Bio fabrication of silver nanoparticles with antibacterial and cytotoxic abilities using lichens

Recently, increase bacterial resistance to antimicrobial compounds issue constitutes a real threat to human health. One of the useful materials for bacterial control is Silver nanoparticles (AgNPs). Researchers tend to use biogenic agents to synthesize stable and safe AgNPs. The principal aim of this study was to investigate the ability of lichen in AgNPs formation and to find out their suppression ability to MDR bacteria as well as their cytotoxic activity. In the current study, lichens (Xanthoria parietina, Flavopunctelia flaventior) were collected from the south of the Kingdom of Saudi Arabia. Lichens methanolic extracts were used for conversion of Ag ions to AgNPs. Prepared biogenic AgNPs were characterized by Ultraviolet–Visible (UV–Vis) Spectroscopy, Transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) and Zeta potential and Energy-Dispersive X-ray Spectroscopy (EDS). Lichens Secondary metabolites were determined by Fourier-Transform Infrared Spectroscopy (FTIR) and Gas Chromatography–Mass Spectrometry (GC–MS). The antibacterial activity and synergistic effect of AgNPs were evaluated against pathogenic bacteria, including gram-positive; Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococcus (VRE), and gram-negative; (Pseudomonas aeruginosa, Escherichia coli) as well as the reference strains (ATCC) using the agar disk diffusion method. Cytotoxic effect of biogenic AgNPs was tested against HCT 116 (Human Colorectal Cancer cell), MDA-MB-231 (Breast cancer cell), and FaDu (Pharynx cancer cell) by MTT test. TEM imaging showed well-dispersed spherical particles of 1–40 nm size as well as zeta size showed 69–145 nm. Furthermore, FTIR and GC–MS identified various lichen chemical molecules. On the other hand, the highest antibacterial activity of AgNPs was noticed against P. aeruginosa, followed by MRSA, VRE, and E. coli. AgNPs influence on gram-negative bacteria was greater than that on gram-positive bacteria and their synergistic effect with some antibiotics was noted against examined microbes. Moreover, higher cytotoxicity for biogenic AgNPs against FaDu and HCT 116 cell line in relation to MDA-MB-231 was noted. Given the current findings, the biogenic AgNPs mediated by lichens had positive antibacterial, synergistic and cytotoxic powers. Therefore, they might be considered as a promising candidate to combat the multi-drug resistance organisms and some cancer cells.

with 100 mL of 80% methanol. The mixtures were left shaking (160 rpm for 24 h) at room temperature. Then mixture was filtered through 'Whatman filter paper Grade 1' and further filtered through 'Whatman filter paper Grade 3' for more extract purification then kept at 4 °C. Furthermore, 100 mL methanol was added to the rest of the extract and left more 24 h on the shaker under the same previous conditions. The mixture was filtered twice in the same way as before and the methanol was removed by a rotary evaporator device. The round bottom flask was weighed before and after extraction, then the difference was calculated, the concentration of the final extract was 10 mg/mL. The extract was closed tightly and saved at 4 °C until used 34 . optimization of Agnps synthesis conditions. The parameters that may affect the nanoparticles biosynthesis were tested such as the ratio of lichen extract to AgNO 3 solution was tested according to (1:9, 1:3, 1:2, 1:1), AgNO 3 concentration (1 mM, 10 mM), incubation time (24 h, 48 h, 72 h) and temperature degree (25 °C; 40 °C). After the color change, the particle size was measured by zetasizer. Thereafter, AgNPs were synthesized by application of the conditions that provided small particle size 27 and considered as an optimum conditions. AgNPs prepared using Xanthoria parietina is known as Xa-AgNPs and those prepared using Flavopunctelia flaventior are Ff-AgNPs. characterization of biogenic Agnps. Different methods for the characterization of the biogenic AgNPs prepared in the current study were used for nanoparticles description such as:

Synthesis of Agnps.
Ultraviolet-Visible Spectroscopy. Ultraviolet-Visible (UV-Vis) Spectroscopy absorption was measured using a spectrophotometer (BIOCHROM Libra S60PC, Serial Number: 119377, England). All measurements were performed for the mixture after 24 h of reaction within the range of 300-600 nm and deionized water was used as a blank 25 .
Transmission electron microscopy (TEM). The size distribution and morphology of AgNPs were investigated at 80 kV voltage by TEM (JEM-1011, JEOL, Japan). samples were prepared by drop-coating on carboncoated (200 mesh) TEM grids 26 .
Dynamic Light Scattering (DLS) and Zeta potential. The size distribution pattern was evaluated by a dynamic light scattering technique and the electrical charge of particles by zeta potential were the measurement with a Zetasizer (NANO ZSP, Malvern Instruments Ltd, Serial Number: MAL1118778, ver 7.11, UK) according to Siddiqi et al. 17 .
Energy-dispersive X-ray spectroscopy (EDS). EDS was used for the elemental analysis and confirmed the presence of the silver element precisely using SEM (JEOL, JED-2200 series, Japan) 35 .
Fourier-transform infrared spectroscopy (FTIR). FTIR measurements were carried out to identify the potential biomolecules in lichen extract responsible for reducing and capping the reduced AgNPs. The spectra were  MHA plates were inoculated by tested strains using a petri plate rotator. The sterile discs were saturated by 20 µL of AgNPs and kept for drying under aseptic conditions. Then dried discs were transported to bacterial cultured agar surface using sterile forceps with pressure for the discs to be closely bonded. Sterile distilled water was used as negative control and antibiotic susceptibility discs were used as positive controls. After 15 min of discs application, plates were inverted and incubated at 35 °C for 16-18 h according to the Clinical and Laboratory Standards Institute 38 . The zone of inhibition around the discs was measured by Vernier caliper. Antibacterial activity was investigated and zone diameter breakpoints (mm) for antibiotics were determined according to M02 and M07 from 39,40 . Such mentioned methods were also applied for lichen methanol extracts and methanol was used as positive control 26 .
MICs and MBCs determination. The minimal inhibitory concentrations (MICs) of the AgNPs were determined by the microdilution method in 96-well microtiter plates. The obtained concentration range of lichen extract was from 2.5 to 0.0098 mg/mL. Positive control (media contains inoculum with antibiotic), negative control (media contain inoculum), and AgNPs solution (media with AgNPs) was applied in the last three columns. All plates were incubated for 18-20 h at 35 °C. MICs were determined by comparing to positive and negative control wells. The lowest concentration with no growth (turbidity or pellet) was defined as MICs. Results expressed as the mean values of two independent replicates. MICs breakpoints (μg/mL) for antibiotics was determined by CLSI 39,40 . The minimum bactericidal concentrations (MBCs) was be determined from broth microdilution by sub-culturing a sample from wells on MHA plates by loop 1 μL. After 24 h of plates incubation, the concentration that kills 99.9% of bacterial growth has been defined as MBC 41 . Furthermore, the tolerance level was calculated as MBCs/MICs ratios to find-out the expected action for AgNPs (bactericidal or bacteriostatic) against tested bacteria 16 .
Synergistic effect of AgNPs and antibiotics against MDR pathogens. The disk diffusion method was used to test the effect of antibiotics in combination with AgNPs against the MDR bacteria. Test plates were inoculated with the microbes in the same way as AST. AgNPs at concentration of 20 μg was added to each disc of Tetracycline (TE), Linezolid (LZD), Gentamicin (CN), and Ampicillin (AMP), then transported to bacterial cultured agar to test the prepared discs activities. Antibiotic discs were used as positive control. The plates were incubated for 18 h at 35 °C 42 then the inhibition zone around discs were assessed. cytotoxicity of Agnps. Cell lines. HCT 116, MDA-MB-231 and FaDu were used ( Table 1). All cells were grown in GIBCO Dulbecco's modified Eagle's medium (DMEM) which containing 10% fetal bovine serum (FBC), 2 mM glutamine, 100 U penicillin and 0.1 mg/mL streptomycin. Culture flasks were incubated for 4 days at 37 °C, 99-100% humidity in 5% CO 2 incubator. The subculture of cells was performed daily to keep the cells from over confluent. After four days of incubation, the media was removed from cell culture flasks T-25 using a sterile pipette. The cells were washed by 5 mL phosphate buffer saline (PBS) for 1 min then added to 1 mL of trypsin. After 2 min, culture flask was checked under the microscope for cells detachment from substrate. 2 mL of media was added to the flask to stop the reaction because the FBS inactivated the trypsin. The cells were placed in tubes and centrifuged for 5 min 6000 rpm to dispose of the trypsin. 2 mL of the medium was put for precipitation then the number of cells was counted 43 . Application of AgNPs on cell culture (Microplate 96-well). 100 µL of media in (Dilution plate) was added for each well except first and last row due to its exposure to air more than the internal rows. 50 µL of Xa-AgNPs was placed in the first well of the second and third row. 50 µL of lichen extract (Xa) was placed in the first well of the fourth and fifth row. 50 µL of methanol was placed in the first well of the sixth and seventh row and methanol was used as control. Using serial dilution method by multichannel micropipette; 50 µL was transferred from the first column to the second column and so on to last column and disposal of the rest 50 µL. 120 µL of cell suspension in cultivation plate was added to each well except first and last row. 60 µL of each column in dilution plate was transferred to the same column in cultivation plate; More precisely 60 µL of (tested material with media) was transferred from the last column of dilution plate to last column of cultivation plate (from lowest concentration to the highest concentration), and so on with all columns. These methods were applied to Ff and Ff-AgNPs and the experiment was performed in duplicate 35 .
MTT assay. 250 mg of MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide was dissolved in 50 mL of PBS and stirred with a magnetic stirrer for homogenization. 20 µL of MTT was added to each well then incubated for 2 h at 37 °C, 99-100% humidity in 5% CO 2 incubator. The medium was removed by suction. Cells were washed with PBS and centrifuged to removed dead cells and cellular debris. 100 µL of isopropanol (C 3 H 8 O) was added to each well then was shaken for 10 min. MTT conversion to purple-colored formazan crystals is due to presence of viable cells with active metabolism. Absorbances were measured at 595 nm by ELISA reader (ANTHOS 2010 Microplate Reader, Biochrom LTD, UK). The cell viability was calculated using following formula: Half-maximal inhibitory concentration (IC 50 ) values represent the concentration of tested materials that required for 50% inhibition of cells growth. IC 50 values were measured from the regression curves 45 .

Statistical analysis.
Means and standard deviations for antibacterial activities were calculated using MICROSOFT EXCEL 2019. AgNPs images were chosen as one of the triplicates. ORIGIN software version 6.1 (ORIGIN Lab Corporation, Northampton, USA) statistical analyses was used for MTT assay for IC 50 assessment.

Biosynthesis of Agnps.
To obtain the best AgNPs size, conditions were optimized and adjusted as follows: the two lichen types were extracted by methanol and each extract was added to AgNO 3 (10 mM) at a ratio of 1:3 at 40 °C for 72 h. It was noticed that increasing the reaction temperature and AgNO 3 concentration reduced Cell viability 100% = OD Sample/OD Control × 100. After 72 h, the mixture was turned to brown and no more colour changes were noted. Such colour stability might point to attainment of maximum reduction time but also it revealed that, colour intensity was highly time dependant. both Xa-AgNPs and Ff-AgNPs assume similar pattern in colour development and stable nanomaterials for more than 4 months were approved. characterization of biogenic Agnps. Reactions between silver ions and both lichen extracts were monitored by UV-Vis Spectroscopy of AgNPs (Fig. 6). The analysis of UV-Vis spectroscopy showed an appearance of surface plasmon resonance peak at 412 and 405 nm for Xa-AgNPs and Ff-AgNPs respectively. TEM imaging for Xa-AgNPs and Ff-AgNPs showed well-dispersed spherical particles of 1-40 nm size, furthermore, clear capping agents around AgNPs as light colour were detected (Fig. 7). Results obtained from DLS for Xa-AgNPs and Ff-AgNPs had average diameters of 145 and 69 nm with a polydispersity index (PDI) of 0.291 and 0.458, respectively (Fig. 8). Zeta Potential values were − 24, − 20 mV (Fig. 9) for Xa-AgNPs and Ff-AgNPs respectively. Analysis through EDS confirmed the presence of the silver element in both AgNPs beside the carbon and oxygen. The results showed strong silver signals Ag-L at 3 keV, along with carbon peak and oxygen peak (Fig. 10). FTIR spectra characterized the lichen extracts from 'Xa and Ff ' as well as after AgNPs prepared by their aid (Fig. 11). Absorbance peaks for both lichen types were at 3421 cm −1 , 2066 cm −1 , 1634 cm −1 and 593 cm −1 . Additionally, the absorbance peaks for Xa-AgNPs and Ff-AgNPs were at 3421-3332 cm −1 , 2070 cm −1 , 1637 cm −1 , and 541 cm −1 , representing the role of various functional groups in the bio-reduction of AgNO 3 .

Identification of Lichens secondary metabolites by (GC-MS).
To find out the chemical constituents that could be involved in conversion of silver ions to AgNPs, Gas chromatography-mass spectrometry was used for lichen methanol extracts. GC-MS analysis of both lichen types showed the presence of 25 compounds (Table 2). Benzenamine, 4-methoxy-N-methyl-compound was noticed for both lichen types.
evaluation of antibacterial activity of Agnps. The antibacterial activities of lichen extracts and the biogenic AgNPs were evaluated against MDR and ATCC bacterial strains. No antibacterial activity was detected for lichen extracts however, AgNPs inhibited the growth of both tested gram-negative and gram-positive strains ( Table 3). The highest antibacterial activity of Xa-AgNPs and Ff-AgNPs was noticed against P. aeruginosa followed by MRSA, VRE and E. coli, respectively. No significant variations were observed in the activity of Xa-AgNPs and Ff-AgNPs against tested ATCC and MDR bacteria. MRSA, VRE, P. aeruginosa MDR, and E. coli ATCC were more sensitive of Ff-AgNPs compared to Xa-AgNPs. Furthermore, the MICs and MBCs of Xa-   (Table 4). For tolerance level of AgNPs against tested bacteria, MBCs/MICs ratios were calculated and determined whether AgNPs action were bacteriostatic or bactericidal. Xa-AgNPs and Ff-AgNPs showed bactericidal effect against all tested bacterial strains since the tolerance levels were ≤ 4 ( Table 5). In addition, the possible synergistic effect was noted against all tested strains when combination between each of Xa-AgNPs and Ff-AgNPs with antibiotics was done. Results showed clear inhibition zone around the discs for all tested bacterial strains (Fig. 12). Such combination of each Xa-AgNPs and Ff-AgNPs and antibiotics provided efficient activity against MRSA and VRE more than P. aeruginosa and E. coli (Table 6). Ff-    cytotoxicity of Agnps. Xa-AgNPs and Ff-AgNPs showed higher cytotoxic effect compared to that of lichen extracts 'Xa and Ff ' ( Table 7). Results from the current study indicated higher cytotoxicity for Xa-AgNPs and Ff-AgNPs against FaDu and HCT 116 cell line in relation to MDA-MB-231. Higher efficiency was observed for Ff-AgNPs compared to Xa-AgNPs.

Discussions
Bioynthesis of Agnps. The present study could be considered as the first report indicating the biosynthesis of AgNPs by the aid of the extracts of lichen types Xanthoria parietina and Flavopunctelia flaventior as well as reporting their antibacterial and cytotoxic activities. Interestingly, methanol has the capability of dissolving polar compounds with a polarity index of 5.1. while water has a polarity index of about 10.2 29 . Our previous study presented the formation of AgNPs by water extract of lichen Parmotrema clavuliferum 46 . However, currently methanol was used for extraction of lichens. Optimizing the conditions for fabrication of AgNPs using both lichens tested were confirmed by color change thereafter, particles size average were measured by zetasizer. Factors affected the synthesis of nanoparticles were the ratio of AgNO 3 to lichen extract, AgNO 3 concentration, temperature and reaction time 27 . Biogenic AgNPs using Cetraria islandica formed in few minutes 27  Zone of inhibition (mm) Figure 12. Synergistic effect of Xa-AgNPs and Ff-AgNPs and antibiotics on MRSA. Table 6. Synergistic effect of Xa-AgNPs and Ff-AgNPs and antibiotics against bacteria strains. a Xa-AgNPs are the AgNPs prepared using Xanthoria parietina and Ff-AgNPs are the AgNPs prepared using Flavopunctelia flaventior.  www.nature.com/scientificreports/ were in accordance with the findings of many researchers who reported 1:3 as the ratio of the extract to the silver nitrate, however, they studied aqueous extract of lichens 26,[28][29][30][31]35 . Siddiqi, et al. 47 confirmed that, color intensity of the mixture increased with temperature increment and quick formation of nanoparticles was obtained. The first sign for AgNPs synthesis using lichen extracts was the change of color from yellow-green to dark-brown in the reaction mixture. Such conversion could be a clear indication for the reduction of silver ions to AgNPs which is related to surface plasmon resonance (SPR) phenomenon 27 . The exact mechanism included in the process of AgNPs formation using biogenic agents was not clearly known, however, several hypotheses of AgNPs formation by the green synthesis was explored in the biological system. The main hypothesis was that, AgNPs are formed underneath the surface of the cell wall and reduced in the presence of biomolecules or enzymes, while intracellular synthesis occurs inside the cells 17,48 . It has been suggested that the silver ions require nicotinamide adenine dinucleotide (NADH) and NADH-dependent nitrate reductase enzymes 49 . In the case of lichens such enzyme is secreted by the fungal partner extracellularly 25 and several secondary metabolites which act as reducing agents to produce AgNPs from AgNO 3 without producing toxic by-product 28,30 . characterization of biogenic Agnps. The SPR peaks for Xa-AgNPs and Ff-AgNPs corresponded to AgNPs production where AgNPs absorb radiation intensely at a wavelength of 400-450 nm due to the transition of electrons 25,26 . The peaks at 400 nm are supposed to be as indicators for spherical shapes of the particles 17 . TEM analysis confirmed the formation of silver nanostructures and visualized of synthesized AgNPs at 100 nm scales. The morphology and size distribution of both Xa-AgNPs and Ff-AgNPs showed spherical particles with nonspecific distribution. Our TEM findings are well matching with reports in the literature where same size range and spherical particles shape were noted 17,[26][27][28]30,31 . It was noticeable that the edges of the particles were lighter than the centres, suggesting that bioorganic molecules such as proteins in lichen and other metabolites capped the AgNPs contributed to reduction of Ag ions to AgNPs 30 . Particle sizes in DLS and Zeta Potential analysis of Xa-AgNPs and Ff-AgNPs with Polydispersity Index (PDI) were in agreement with the particle size early mentioned 17,30 . Zeta Potential provided high negative value of zeta potential for both AgNPs confirming the repulsion among the particles, and the negative value also indicates that nanoparticles have high degrees of stability 50 . The negative potential values could also be due to the presence of bio-organic components in the lichen extract that acted as capping agents. Generally, AgNPs synthesized using biological materials has good mono-dispersity as well as stability 51 . The particle size differed in TEM and DLS because the principles of the techniques in both analyses were different. The particles size in DLS was larger than those detected by TEM micrographs which could be due to the presence of impurities of bio-active molecules of lichen on the AgNPs surface. In addition to above mentioned points, DLS mainly measures the hydrodynamic radius of the nanoparticles 30 . The EDS results are consistent with previous studies that reported a strong peak for Xa-AgNPs and Ff-AgNPs at (3 keV), which is typical for the absorption of metallic silver nano-crystallites. Two impurity peaks were detected below 1 keV, which corresponded to carbon peak (CK) and oxygen peak (OK), that might be originated from the lichen extract 26,28,35 . It is well documented that, lichen extracts contains several metabolites, including antranorin as (+)-praesorediosic acid, and (+)-protopraesorediosic acid that has important roles in the synthesis of AgNPs 28 . The functional groups of Xa and Ff lichen extracts responsible for the reduction of Ag + from AgNO 3 and stabilization of AgNPs was studied by FTIR 30 . FTIR is an analytical technique to identify organic and inorganic materials that used to obtain an infrared spectrum of absorption or emission of a solid, liquid and gas 29 . The spectrum was observed at 3300-3500 cm −1 , indicating the presence of polyphenolic -OH group and N-H stretching of amine 17,25 . On the other hand, it was observed that the ratio between the intensity of the bands at 2066-2070 cm −1 could be attributed to the -C=C-stretch; alkynes 52 . Intense absorption bands in FTIR at 1600-1650 cm −1 might be attributed to amide I due to carbonyl stretch in proteins C=O stretch 25,27,29,30 . A comparison between the spectra of lichen extracts and AgNPs displayed little alterations in the position and the magnitude of the absorption bands indicating using the lichen secondary metabolites in nanoparticle formation.

Identification of Lichens secondary metabolites by (GC-MS).
The efficiency of lichens might be ascribed to a unique chemo-diversity having secondary metabolites 80% more than those produced by other organisms 29 . In addition, lichens have distinctive chemical compounds that is totally different from those produced by fungi, algae and plants 53 . At least 1050 different compounds have been identified from lichen species. Such compounds may act as antibacterial, antifungal, antiseptic, anti-inflammatory, antioxidant, antiviral, anticancer and antiproliferative agents as well as healing properties has been also confirmed. However, a major obstacle of lichen metabolites to be incorporated into medical applications is the natural toxicity of some secondary metabolites 54 . In Saudi Arabia, a large number of lichen species are present however, they were not well identified which could be related to the complexity of lichen cultivation. GC-MS results are illustrated in Table 2 displaying similarity with reported literature that detected phenols, amines, aldehydes and ketones, besides many other compounds that could be responsible for the reduction and stabilization of AgNPs 17,25,30,31 . Lichens of same species may identified different chemical compounds which could be related to substrate types and concentrations that used for lichen extraction 54 . Compared to previous researches, the chemical compounds detected in both lichen types tested in the current study were [undecanal 55,56 , Piperidine 29 , and Colchicine 57 ]. Benzenamine, 4-methoxy-N-methyl-compound was observed in both lichen types of Xa and Ff, it is a form of amine group that possibly contribute in reducing silver ions to AgNPs 58 . Some identified compounds from Xa in the current study had antibacterial activity such as (Benzyl bromide 59 , Hexachlorophene 60 , Pyrrolidine, 1-methyl-61 , 2-Amino-2-ethyl-1,3-propanediol 62 , undecanal 63 63 . On the other hand, Oxacyclododecan-2-one 70 , Piperidine 65 , Ricinoleic acid 71 , 2′,5′-Dihydroxypropiophenone 68 , 2,5-Cyclohexadiene-1,4-dione, 2,6-bis(1,1-dimethylethyl) 72 , and Colchicine 69 were identified from lichen type Ff and Dichloran 73 , Hexachlorophene 74 were found in lichen type Xa, all were characterized by their antifungal activities. Some compounds identified in lichen type Ff showed therapeutic activities such as Beclomethasone that used to treatment of persistent asthma 75 , Colchicine used to treatment of gout 76 , Atropine used to treatment of Arrhythmias 76 Ff include Beclomethasone 75 that has anti-inflammatory activity and Octadecylamine 77 involved with other compounds in materials manufacture that used in drug delivery.
evaluation of antibacterial activity of Agnps. Biogenic AgNPs by the aid of both lichens were examined in the current study for antibacterial activity against different bacteria, however, no antibacterial activity was detected for lichen extracts alone; suggesting that 10 mg/mL concentration was not enough for antibacterial efficacy 26,[28][29][30] . Some investigations reported the antibacterial activity of AgNPs prepared using lichen extracts influenced gram-negative bacteria more than gram-positive bacteria 26,30,31 ; however, our study didn't show the same pattern which is in accordance with some other investigations 17,28,35 . Noticed activity of the AgNPs against Pseudomonas aeruginosa; might be due to the cell wall structure of gram-negative 31 since MRSA and VRE showed less sensitivity. Penetration of the AgNPs in gram-negative bacteria could be much easier than that in gram-positive due to the cell wall that composed of a thinner layer of peptidoglycan compared to grampositive 28 . Ff-AgNPs showed more efficacy against some bacteria strains (MRSA, VRE, P. aeruginosa MDR, E. coli ATCC) compared to Xa-AgNPs which might be due to the size of the particles that was 69 nm for Ff-AgNPs, while that for Xa-AgNPs was 145 nm. The chemical components were identified from Ff lichen type were more than those identified in Xa which could also be a reason for high antibacterial activity since some of had antibacterial and antioxidant activities. Recent studies have also reported similar trend of observations regarding antibacterial activity of AgNPs against P. aeruginosa in relation to S. aureus and E. coli 30,31 . E. coli in the current study showed less sensitivity to biogenic AgNPs which might be related to the fact that, such antibacterial agents are bacterial specific therefore, AgNPs mechanism against bacteria should be further investigated since no special trend was observed regarding microbial species. It has been noticed that the higher concentration of biogenic AgNPs, the greater the antibacterial activity was detected 30 . Also, smaller AgNPs with a large surface area possesses antibacterial effects greater than that of larger AgNPs size because the smaller particles can easily anchor to bacterial cell wall and penetrate 28 . However, several studies proposed that AgNPs mode of action against bacteria could be related to following points (a) interact with the membrane surface during cell wall synthesis (b) suppression during protein synthesis (c) disruption of transcription process (d) perturbation of metabolic pathways where interact of Ag with the thiol groups of respiratory enzymes of bacteria 17,30 . Furthermore, MICs and MBCs of biogenic AgNPs exhibited good bactericidal activity at low concentrations against tested bacterial strains 78 . No available reports for MICs and MBCs for AgNPs prepared by lichens. The present investigation is in consistence with the other study that revealed the tolerance level was bactericidal for biogenic AgNPs prepared by some plant extracts 16 . Synergistic effect of antibiotics and AgNPs was examined and their combination increased the activity against all tested bacterial strains 79 . Tetracycline, linezolid and gentamicin are protein synthesis inhibitors antibiotics. Tetracycline and gentamicin inhibit protein synthesis by binding the 30S subunit of bacterial ribosome, whereas linezolid binds to 50S subunit. Ampicillin is part of beta-lactam antibiotics group and considered as cell wall synthesis inhibitor 80 . Synergistic results represented a significant consensus with previously findings reported the AgNPs using biogenic extracts [80][81][82][83][84][85] . Such activity could be of interest to overcome the antibiotic resistant microbes since combination increased the antibiotic activity suggesting easy penetration of the antibiotic active ingredients by the aid of the nanoparticles. Such enhancement of antibiotic activity could be related to the fact that, AgNPs facilitate good transfer to the antibiotic and bind it to the interaction site with the microbe 86 . Same trend of observation was also recorded in various studies 87 . cytotoxicity of Agnps. To investigate the biogenic AgNPs cytotoxicity, MTT test was done against HCT 116, MDA-MB-231and FaDu cell line that showed high activity since the IC 50 was < 100 µg/mL; while MDA-MB-231 was less sensitive and showed IC 50 > 100 µg/mL. It has been reported that, biogenically synthesized AgNPs using lichen extracts had anticancer activity against human lung cancer cell line 35 . In their study, lower IC 50 of 3.8 and 6.6 µg/mL were found compared with our results when they used AgNPs synthesized using Ganoderma lucidum and Phellinus igniarius respectively. Generally, anticancer activity of AgNPs prepared using green synthesis was well documented 16,35,58,88,89 . Further studies stated the IC 50 of AgNPs synthesized using biogenic extract were ranged between 10.9 to 21.4 against HepG2, LoVo, and MDA-MB 231cell 90 and another study showed IC 50 ranged between 35.15-56.73 μg/mL against a LoVo cell with 16 . These differences in cytotoxicity results could be related to cell lines variations and AgNPs size, shape and concentrations. Ff-AgNPs showed more efficacy against all cell lines compared to Xa-AgNPs which might be due to the small size of the particles in Ff-AgNPs. Also, it could be related to the chemical components detected by GC-MS since higher chemicals components were identified from Ff-AgNPs compared to Xa-AgNPs. Differences in sensitivities toward different AgNPs might be due to their physiological behaviour and structural characteristics. Biogenically synthesized AgNPs may inhibit growth of cancer cell lines by various modes of actions. They may cause cellular damage in cancer cell line through the generating of ROS which leads to DNA damage by activation of Caspase-3 molecule, thus undergone apoptosis 17 . It could be concluded that, AgNPs may produce free radicals that lead cell oxidative stress, subsequently programmed cell death via caspase cascade pathway 58 .  In the light of our current findings, it might be concluded that methanol extracts of lichen could be potential candidate in reducing and capping of AgNPs in the context of eco-friendly approach for extracellular synthesis in one step. The FTIR analysis showed participation of different lichen biomolecules in reducing and capping of AgNPs. The biogenic AgNPs were well-dispersed spherical of 1-145 nm which was confirmed by TEM and zeta size analysis. The eco-friendly technique presented here might be practical for AgNPs production in largescale. Encouragingly, the biogenic AgNPs showed promising materials in antibacterial applications against P. aeruginosa, MRSA, VRE, and E. coli. Additionally, AgNPs influence on gram-negative was greater than that on gram-positive bacteria and higher cytotoxicity against FaDu and HCT 116 cell line in relation to MDA-MB-231 was found. Furthermore, substantial antibacterial and a good synergistic effect for AgNPs when associated with antibiotics might help in antibiotic-resistant microorganism treatment. Therefore, AgNPs prepared in the current study might be recommended for biomedical and pharmaceutical applications. Our study is considered as the first report that studied AgNPs fabricated using lichen types Xanthoria parietina and Flavopunctelia flaventior and approved their activity and synergistic impact with some antibiotics against pathogenic bacteria as well as cytotoxicity against three cell lines. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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