectively 51%, 28%, 39%, 40%, and 20%. MNPs/MONPs concentration was increased slowly within 96 hrs (f).



3.2.

XRD analysis

The crystal structure and phase purity of all MNPs/MONPs powders were examined by XRD technique. Diffraction peaks with miller indices of each MNP/MONP are presented at Figure 2ae. Ag NPs demonstrated 2 theta degrees of 35.5°, 43.6°, 65.6°, and 72.1° for (111), (200), (220), and (311) indices respectively (JCPDS card no: 65-2871)40. 35.9°, 39.6°, 44.3°, 54.3°, and 57.2° of diffractive peaks were respectively observed for (002), (111), (202), (020), and (202) crystal planes of Cu NPs (JCPDS card no: 04-0836)41. Fe3O4 NPs had (200), (311), (400), (422), (511), and (440) indices in the case of 29.9°, 35.1°, 44.2°, 50.6°, 54.3°, and 64.8° degrees (JCPDS card no. 85-1436)42. Similar to other green synthesized Fe3O4 NPs by Kappaphycus alvarezii seaweed and C. guianensis fruit extracts, these crystal planes are corresponding to the cubic spinel phase structure38, 43. Based on JCPDS card numbers 21-1272 (anatase phase) and 21-1276 (rutile phase), prominent diffractive peaks for TiO2 NPs including (101), (004), (200), (105), (211), (204), and (215) were at degrees of 25.2°, 37.6°, 47.8°, 53.5°,54.7°, 62.2°, 74.4° respectively44. In addition, 32°, 35.3°, 37.6°, 48.3°, 57.2°, 64.6°, 65.6°, 66.8°, 68.8°, and 64.5° peaks were respectively belonging to (100), (002), (101), (102), (110), (103), (200), (112), and (201) indices of ZnO NPs. In the case of ZnO NPs, the (100), (002), (101), and (102) indices can be associated with hexagonal structure of wurtzite structure (JCPDS data card no: 79-2205) 37, 45. Crystallite size calculations of Ag, Cu, Fe3O4, TiO2, and ZnO NPs by the Scherrer equation showed 44.87, 34.38, 45.84, 35.1, and 45.35 nm. Also, monoclinic and tetragonal crystal systems were observed for (Ag, Cu, Fe3O4, and ZnO NPs) and (TiO2 NPs) respectively. Similar to previous study, Ag and Cu NPs showed face centered cubic (FCC) crustal structure. In addition, rutile form was observed for TiO2 NPs30. Comparatively, the mean crystallite size for phytosynthesized Cu NPs by the fruit extract of D. erecta was 76 nm larger than our synthesized NPs by lichen extract (34.38 nm)36. Also, the average


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crystallite sizes for green synthesized TiO2, Cu, and Ag NPs by A. haussknechtii leaves were 66, 51, and 47nm30. 120

(a)

40

A B

20

(b)

100

A=(111), B=(200), C=(220), D=(311)

60

Intensity (a.u)

Intensity (a.u)

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A= (002), B= (111), C=(202), D=(020), E=(202)

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A= (101), B= (004), C= (200) D= (105), E= (211), F= (204), G= (215)

60

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40 20

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(d)

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A= (220), B= (311), C= (400), D= (422), E= (511), F= (440)

100

40

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(c)

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Intensity (a.u)


140

(e)

120

A= (100), B= (002), C= (101), D= (102), E= (110), F= (103), G= (200), H= (112), I= (201)

100 80

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F D E GHI

20 0 20

30

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50 60 2θ (deg)

70

80

Fig. 2. Diffraction peaks with miller indices of Ag (a), Cu (b), Fe3O4 (c), TiO2 (d), and ZnO (e) NPs.

3.3.

FT-IR analysis

Detection of functional groups contributing in green synthesis of MNPs/MONPs can be possible by FT-IR analysis. Resulted spectra of this analysis illustrated three major functional groups involving C=C, S=O, and C-Br in all samples (lichen aqueous extract and five MNPs/MONPs) (Figure 3a-f). Lichen aqueous extract showed different peaks; 630.39: C-Br stretching bond; 1039.46: S=O stretching bond related to sulfoxide; 1318.53: O-H bond-bending (phenol); 1410.87: S=O stretching bond (sulfonyl chloride); 1617.53: C=C stretching bond (α,β-unsaturated ketone). Striking peaks for Ag NPs were 795.72: C=C bond-bending (alkene); 1366.23: S=O (sulfonamide); 1752.11: C=O stretching (carboxylic acid); 2354.43: O=C=O stretching (carbon dioxide). Cu NPs had more peaks than other NPs at 629.39 and 676.90: C-Br stretching bond (halo compounds); 1017.06: C-O stretching (vinyl ether); 1126.80: S=O stretching (sulfone); 1517.72: N-O stretching (nitro compound); 1630.03: C=C stretching (α,β-unsaturated ketone). In addition, MONPs (Fe3O4, TiO2, and ZnO NPs) illustrated almost similar peaks indicating of participating


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of identical functional groups of P. muralis extract in MONPs green synthesis. There was common broad peak at about 3500 cm-1 wavenumber for all samples which can be associated with O-H stretching bond. This peak may be caused by presence of alcohols or carboxylic acids in samples46. In our previous study, we showed several functional groups such as C–H stretch (alkane), C=C stretch (aromatic), C–O stretch (alcohol), and amine N–H stretch for phytosynthesized Ag, Cu, and TiO2 NPs by A. haussknechtii leaf extract

30.

Similarly, the stretching vibrations related to

carbonyl (C=O), C-N, C-O and C-O-C groups were observed for green synthesis of Cu NPs36. This result approves role of secondary metabolites such as phenol with O-H bon-bending as reducer and stabilizer agents47.

Fig. 3. FT-IR spectra of P. muralis aqueous extract (a), silver (b), copper (c), Fe3O4 (d), titanium dioxide (e), and zinc oxide (f) NPs.

3.4.

TEM, SEM, and EDAX analyses



Based on TEM images, different shapes were observed for each MNP/MONP (Figure 4a-e). Spherical shape was common shape for all five MNPs/MONPs. This shape was very clear in the case of Fe3O4 NPs (Figure 4 e). It is worth noting that presence of extract components of lichen was more obvious for Cu, Fe3O4, and ZnO NPs (Figure 4 b, e, and d). Average diameter of Ag, Cu, Fe3O4, TiO2, and ZnO NPs were respectively 33.49±22.91, 253.97±57.2, 307±154, 133.32±35.33, and 178.06±49.97 (Figure 4f). Moreover, higher size uniformity of Cu NPs than other MNPs/MONPs can be resulted from this technique. In comparative way, both SEM and TEM images illustrated approximately spherical shapes for all NPs. Green synthesized Ag NPs by Ramalina dumeticola and Usnea longissima lichens showed respectively average diameters of 13 nm and 10.49 nm with spherical shape48-49. There were spherical shape and 70, 75, and 17 ± 10 nm mean diameter sizes respectively for biosynthesized Cu, ZnO, and Fe3O4 NPs by aqueous extracts of plant sources36-38.


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(f) 500 400 Diameter (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


300 200 100 0 Ag

Cu

Fe3O4 TiO2 NPs


ZnO


Fig. 4. TEM images of Ag (a), Cu (b), TiO2 (c), ZnO (d), and Fe3O4 (e) NPs with partilces size mean (f).

As it is obvious from figure 5a-e, SEM images demonstrated similar results in the case of lower size of Ag NPs than other MNPs/MONPs. Shapes of MNPs/MONPs were different in solid state compared to colloidal state. Presence of Ag (87.72%), Cu (26.42%), Fe (84.07%), Ti (66.41%), and Zn (25.61%) were respectively higher in 3.03, 8.1, 6.45, 4.5, and 8.7 keV on the basis of results of qualitative and quantitative analysis of EDAX. Peaks at 2.3 and 8 keV were indicating of biosynthesized Ag and Cu NPs50. In addition, this analysis showed chlorine (Cl) as the common element in Ag, Fe3O4, TiO2, and ZnO NPs formation with weight percentages of 1.65, 11.96, 1.96, and 10.34 respectively (Figure 5f, h, i, and j). Cu and ZnO NPs had respectively 15.46% and 25.14% weights of sulfur (S) element in their structures. Carbon (C) element was observed only with regard to Fe3O4 NPs. Also, TiO2 NPs demonstrated existence of calcium (Ca), silicon (Si), and iron (Fe) elements. Complex of MNPs/MONPs with Cl and S may be resulted from higher electronegative property of this element by values of 3.16 and 2.58. EDAX spectrum in the case of biosynthesuzed Ag NPs by P. praesorediosum lichen extract illustrated C and O elements in addition to Ag element51. Presence of O, S, and Cl elements was reported as complementary elements in green synthesis of Ag, Cu, and TiO2 NPs30. There were copper and oxygen elements for phytofabricated Fe3O4 NPs by fruit extract of C. guianensis plant38. Furthermore, green synthesized ZnO NPs via leaf extract of Tabernaemontana divaricata demonstrated only O and Zn elements52.


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Fig. 5. SEM images of Ag (a), Cu (b), Fe3O4 (c), TiO2 (d), and ZnO (e) NPs. EDAX analysis demonstrates amounts of each elements in green synthesized MNPs/MONPs of Ag (f), Cu (g), Fe3O4 (h), TiO2 (i), and ZnO (j).

3.5.

Disc diffusion and MIC-MBC assays



Two assays including disc diffusion and MIC-MBC were used to initially evaluate antibacterial activities of five MNPs/MONPs and detect one type of MNP with higher antibacterial ability than others. Results illustrated concentration-dependent behavior of MNPs/MONPs against E. coli, S. aureus, and P. aeruginosa species (Figure 6 and Table 1). For example, Ag NPs showed 18±0.89, 14±1.78, and 8±0.89 mm diameter of inhibition zones (DIZs) respectively at 0.1, 0.01, and 0.001M metal salt concentrations for E. coli. At highest concentration of metal salt, order of antibacterial strength against E. coli was Ag > Cu = ZnO > Fe3O4 > TiO2 NPs. Moreover, with regard to S. aureus and P. aeruginosa, arranges of Ag > ZnO > Fe3O4 > Cu ˃ TiO2NPs and Ag > Cu > Fe3O4 > ZnO >TiO2NPs were observed respectively. Ag and Cu NPs had respectively more antibacterial effect on P. aeruginosa by 30±0.89 and 24±0.89 mm which shows higher sensitivity of this pathogen than E. coli and S. aureus to these MNPs. In total, E. coli had more resistance than S. aureus and P. aeruginosa species to all five MNPs/MONPs. Also, among the NPs, TiO2 demonstrated weaker antibacterial properties.


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Fig. 6. Disc diffusion assay of (a) E. coli ATCC 25922, (b) S. aureus ATCC 43300, and (c) P. aeruginosa ATCC 27853 under treatment of green synthesized Ag, Cu, TiO2, ZnO, and Fe3O4 NPs.

Table 1. Antibacterial effect of Ag, Cu, TiO2, ZnO and Fe3O4 NPs on E. coli ATCC 25922, S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 as inhibition zone diameter. Diameter of inhibition zone (mm)±SD Bacterial strains

NPs

0.1M

0.01M

0.001M

E. coli ATCC 25922

Ag

18±0.89

14±1.78

8±0.89

Cu

14.33±1.36

14±0.89

7.66±1.86

TiO2

11.66±0.51

10.33±1.36

10±1.78

ZnO

14.33±0.51

7.33±0.51

7±0.89



S. aureus ATCC 43300

P. aeruginosa ATCC 27853

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Fe3O4

12±0.89

10.33±1.36

10±0.89

Ag

22±0.89

20±1.54

14±1.54

Cu

12.66±2.73

10±0.89

6±0.89

TiO2

10±1.78

9.66±0.51

9.33±1.86

ZnO

15.66±1.03

10.33±1.36

8±0.89

Fe3O4

15±0.89

11±0.89

10.33±1.36

Ag

30±0.89

24±1.78

16±0.89

Cu

24±0.89

14±1.78

11.66±2.25

TiO2

9.66±0.51

9.33±1.36

7±0.89

ZnO

7±0.89

12±1.78

8±0.89

Fe3O4

13.66±1.36

13±0.89

4±1.78

Disc diffusion results about antibacterial activities of green synthesized silver, copper, and titania NPs were somewhat similar to previous study. In regards to E. coli, green synthesized Ag and Cu NPs by A. haussknechtii plant species had higher antibacterial properties as values of 36±2 and 34±2.64 mm respectively

30.

ZnO NPs had antibacterial activity against sensitive and MDR

bacteria containing E. coli and S. aureus at 0.1M concentration of metal salt (ZnCl2)39. More antibacterial activities against E. coli and S. aureus compared to Salmonella paratyphi bacteria were observed for green synthesized ZnO NPs52. In addition, ecofriendly fabricated CuO NPs by fruit extract of Terminalia belerica showed IZDs values of 24, 18, 18, 24, and 17 mm against S. aureus, Bacillus subtilis, E. coli, Klebsiella pneumoniae, and Salmonella enterica bacteria respectively53. Green synthesized Fe NPs by flower aqueous extract of Hibiscus sabdariffa (with mean diameter size of 18 nm) had high antibacterial effects on E. coli MTCC405 and S. aureus MTCC3160 strains54.


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(a)

(b)

100

120

80

100

60

80

E. coli

40

S. aureus

20

P. aeruginosa

0

(μg/mL)

60

E. coli

40

S. aureus

20

P. aeruginosa

Cu Ti O 2 Zn O Fe 3O 4

NPs

g

Zn O Fe 3O 4

O 2

Ti

Cu

A g

0

A

(μg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NPs

Fig. 7. Values (μg/mL) of MIC (a) and MBC (b) of five NPs against E. coli, S. aureus, and P. aeruginosa.

In order to indicate bacteriostatic and bactericidal effects of MNPs/MONPs, MIC-MBC method at concentration range of 0.5, 1, 2, 4, 10, 20, 40, 60, 80, and 100 μg/mL was used. As illustrated in figure 7, values of MIC-MBC assay approved results of disc diffusion assay. MIC amounts for Ag NPs were 60, 20, and 10 μg/mL for respectively E. coli, S. aureus, and P. aeruginosa. In addition, MBC values with 80, 60, and 20 μg/mL were observed for this MNP. Overall, lowest and highest MIC/MBC results were related to Ag and TiO2NPs respectively. More antibacterial results of Ag and Cu NPs than MONPs were similar to disc diffusion assay. In previous study, 10 and 60 μg/mL amounts of MIC and MBC were reported for E. coil ATCC 25922 and S. aureus ATCC 43300 under stress of biosynthesized Ag and Cu NPs30. Green synthesized Ag NPs by Penicillium polonicum by average diameter size of 10–15 nm had MIC and MBC values by 15.62 μg/mL and 31.24 μg/mL against Acinetobacter baumanii as MDR bacterium55. In addition, in synergism effect, silver NPs with Stachys Lavandulifolia-modified magnetic NPs showed bacteriostatic concentrations of 43 and 16 μg/mL against E. coli and S. aureus

56.

Several antibacterial mechanisms including ions release of Ag+ and Cu2+ from



respectively Ag and Cu NPs, interaction of these ions with cell membrane and biological macromolecules belonging to cytosolic part of bacteria as well as production of oxygen reactive species (ROSs) were presented for these nanomaterials57-59. Fe3O4 NPs can kill bacteria by formation of OH° and HO°2 free radicals resulted from reaction of these NPs with H2O2 in bacterial medium60-61. In the case of ZnO and TiO2 NPs, electron-hole pair generation under ultraviolet or visible radiation can disrupt cell membrane, functional or structural proteins, and nucleic acids of bacterial cell34, 39, 62. 3.6.

Bacterial growth kinetic under Ag NPs stress

Bacteria growth of three bacteria (E. coli, S. aureus, and P. aeruginosa) in planktonic situation was evaluated upon different concentrations of Ag NPs stress during 6 hrs of follow-up (Figure 8). Two initial optical densities of bacteria cultures (0.1 and 0.2) were selected in order to measure effect of initial microorganism amount on dynamic response of bacteria to MNPs/MONPs presence. All bacteria in low initial OD value had less resistance to MNPs/MONPs than higher OD. Also, significant growth inhibition was observed in MNPs/MONPs concentrations of 10, 20, 40, and 60 μg/mL. In comparative way, at highest concentration of MNPs/MONPs (60 μg/mL), lower initial OD, and at sixth hour, ODs of E. coli, S. aureus, and P. aeruginosa were respectively 0.102±0.041, 0.047±0.021, and 0.037±0.015. Similar investigation demonstrated growth inhibition of S. aureus and Bacillus subtilis at 100 μg/mL amount of p-ZnO NPs63.


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0

1.2

0.5

1 E. coli

2

4

0

1.5

0.5

0.8 0.6 0.4

1 E. coli

2

4

1

OD600

OD600

1

0.5

0.2 0

0 0

1

0

1.5

2 3 4 Incubation time (hrs)

0.5

1 S. sureus

5

2

0

6

4

1


0

1.2

0.5

1

5

6

2

4

S. aureus

1

OD600

OD600

1

0.5

0.8 0.6 0.4 0.2

0 0

1

0

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0


5

0.5 1 2 P. aeruginosa

0

6

0

4

OD600

OD600

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

1


5

6

1

0

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0


5

0.5 1 2 P. aeruginosa

1


6

4

5

6

Fig. 8. Growth kinetics with initial optical densities (ODs) of 0.1 and 0.2 in the presence of various amounts of Ag NPs (0.5, 1, 2, 4, 10, 20, 40, and 60 μg/mL) for E. coli ATCC 25922, S. aureus ATCC 43300, and P. aeruginosa ATCC 27853.



3.7.

Changes of bacterial morphology at Ag NPs presence

Antibacterial activity of green synthesized Ag NPs as deformation of bacteria cell wall was surveyed precisely by SEM technique (Figure 9). For this case, P. aeruginosa as gram negative bacterium was selected based on previous antibacterial results. As illustrated in Figure 9, Ag NPs absorbance on cell surface as aggregation form and wrinkled cell wall of bacteria were two striking results of this assay. In our previous investigation, gram negative bacterium, E. coli showed Ag NPs adsorption on the bacterial cell membrane, membrane blebs, membrane clumping, and bacterium death which can be approximately similar with present results30. Deformation as wrinkled cell wall of P. aeruginosa proves interaction of Ag NPs with this section of bacterium as first antibacterial mechanism of MNPs. Higher antibacterial abilities of Ag NPs can be resulted from Ag+ ions release from Ag NPs following by damage of biological macromolecules such as phospholipids, proteins, and nucleic acids64. Electrostatic bond between positive charge of Ag+ ions and negative charge of bacteria cell envelope is a first probable interaction to increase permeability of bacterial membrane or cell wall via making of pits or holes65.


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Fig. 9. SEM images of green synthesized Ag NPs on the surface of P. aeruginosa cell wall as wrinkled deformation result.

3.8.

Biofilm formation assay

Biofilm formation of three bacteria E. coli ATCC 25922, S. aureus ATCC 43300, and P. aeruginosa ATCC 27853 were evaluated by CV assay at six concentrations (3.12, 6.25, 12.5, 25, 50, and 100 μg/mL) of five biosynthesized NPs. As shown in figure 10, there was reduction in biofilm formation by increasing of MNPs/MONPs concentrations for three bacteria species. This inverse relationship was intense for Ag, Cu, and ZnO NPs. As, at 100 μg/mL, there was more reduction of biofilm biomass of three bacteria than to controls (p≤0.05). In contrast, a lower antibiofilm activity was displayed by TiO2 and Fe3O4 NPs (Figure 10 c and e). Antibiofilm effect of Ag, Cu, and ZnO NPs at highest concentration on E. coli was respectively values of 0.222± 0.041, 0.312±0.058, and 0.295± 0.039 which intensity of antibiofilm activity was Ag > ZnO > Cu NPs. In respect of S. aureus, reduction amounts of 0.241±0.067, 0.283±0.057, and 0.331± 0.036 were observed for respectively Ag, Cu, and ZnO NPs. Moreover, P. aeruginosa with higher sensitivity than other bacteria had biofilm reduction values by 0.184±0.043, 0.254±0.088, and 0.244±0.022. Totally, it can be result that ordering of antibiofilm activity was Ag > ZnO > Cu > TiO2 > Fe3O4 NPs.



E. coli Biofilm formation (OD570nm)

2

(a)

S. aureus

(b)

P. aeruginosa

(c)

(d)

(e)

1.5

0.5

***

***

3.12 6.25 12.5 25 50 100

1

0 3.12 6.25 12.5 25 50 100

***

3.12 6.25 12.5 25 50 100

3.12 6.25 12.5 25 50 100

0 3.12 6.25 12.5 25 50 100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 46

Concentrations (μg/mL)

Fig. 10. Effects of different concentrations of Ag (a), Cu (b), TiO2 (c), ZnO (d), and Fe3O4 (e) NPs on the biofilm formation by E. coli ATCC 25922, S. aureus ATCC 43300, and P. aeruginosa ATCC 27853 after 24 hrs incubation without shaking at 37 C. Data are presented as Mean ± SD and (*) sign is P≤0.05 value versus control samples.

Higher antibiofilm ability of phytosynthesized Ag NPs via red ginseng root extract against S. aureus and P. aeruginosa was observed at 4 μg/mL66. It is worth mentioning that lower effect of Fe3O4 NPs on biofilm formation may be resulted from chelation mechanisms of iron by biofilm structure67-68. Antibiofilm activity of copper NPs was reported as higher values of 90.58 % for P. aeruginosa PACI02 clinical pathogens69. Nanocomposite by 1.5 wt% of Ag-TiO2 had complete reduction in biofilm of S. aureus and E. coli during 24 hrs treatment70. ZnO NPs demonstrated antibiofilm property on E. coli O157:H7, methicillin-resistance S. aureus (MRSA), methicillinsensitive S. aureus (MSSA), and P. aureuginosa PAO1 strains at 1, 5, 5, and 1 mM concentrations71. Also, for the case of Fe3O4 NPs, viable bacteria in biofilms of S. aureus ATCC 25923 and E. coli 15224 were reduced significantly on silicon and glass surface with Fe3O4 NPs coating after 72 hrs incubation at 37°C72.


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3.9.

Biofilm morphology

Bacteria including E. coli, S. aureus, and P. aeruginosa can produce biofilm structure in extreme conditions which has more resistance to conventional antibiotics. In this way, light microscopy was used to observe changes of biofilm structure under different concentrations (0, 3.12, 6.25, 12.5, 25, 50, and 100 μg/mL) of green synthesized Ag NPs. As it is observable in Figure 11, higher concentrations of MNPs had more antibiofilm effect on all bacteria than lower one. Maximum reduction (72%) was in the case of P. aeruginosa biofilm than S. aureus (68%) and E. coli (66%). Results demonstrated higher biofilm inhibition by Ag NPs amount of 100 μg/mL. Similar study showed concentration of Ag NPs (15μg/mL) for E. coli (75%) and S. aureus (89%)73.



Fig. 11. Biofilm images of E. coli, S. aureus, and P. aeruginosa in presence of different amounts of biosynthesized Ag NPs: control (a), 3.12 (b), 6.25 (c), 12.5 (d), 25 (e), 50 (f), and 100 μg/mL (g) (by a magnification of 40×). Blue images show negative form of biofilm formation for affirmation of reduction amount.

3.10.

Motility assay

Motility of E. coli and P. aeruginosa on semi-solid surface was defined as swarming mode which requires energy source in medium for movement of flagella as active form. In contrast, S. aureus has passive motility named spreading by synthesis of Phenol Soluble Modulin (PSM) surfactant. Bacterial motility is associated to virulence ability and biofilm formation which can be affected by medium conditions74. In this study, motility of three bacteria was tested upon green synthesized Ag NPs stress at six serial concentrations (3.12, 6.25, 12.5, 25, 50, and 100 μg/mL). As illustrated in Figure 12f and g, there was meaningful reduction of colony forming in higher concentrations (50 and 100 μg/mL). Inhibition of bacterial motility may be resulted from Ag NPs interaction with active and passive motion mechanisms of these bacteria75.


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Fig. 12. Reduction in motility ability of E. coli, S. aureus, and P. aeruginosa under 0 (a), 3.12 (b), 6.25 (c), 12.5 (d), 25 (e), 50 (f), and 100 μg/mL (g) amounts of lichen synthesized Ag NPs.

3.11.

Pyocyanin measurement

Pyocyanin, type III secretion system (T3SS), exotoxin A, and hydrogen cyanide (HCN) are virulence factors of P. aeruginosa which can resulted in cytotoxicity effects in infectious diseases such as cystic fibrosis76. Resistance to Ag ions (Ag+) in P. aeruginosa (PA14) strain was reported previously. In this study, P. aeruginosa illustrated significant sensitivity at higher concentrations (50 and 100 μg/mL) of lichen synthesized Ag NPs by reduction in pyocyanin production (Figure 13). Values of 1.17±0.021 and 1.31±0.025 μg/mL were observed for 100 and 50 μg/mL compared to control with 2.4±0.022 μg/mL (p≤0.05). Therefore, decrease of virulence strength of P. aeruginosa in presence of Ag NPs can be result from this assay.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Pyocyanin Concentrations (μg/mL)


Page 34 of 46

3 2.5 2

___*___

1.5 1 0.5 0 0

3.12 6.25 12.5 25 MNPs Concentrations (μg/mL)

50

100

Fig. 13. Significant decrease of pyocyanin concentration as virulence factor of P. aeruginosa ATCC 27853 under Ag NPs treatment in higher amounts (50 and 100 μg/mL). (meaningful difference, Turkey’s test, *p ≤ 0.05). Data were presented as average values of three replicates with error bars of standard deviation.

3.12.

Total phenol, flavonoid, flavonol, and tannin contents

Amounts of important secondary metabolites by lichen aqueous extract and five MNPs/MONPs were measured as total contents of phenolic, flavonoid, flavonol, and tannin (Table 2). TPC, TFC, TFLC, and TTC were highest for Ag NPs than other MNPs/MONPs and lichen extract. After this type of MNP, Cu NPs showed more TPC, TFC, TFLC, and TTC by 6.51±1.14 GAE/g DW, 1.73±0.65 RE/gDW, 0.77±0.08, and 0.61±0.1 CE/gDW values respectively. Lichen aqueous extract had higher amounts of TPC, TFLC, and TTC than Fe3O4, TiO2, and ZnO NPs. In addition, Fe3O4 > ZnO > TiO2 NPs sequence was observed in regards to TPC, TFC, TFLC, and TTC parameters. There was no significant difference between lichen extract and MNPs/MONPs for these assays. Furthermore, flavonoid/phenol ratio was in the range 0.25 - 0.28. Higher physicochemical activities of Ag NPs than other MNPs/MONPs and lichen extract can be reason


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of these results. As reported previously, copper NPs similar to silver NPs illustrate approximately more adsorption of secondary metabolites30. These properties may have major effects on NPs formation, antibacterial, and antioxidant activities77-78. Based on previous study, three important secondary metabolites named Cedran-diol, (8S,14)- (C15H26O2), usnic acid (C18H16O7), and 3,9Dimethyltricyclo[4.2.1.1(2,5)]dec-3-en-9-ol (C12H18O) had respectively area percentages of 34.38, 16.60, and 6.26 79. As illustrated in Figure 14, these materials having hydroxyl groups can contribute in MNPs/MONPs formation by reduction of metal ions and also stabilization of MNPs/MONPs 80. It is worth to note that tannins having several hydroxyl groups can contribute in reduction of metal salts and stabilizing of MNPs or MONPs in colloidal suspensions53. Table 2 . Total phenolics, flavonoid, flavonol, tannin contents, and flavonoid/phenol ratio of green synthesized Ag, Cu, Fe3O4, TiO2, and ZnO NPs and aqueous extract of P. muralis lichen. Samples

TPC*

TFC**

TFLC***

TTC***

Flavonoid/Phenol

Lichen extract

6.24±0.9

1.56±0.39

0.71±0.12

0.56±0.12

0.25

Ag NPs

9.48±1.08

2.67±0.42

1.07±0.23

0.86±0.09

0.28

Cu NPs

6.51±1.14

1.73±0.65

0.77±0.08

0.61±0.1

0.26

Fe3O4 NPs

6.04±0.63

1.68±0.15

0.7±0.25

0.54±0.09

0.27

TiO2 NPs

5.33±0.98

1.36±0.31

0.5±0.18

0.39±0.15

0.26

ZnO NPs

5.7±1.69

1.52±0.43

0.6±0.11

0.46±0.13

0.25

*mg

gallic acid equivalent (GAE)/gDW; **mg rutin equivalent (RE)/gDW; ***mg (+)-catechin equivalent (CE)/gDW;

Values are averages of three independent analyses ± standard deviation (n = 3).



Fig. 14. Chemical structures of three major secondary metabolites related to P. muralis extract (PebChem; Open Chemistry Database).

3.13. Total antioxidant capacity (TAC) and DPPH assay TAC and DPPH assays were used to determine antioxidant property of P. muralis lichen and all MNPs/MONPs. As illustrated in Figure 15, both tests showed higher antioxidant activity for ascorbic acid. For the case of treatments, there was similar antioxidant serialization with results of secondary metabolites contents (lichen extract > Ag NPs > Cu NPs > Fe3O4 NPs > ZnO NPs > TiO2 NPs). In highest concentration (500 μg/mL), percentages of DPPH assay were 76.99 ± 3.72, 48.57 ± 3.22, 37.31 ± 2.51, 31.02 ± 2.73, 28.43 ± 1.72, 25.26 ± 3.28, and 14.22 ± 2.88 for respectively ascorbic acid, lichen extract, Ag NPs, Cu NPs, Fe3O4 NPs, ZnO NPs, and TiO2 NPs. Similarly, this sequence was observed in this concentration for TAC experiment. These results show silver and copper NPs have good antioxidant with antibacterial properties compared to MONPs. It is noteworthy to emphasize that antioxidant property is irrevocable factor in selection of suitable antibacterial agents with biocompatibility in living organism81. Direct relationship between higher antioxidant activities and total poly-phenolic content was indicated in the case of bark aqueous extract of Phellodendron amurense tree82. In this way, synergistic antioxidant and


Page 36 of 46

Page 37 of 46

antibacterial impacts may be resulted from various secondary metabolites adhering on the surface of MNPs or MONPs 83 84. 90

(a)

0.6 0.5 0.4

100 200

0.3

300

0.2

400

0.1

500

DPPH free radical scavenging activity (%)

70 60 50

100

40

200

30

300

20

400

10

500

0

N Cu Ps Fe N 3O Ps 4 Ti NP O s 2 N Zn Ps A O sc or NP bi s ca ci d

ct tra

g

ex en Li ch

A

ex

tra c g t N Cu Ps Fe N 3O Ps 4 Ti NP O s 2 N Zn Ps A sc O N or bi Ps ca ci d

0

en

(b)

80

A

Total antioxidant capacity (OD)

0.7

Li ch

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 15. Total antioxidant capacity (a) and DPPH free radical scavenging activity (b) of lichen aqueous extract and biosynthesized Five MNPs/MONPs in various concentrations (100, 200, 300, 400, and 500 μg/mL). Ascorbic acid solution was presented as standard control. Values are means of three independent analyses ± standard deviation (n = 3).

4. Conclusions Using of ecofriendly substances to prevent entering of toxic materials into the environment is important duty of green chemistry. Most chemical and physical approaches in MNPs/MONPs synthesis do not follow this affair. In this way, emerging of green methods by application of herbal, algae, fungus, lichen, and bacterial materials has been promising. There are few investigations about MNPs/MONPs synthesis by lichen species. This study attempted to show simple green synthesis of Ag, Cu, MNPs and TiO2, ZnO, and Fe3O4 MONPs by P. muralis lichen aqueous



extract with comparison of their main physicochemical properties. In according to results by antioxidant assays, roles of secondary metabolites including phenol, flavonoid, flavonol, and tannin were efficient in MNPs/MONPs formation. Moreover, meaningful results that were observed in antibacterial, antibiofilm, and antivirulence activities of green synthesized Ag NPs against MDR and sensitive bacteria are valuable in the field of infections therapy. Motility ability of P. aeruginosa, E. coli, and S. aureus as well as pyocyanin production by P. aeruginosa as virulence factors were decreased under Ag NPs stress. These results can be more important to formulate new drug delivery system based on green synthesized MNPs or MONPs due to remove colonization and infections of these bacteria in respiratory, gastrointestinal, and urinary tracts. However, in addition to suitable results about antioxidant capacity of these MNPs/MONPs, complementary and precise studies as in vivo are needed to evaluate other nontoxicity aspects such as cytotoxicity. It is noteworthy to highlight that one of the prominent disadvantages of green synthesis is non-uniformity of NPs size and shape in reaction medium. In this way, high polydispersity of NPs can impact on biological and medicinal effects specifically in physiological conditions. Therefore, more investigations are required to improve this hindrance such as utilization of other physical and chemical methods. Acknowledgments We are grateful to microbiology laboratory of Razi University for providing sensitive and MDR strains of E. coli ATCC 25922, S. aureus ATCC 43300, and P. aeruginosa ATCC 27853.


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For Table of Contents Use Only Title: Antibacterial, antibiofilm, antiquorum sensing, antimotility, and antioxidant activities of green fabricated Ag, Cu, TiO2, ZnO, and Fe3O4 NPs via Protoparmeliopsis muralis lichen aqueous extract against multi drug resistant bacteria Authors: Mehran Alavi, Naser Karimi, Tahereh Valadbeigi




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Antibacterial, antibiofilm, antiquorum sensing, antimotility, and

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