1 Introduction

Silver nanoparticles (AgNPs) have been extensively employed in different commercial products such as healthcare products, in cleaning agents, textile coatings, food packing and storage and medical devices (Verma and Maheshwari 2019). AgNPs find important biomedical, environmental and commercial applications mainly due to their effective and broad-spectrum antimicrobial activity against bacteria, including resistant strains, pathogen fungi and viruses (Durán et al. 2016a, b). The successful uses of AgNPs in several commercial products, such as in antiseptic sprays, wound dressing bandages and dermatological creams, are derived from the ability of AgNPs to disrupt pathogenic membrane, hence impairing the microorganism’s enzymatic activities (Flores-López et al. 2019).

In addition, the overuse of antibiotics has led to the emergence of antibiotic resistance that becomes a serious health issue worldwide (Furusawa et al. 2018). The major problem related to bacteria and drug resistance is the formation of biofilm, which is a complex bacteria community covered by polysaccharide matrix. This matrix protects the bacteria from antibiotic treatment and retains the nutrients required for the growth of the bacteria community (Parrino et al. 2019). Thus, bacteria biofilm contributes to antibiotic resistance, since 1000 times more antibiotics are required to kill bacteria, in comparison with planktonic cells (Smith 2005). Therefore, there is an increasing interest in the design of effective and low-cost strategies to combat resistant bacteria, including biofilms. Several works describe that due their small size and large surface area to volume ratio, AgNPs have potent antimicrobial activities against Gram-negative, Gram-positive bacterial strains and also toward established biofilms (Durán et al. 2016a, b; Ali et al. 2018). Moreover, naturally occurring bacteria do not develop resistance to AgNPs (Mühling et al. 2009). Thus, AgNPs are considered one of the most effective engineered nanoparticles acting as broad-spectrum antimicrobial agent (Durán et al. 2016a).

AgNPs can be synthesized by chemical, physical and biological routes. Chemical reduction is one of the most traditional methods to synthesize AgNPs, and sodium borohydride and sodium citrate are usually employed as reducing agents (Baber et al. 2017). Usually, these methods also required the addition of capping/stabilizer agent to coat the surface of AgNPs avoiding nanoparticle agglomeration and aggregation (de Lima et al. 2012). Although chemical and physical routes are able to synthesize size controllable engineering nanoparticles with good reproducibility, these strategies might involve the presence of toxic chemicals, demand high-energy input, in some cases, controllable pressure and temperature, which might result in high costs, and the production of environmental contamination (Duran and Seabra 2018).

Biological routes to synthesize metallic nanoparticles, including AgNPs, involve the uses of plant extracts (green synthesis) or microorganisms, such as bacteria, fungi, and algae, as reducing and capping agents (de Lima et al. 2012; Ali et al. 2018; Duran and Seabra 2018). Biological methods have gained considerable importance in the last years due to their cost-effective and eco-friendly features. Usually, biological protocols to synthesize AgNPs are performed in aqueous medium (absence of organic solvents), at ambient temperature and pressure, in an easy protocol, free of toxicity (Alsammarraie et al. 2018; Asghar et al. 2018). The biological source acts not only as a potent reducing agent (reducing Ag+ to Ag0, leading to the formation of AgNPs), but also as capping/stabilizing agent on the nanoparticle surface, avoiding nanoparticle agglomeration/aggregation, increasing the stability of the colloidal suspension (de Lima et al. 2012; Cuevas et al. 2015; Duran and Seabra 2018).

Among the biological routes to synthesize AgNPs, the use of plant extract is considered efficient, versatile, low cost, simple, fast and easy to scale-up (Ebrahiminezhad et al. 2018). Nowadays, several different parts of plants (leaf, seed, fruit, bark, peel, flower, bran) have been used for the green synthesis of metallic nanoparticles (Niraimathi et al. 2013). In this sense, plant phytochemicals are strong reducing agents, leading to the formation of capped nanoparticles in a single- and one-pot reaction. In addition, plant extracts have their own medical value, due to the presence of phenolic compounds (polyphenols, flavonoids, tannic acid and terpenoids), vitamins, minerals and amino acids giving an additional functionality for the capped nanoparticle (Fierascu et al. 2010; Fahimirad et al. 2019).

Among the plant extracts that can be used for the green synthesis of AgNPs, green tea (Camellia sinensis) extract has been successfully employed for the biological synthesis of metallic nanoparticles (de Oliveira Silva and Seabra 2016; Rolim et al. 2019a, b). Green tea is rich in polyphenolic compounds, which is mainly comprised by epigallocatechin-3-gallate (EGCG), epigallocatechin, epicatechin-3-gallate and epicatechin (Ramiréz-Aristizabal et al. 2015). The green tea phytochemicals have important medical benefits as effective antioxidant and anti-inflammatory agent, acting as protector agent of the immunological system, in the protection of intestine against food-borne pathogenic bacteria, and also in cosmetic and therapeutic applications (Singh et al. 2016).

The herb dill belongs to the family Apiaceae, genus Anethum and species A. graveolens. As green tea, dill is rich in polyphenols, minerals, vitamins (C, A, β-carotene, folic acid, niacin, riboflavin) and flavonoids (Ramadan et al. 2013; Kalangi et al. 2016). Dill has medical importance as an anti-hyperlipidemic agent, diuretic sedative and antispasmodic (Zayed et al. 2012; Kalangi et al. 2016). Recently, dill extract was used for the synthesis of AgNPs for antileishmanial activity (Kalangi et al. 2016). The authors reported that AgNPs enhanced the effect of meltefosine alone (traditional drug used to treat leishmaniasis), decreasing the IC50 of miltefosine about to half (Kalangi et al. 2016).

In addition to the mentioned above, the synthesis of AgNPs can also be obtained by mycelium-free extract of fungi. Microorganisms such as Fusarium oxysporum can be used in the biosynthesis of inorganic nanomaterials (Durán et al. 2005). The nonpathogenic white-root fungus Stereum hirsutum (S. hirsutum) has been used to biogenically synthesize copper and copper oxide nanoparticles (Cuevas et al. 2015). S. hirsutum is a native white-root fungus extensively found in Chilean forests. To our best knowledge, this is the first report to describe the use of S. hirsutum for synthesis of AgNPs.

Therefore, the purpose of this study was to synthesize AgNPs by following biological routes by using plant extracts of green tea and dill and from mycelium-free extract produced by S. hirsutum and compare their antimicrobial and antibiofilm activities. The obtained nanoparticles were extensively characterized and showed potent antioxidant, antibacterial and antibiofilm activities. To our best knowledge, this is the first report to compare the antioxidant and antibacterial effects of AgNPs synthesized by different biological entities (green tea, dill and S. hirsutum) with great importance in the combat of resistant bacteria and biofilms.

2 Materials and methods

2.1 Materials

Silver nitrate (AgNO3), phosphate buffer saline (PBS, pH 7.4), 2,2-diphenyl-1-picryl-hydrazyl- hydrate (DPPH), L-ascorbic acid, ferrozine, iron nitrate nonahydrate (Fe(NO3)3 9H2O, buffer acetate (pH 5.5) and Folin–Ciocalteu reagent were obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH), bromide potassium (KBr), hydrochloric acid (HCl), gallic acid, sodium bicarbonate and ethanol were obtained from Labsynth (Diadema, SP, Brazil). Green tea powder (Camellia sinensis) was obtained from Sumioka Shokuhin Kabushikikaisha, Hiraguti, Japan. Commercial dill (Anethum graveolens) was purchased from local market at São Paulo, Brazil. Stereum hirsutum, a native fungus from southern Chilean forests, was obtained from the Chilean Collection of Culture Type (code CCCT16.03) of Universidad de La Frontera, Chile. Mueller–Hinton (broth and agar), glucose, potato peptone and yeast extract were obtained from Difco (Franklin Lakes, EUA). All experiments were carried out using analytical grade water from a Millipore Milli-Q Gradient filtration system (Millipore, 18.2 MΏ, USA).

2.2 Preparation of plant and mycelium-free extracts

To prepare the aqueous plant extracts of green tea (GT) and dill (D), the mass of 2 g of each plant powder was separately dissolved in 100 mL of water, under stirring and heating at 60 °C for 15 min. The extract suspensions were filtered using the Whatman No. 1 filter paper and cooled for later use. For the preparation of mycelium-free extract from fungus (F), five circular agar plugs (4 mm in diameter) taken from the margin of a growing colony of the S. hirsutum growing on malt extract agar were put into 500-mL Erlenmeyer flasks with 100 mL of growth medium (pH 5.5) containing in g L−1: glucose (15), potato peptone (5) and yeast extract (2.5) and incubating in dark for 14 days and 25 °C. The fungal biomass obtained at the end of the incubation period was filtered through Whatman No. 1 filter paper and washed thoroughly with deionized water. The fungal biomass was transferred into 100-mL Erlenmeyer flasks containing 50 mL of sterilized deionized water and incubated in an orbital shaker (150 rpm) for 24 h at 25 °C.

2.3 Characterization of the prepared extracts

Plant extracts (GT and D) were characterized as described below and used for the green synthesis of AgNPs.

2.3.1 Determination of total phenolic content

Folin–Ciocalteu procedure was used to determine the total phenolic compounds in plant extracts used to prepare AgNPs, as previously reported (San et al. 2013). Firstly, Folin–Ciocalteu reagent was diluted with deionized water (1:10). The extracts (green tea or dill) (20 mg mL−1) or standard gallic acid solution (10–150 μg mL−1), as positive control, was mixed with 0.8 mL of sodium bicarbonate solution (7.5% w/v) in the presence of 0.5 mL of diluted Folin–Ciocalteu reagent. Absorbance values of final mixtures were measured with UV–Vis spectrophotometer (Agilent, model 8453, Palo Alto, CA, USA) at λ = 765 nm. The total phenolic content in the different plants (GT, D) was calculated and expressed as milligrams of gallic acid equivalent (GAE) in 1 g of the dried powder. The results were obtained as mean ± SD (n = 3).

2.3.2 Ferric reducing antioxidant power method (FRAP): Antioxidant activity

The antioxidant capacity of the plant extracts (green tea and dill) was evaluated by the FRAP assay, as previously described with some modifications (Vázquez et al. 2008). Briefly, 0.0202 g of Fe(NO3)3 · 9 H2O and 0.1231 g of ferrozine were dissolved in 1.0 mL of HCl (1.0 mol L−1) and brought to 25.0 mL with deionized water. The final concentrations of Fe(NO3)3 · 9 H2O and ferrozine were 2.0 × 10−3 mol L−1 and 1.0 × 10−2 mol L−1, respectively. A volume of 0.25 mL of previously prepared extracts (GT or D) was added to 0.25 mL of water, 0.75 mL of ferrozine-Fe(III) solution and 1.0 mL of 0.2 mol L−1 acetate buffer at pH 5.5. The final mixtures were incubated for 30 min at 25 °C, and the intensities of the absorption bands at 562 nm were recorded by using the UV–Vis spectrophotometer. The relative activities of samples were calculated from the calibration curve of L-ascorbic acid (0.025–0.2 mmol L−1) (as positive control), and the results are expressed as nmol ascorbic acid equivalent (AAE) mg−1 of extract (on a dry basis), as previously reported (Vázquez et al. 2008). The results were obtained as mean ± SD (n = 3).

2.4 Synthesis of AgNPs by plant extracts

The volume of 75 mL of plant extracts (GT or D) was mixed with 0.1 mol L−1 of AgNO3, and the pH of the final suspensions was adjusted to 10.5 by dropping NaOH (1.0 mol L−1). The obtained suspensions were stirred for 15 min and centrifuged at 9660 × g for 10 min. The supernatant was discarded, and the obtained AgNPs were washed twice and freeze-dried. This process led to the formation of plant extract synthesized AgNPs by GT and D referred as AgNPs-GT and AgNPs-D, respectively.

2.5 Synthesis of AgNPs using Stereum hirsutum

The synthesis of AgNPs using the fungus strain S. hirsutum was prepared as described by Cuevas and coworkers (2015). A volume of 242.5 mL of S. hirsutum extract was transferred into 250-mL Erlenmeyer flasks. Then, 7.5 mL of a stock solution (100 mmol L−1) of AgNO3 was added to obtain a final concentration of 3.0 mmol L−1 and the pH was adjusted to 12.0 (NaOH 0.1 mol L−1). The flasks were incubated for 24 h on an orbital shaker (150 rpm) at 25 °C in the dark. After, the synthesized nanoparticles were isolated by centrifugation, washed twice with water and lyophilized (Cuevas et al. 2015). The obtained AgNPs biogenically synthesized by the fungus S. hirsutum are referred as AgNPs-F.

2.6 Characterization of synthesized AgNPs

The plant extracts and fungus-synthesized AgNPs (AgNPs-GT, AgNPs-D and AgNPs-F) were characterized by different techniques, as described below.

2.6.1 UV–Visible spectroscopy analysis

All prepared AgNPs were characterized by UV–Visible spectroscopy analysis using a UV–Vis spectrophotometer, in the wavelength ranged from 200 to 800 nm with a resolution of 1 nm. AgNPs were dispersed in aqueous media with an ultrasound bath for 1 h, and the suspensions were transferred to quartz cuvettes for analysis.

2.6.2 X-ray diffraction (XRD)

AgNPs prepared by different extracts were characterized by XRD. A STADI-P (Stoe®, Darmstadt, Germany) diffractometer was used to record X-ray powder diffraction operating at room temperature, 50 kV, 40 mA and using CuKα (λ = 1.54060 Å. The X-ray photons were collected by a Mythen 1 K (Dectris®, Baden, Switzerland) detector. Data were recorded in the 2θ range from 15° to 90°, with step sizes of 0.015º and a counting time of 100 s at each 0.785°. In addition, the grain size of the synthesized AgNPs was calculated with the Debye–Scherrer equation (Eq. 1) (Naika et al. 2015; Rolim et al. 2019a).

$$D \, = \, (k\lambda )/(\beta \,cos \, )$$
(1)

where D is the diameter of crystallite size, λ is the wavelength for Cukα, β is the full width at half maximum (FWHM) of the peak, θ is the Bragg diffraction angle, and K is a constant. (0.94 is used to correspond spherical crystallites with cubic symmetry.)

2.6.3 Fourier-transform infrared spectroscopy (FTIR)

The synthesized AgNPs and the pure biological extracts (GT, D and F) were triturated with pure potassium bromide (KBr) powder. These mixtures were ground into fine powders, pressed in a mechanical press to generate translucent pellets and analyzed using an Agilent Cary 630 spectrometer (Palo Alto, CA, USA). A pure pellet of KBr was used for background. The FTIR spectra were recorded from 700 to 4000 cm−1 at a resolution of 4 cm−1.

2.6.4 Atomic force microscopy (AFM)

The size of AgNPs obtained by different extracts was determined by atomic force microscopy, using an AFM/STM Series 5500 dynamics (Agilent Technologies, Palo Alto, CA, USA). The non-contact mode was employed using silicon tips of 4 nm thickness, 125 µm length, 30 µm width, resonance frequency of 320 kHz and force constant of 42 N m−1. Nanoparticles were dispersed in an ultrasound bath (0.5 mg mL−1), diluted (1:999), and 10 µL was drop-casted onto a silicon wafer and dried in room temperature. Images obtained were analyzed using WSxM 5.0 software, and average size and standard error were evaluated using OriginPro 8.0 software.

2.6.5 X-ray photoelectron spectroscopy (XPS)

XPS (Thermo K-alpha spectrometer, MA, USA) was carried out to measure the elemental composition of the AgNPs prepared via different extracts. The measurements were taken in powdered samples where the instrument utilized a 72 W monochromated Al K-alpha+ source (E = 1486.6 eV) with energy adjusted to 3000 eV, medium current, a spot size of 400 µm and depth of 10 nm was set. The analyses were performed in two different points of the samples, and the data were analyzed using CasaXPS software, representing the average of all measurements.

2.6.6 Dynamic light scattering (DLS)

A Zetasizer Nano ZS (Malvern Instruments Co., UK) was used to analyze the average hydrodynamic size (evaluated by % of number), and polydispersity index (PDI), and zeta potential of the obtained nanoparticles in aqueous media at 25 °C. Measurements were taken using disposable folded capillary zeta cell (10 mm path length) and using a fixed angle of 173°.

2.7 Antioxidant activity of AgNPs prepared by plant and fungus extracts

The antioxidant activity of AgNPs-GT, AgNPs-D and AgNPs-F was measured using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical photometric assay, indicating antioxidant activity through its discoloration (Guo et al. 2013; Nakurte et al. 2017). A volume of 500 µL of 0.1 mmol L−1 DPPH ethanolic solution was added to 500 µL of aqueous suspension of AgNPs-GT, AgNPs-D or AgNPs-F, with different final concentrations of 2.0, 20, 40 or 80 µg mL−1 diluted in PBS. A control group was prepared by using PBS and DPPH ethanolic solution. The final mixtures were kept at room temperature, protected from light for 30 min and placed into quartz cuvettes. The absorbance intensities at 517 nm for all samples were recorded by using an UV–Vis spectrophotometer. The obtained absorbance values were converted to the percentage of antioxidant activity (AA) through the following equation (Sudha et al. 2017):

$${\text{AA}}\% = \, \left[ {\left( {Abs_{\text{control}} - \, Abs_{\text{sample}} } \right) \, / \, Abs_{\text{control}} } \right] \, \times \, 100$$
(2)

where Abssample is the absorbance of the sample in ethanolic DPPH and Abscontrol is the absorbance of ethanolic DPPH, in the absence of the nanoparticles. The results were plotted as sample concentration on the x-axis and the respective percentage of inhibition on y-axis. The assay was performed in triplicate.

2.8 Antibacterial activity

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for AgNPs-GT, AgNPs-D and AgNPs-F were carried out by microdilution assay as described by the Clinical and Laboratory Standards Institute (CLSI 2012). The bacterial strains evaluated were the Gram-positive strain S. aureus ATCC 29213 and Enterococcus faecalis ATCC 3229 (standard CLSI); and the Gram-negative bacterial strains (standard CLSI) Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13803 and the multidrug-resistant (MDR) Pseudomonas aeruginosa KPC 37. Initially, the bacteria were grown in Mueller–Hinton solid medium at 37 °C to obtain the isolated colonies. Subsequently, the colonies were solubilized in a Mueller–Hinton and adjusted to the 0.5 index of the MacFarland scale (1.5 × 108 colony-forming units (CFU) mL−1). This solution was diluted 10x in Mueller–Hinton broth and distributed in a 96-well plate. Each well was treated with different concentrations of AgNPs (1.56 to 100.0 µg mL−1 µg mL−1). The plates were incubated for 18 h, and the bacterial growth was measured after this period. The MBC tests were performed after the MIC tests. After 24 h of incubation, drops of bacterial broth from the cavity with no visual growth were added to a Petri dish with the Mueller–Hinton solid medium, and bacterial growth was observed after 24 h. After, the tolerance level of S. aureus, E. faecalis, K. pneumoniae, P. aeruginosa and E. coli against the biosynthesized AgNPs was determined according to the method of Neethu and coauthors (2018).

2.9 In vitro biofilm formation assay: Antibiofilm activity of the nanoparticles

The antibiofilm activities of AgNPs-GT, AgNPs-D and AgNPs-F were evaluated as described by Stepanović and coworkers (2000). After the MIC and MBC tests, the obtained 96-well plates with S. aureus, E. Faecalis, E. coli, K. pneumoniae and P. aeruginosa KPC 37 were washed three times with 200 µL of distilled water. Then, 200 µL of methanol was added to for 15 min and left to dry at room temperature. After, the adhered cells were stained with 200 µL of 1% crystal violet for 20 min and washed with distilled water three times. The biofilm-associated/crystal violet was solubilized with 33% v/v glacial acetic acid per well, and the optical density (OD) was measured at 570 nm using a microplate reader (Stepanović et al. 2000).

2.10 Imaging of MDR P. aeruginosa KPC 37 biofilm using scanning confocal laser microscopy

An overnight culture of MDR P. aeruginosa KPC 37 was diluted in Mueller–Hinton medium, and after that the cells were grown on glass slides during 24 h at 37 °C forming the biofilms. The culture supernatant was removed, and a fresh Mueller–Hinton medium was added with AgNPs-GT with concentrations of 50 and 100 µg mL−1. The cells were further grown during 24 h. The biofilm viability was assessed with Live/Dead BacLight Bacterial Viability and Counting Kit (Invitrogen, Molecular Probes) provided for scanning confocal laser microscopy (Olympus, FV1000, Japan) with an excitation wavelength at 488 nm. The images were processed using Image Processing software (FV10-ASW v.0.2c).

3 Results and discussion

3.1 Total phenols contents and antioxidant activity of plant extracts

The phenolic compounds present in plant extracts have antioxidant activity and the capacity of reduced ions, in this case, to reduce Ag+ ion into Ag0 (Ignat et al. 2011; Machado et al. 2013). The phenolic content and the antioxidant activity of the plant extracts used to synthesize the AgNPs were measured by the Folin–Ciocalteu method (Fig. 1a) and by the FRAP method (Fig. 1b), respectively. The results demonstrated a higher phenolic content in green tea extract (164.9 ± 6.9 mg GAE g−1 of extract) compared with dill extract (54.9 ± 5.0 mg GAE g−1 of extract) (Fig. 1a). Commercials green tea is known to have ca. 11–130 mg GAE g−1 (Pal et al. 2012; Ramiréz-Aristizabal et al. 2015).

Fig. 1
figure 1

Total phenolic content (a) and ferric reducing antioxidant power (b) of green tea and dill extracts

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In the case of antioxidant activity, the green tea extract demonstrated a superior iron-reducing capacity, with a reduction of 4.2 ± 0.1 mmol AAE mg−1 of extract, compared with dill extract (2.0 ± 0.1 mmol AAE mg−1 of extract) (Fig. 1b). Thus, green tea extract showed higher total phenolic content and antioxidant ability, compared with dill extract. As expected, antioxidant activity of the compound depends on the amount of phenolic compounds in the plants. It should be noted that a different antioxidant capability of polyphenols depends on their chemical structure, regarding the number and position of phenolic hydroxyl groups (Ramiréz-Aristizabal et al. 2015). Similar results have been described by several papers in the measurements of total phenolic content and/or antioxidant activity of different plant extracts, employed for the biosynthesis of metallic nanoparticles (Vázquez et al. 2008; Pal et al. 2012; Kumar et al. 2016). Green tea and dill demonstrated antioxidant capability due to the presence of polyphenol groups, and thus ability to synthesize AgNPs, as described below.

3.2 Green synthesis of AgNPs

In this work, the plant extracts (green tea and dill) and the mycelium-free extract of the fungus S. hirsutum were screened for their bioreduction in the synthesis of AgNPs. Indeed, plant extracts are rich in polyphenols that act as reducing and capping agents of the nanoparticles (Alsammarraie et al. 2018; Asghar et al. 2018; Duran and Seabra 2018). Moreover, due to the intrinsic features of the plant extract, its presence on nanoparticle surface might gain additional biological importance to the nanomaterial (Fierascu et al. 2010; Fahimirad et al. 2019). In addition, a mycelium-free extract of S. hirsutum was employed for the synthesis of AgNPs. The main biological entity responsible for the bioreduction in Ag+ is reported to be electron shuttle in conjugation with nitrate reductase (Durán et al. 2005).

3.3 Characterization of green synthesized AgNPs

3.3.1 UV–Vis spectroscopy analysis

Firstly, the formation of AgNPs was confirmed by color changes in the starting material (Ag+) from colorless to brownish black, characteristic of colloidal AgNPs (Saeb et al. 2014; Asghar et al. 2018). In addition, the formation of AgNPs was confirmed by the detection of the surface plasmonic resonance (SPR) band that typically occurs between 390 and 510 nm (Oliver et al. 2018). This band is associated with the oscillation of the electrons on the surface of the nanoparticles, upon excitation in a determined wavelength (Dubey et al. 2010). Figure 2 shows the SPR bands are centered at 404, 416 and 404 nm for AgNPs-GT, AgNPs-D and AgNPs-F, respectively. It is possible to observe a similar absorption profile for all synthesized AgNPs with no significant shiftiness of wavelengths. Similar profile for SPR bands indicates a similar shape for all green synthesized AgNPs. The higher intensity of AgNPs-F observed in Fig. 2 is due to the higher concentration of AgNPs in the suspension, compared with AgNPs synthesized by plant extracts. Our results suggest the formation of small and uniformly spherical nanoparticles (Rolim et al. 2019a).

Fig. 2
figure 2

UV–Vis absorption spectra of AgNPs-GT (black line), AgNPs-D (red line) and AgNPs-F (blue line)

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3.3.2 X-ray diffraction (XRD)

The crystalline nature of AgNPs was confirmed by XRD (Fig. 3). The XRD analyses for AgNPs-GT, AgNPs-D and AgNPs-F revealed similar 2θ  peaks values at 38.1°, 44.4°, 64.6° and 77.4°, which is associated with the planes [111], [200], [220] and [311] facets of AgNPs (Vázquez et al. 2008; Rolim et al. 2019a, b). In addition, the XRD pattern of AgNPs-D shows an additional peak at 32.7º that might be attributed to the presence of silver chloride (AgCl) (Durán et al. 2016b). This result suggests surface oxidation on these nanoparticles. Similar results were obtained by Oliver and coauthors (2018). Moreover, the crystallite-sized nanoparticles calculated by the Debye–Scherrer equation for AgNPs-GT, AgNPs-D and AgNPs-F were found to be 4.52, 11.11 and 18.97 nm, respectively. Interesting, it is possible to observe broader peaks for AgNPs-D and AgNPs-GT and this behavior can be associated with small crystallite sizes (Oliver et al. 2018). In contrast, it is possible to observe a narrow peak of the plane [111] for AgNPs-F, which corresponds to a higher crystallite size. The obtained results indicate the successful formation of AgNPs with a face-centered cubic structure with crystallite at the nanoscale (Durán et al. 2016b).

Fig. 3
figure 3

XRD patterns of the synthesized AgNPs using a Cu source (1.5418 Å). Miller indexes for all Bragg reflections are indicated in the figure

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3.3.3 Fourier-transform infrared spectroscopy (FTIR)

FTIR analyses were performed to confirm the presence of phytochemicals and fungus extract on the surface of AgNPs, which act as reducing and capping agents. Biomolecules derived from plant and fungal extracts play major roles in the reduction of Ag+ and the stabilization of the obtained AgNPs (Singh et al. 2018). Figure 4 shows the FTIR analyses for all plant and fungus-synthesized AgNPs, and their corresponding plant extracts. As can be observed in Fig. 4a, b, plant-mediated AgNPs have similar profile compared with their corresponding plant extract, indicating the presence of phytochemicals on the surface of plant-mediated AgNPs.

Fig. 4
figure 4

FTIR spectra of synthesized AgNPs-GT a, AgNPs-D b and AgNPs-F c and their respective powder extracts

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Figure 4a, b shows the spectra of pure green tea and the AgNPs-GT, and for pure dill and AgNPs-D, respectively. It is possible to observe five major peaks at 3443 cm−1, 2931 cm−1, 1640 cm−1, 1390 cm−1 and 1054 cm−1. The peaks at 3443 cm−1 and 2931 cm−1 are attributed to hydroxyl groups (O-H stretching) assigned to − OH group of polyols such as catechins, and C–H/CH2 vibration of aliphatic hydrocarbons, respectively (Rolim et al. 2019a, b; Kalangi et al. 2016; de Oliveira Silva and Seabra 2016). In addition, the peaks at 1640 cm−1 and 1390 cm−1 are attributed to C=O groups from carboxylic acids and C–N stretching, derived from caffeine present in green tea (de Oliveira Silva and Seabra 2016; Rolim et al. 2019a, b). The peak at 1054 cm−1 could be attributed to C–O–C stretching vibration (de Oliveira Silva and Seabra 2016). Comparing the spectra of AgNPs with the corresponding powder extract, it is possible to observe a decrease in the hydroxyl band. This behavior is related to the possibility that the hydroxyl groups of dill extract might undergo oxidation reaction of the phenols and subsequently, reduction of the Ag+ ions into Ag0 (Seabra et al. 2017).

Furthermore, the FTIR spectrum of AgNPs-F (Fig. 4c) revealed bands in the regions of 3437 cm−1 and 2944 cm−1, which might be attributed to movements of stretching vibrations in primary and secondary amines (Cuevas et al. 2015). The peak at 1640 cm−1 is associated with bending vibrations, and the bands near 1069 cm−1 are attributed to aliphatic amines (Cuevas et al. 2015). These results are in accordance with the previous reports that demonstrated the presence of biomolecules, such as proteins and polyphenols in the biological extracts that act as reducing and capping agents on the surface of the synthesized nanoparticles (Zayed et al. 2012; Oves et al. 2013; Rajasekharreddy and Rani 2014; Sarsar et al. 2015). These biological capping agents could possibly form a layer covering the metal nanoparticles preventing nanoparticle agglomeration and thereby stabilizing the medium.

3.3.4 Atomic force microscopy (AFM)

AFM was used to characterize the morphology and size distribution of green synthesized AgNPs, at solid state. Topography, phase contrast and amplitude micrographs were simultaneously recorded, and the average size distribution of AgNPs was calculated. Figure 5 shows the representative micrographs and the estimated size distribution of AgNPs-GT (a), AgNPs-D (b) and AgNPs-F (c). The micrographs indicated the formation of spherical and well-dispersed AgNPs. The values of average size distribution were found to be 9.67, 11.3 and 21.7 nm for AgNPs-GT, AgNPs-D and AgNPs-F, respectively (Fig. 5). The results indicated a larger size distribution for AgNPs synthesized via fungus extract, compared with AgNPs synthesized by plant extracts. These results are in accordance with the previous reports based on fungi-mediated biosynthesis of AgNPs (Basavaraja et al. 2008; Krishna et al. 2015; Garg and Garg 2018).

Fig. 5
figure 5

Atomic force microscopy (AFM) images (left) and size distribution at solid state (right) of synthesized AgNPs-GT (a), AgNPs-D (b) and AgNPs-F (c)

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Comparing the average size distribution of AgNPs synthesized by plant extracts, it is possible to notice that the average size was gradually increased from green tea to fungus extracts employed in the synthesis. According to the crystallization theory, the final particle size depends on several parameters including nucleation, growth rates and the nature of the reducing agent. Generally, slow nucleation process leads to a small number of nuclei that may grow and lead to larger nanoparticles, while fast nucleation tends to yield a large number of nuclei, which may result in smaller nanoparticles (Mehta et al. 2010). In this sense, the average size distribution prepared AgNPs might be intrinsically related to the nature of the biological extract chosen for the synthesis. In addition, Fig. 1 shows a higher total phenolic content (Fig. 1a) and a superior antioxidant activity (Fig. 1b) for AgNPs-GT, compared with nanoparticles synthesized by dill extract. These results suggest that nucleation process might be faster for green synthesis using green tea extract, which possesses a higher capability of reducing Ag+ ions to Ag0 leading to large number of nuclei and smaller nanoparticles. Similarly, Mehta and coauthors reported different size distributions of AgNPs when prepared by different plant extracts (Mehta et al. 2010).

3.3.5 X-ray photoelectron spectroscopy (XPS)

The XPS characterization was carried out to reveal the actual oxidation state of silver at the prepared nanoparticles. The survey spectra (data not shown) of AgNPs-GT, AgNPs-D and AgNPs-F indicated the presence of carbon (C), silver (Ag), oxygen (O) and nitrogen (N) for all samples. Figure 6 shows the spectra in Ag 3d region for AgNPs-GT (a), AgNPs-D (b) and AgNPs-F (c). It was possible to observe two characteristic peaks at 374.3 eV and 368.2 eV, which can be assigned, respectively, to Ag 3d3/2 and Ag 3d5/2 core-level binding energies, well corresponded to metallic silver, being consistent with XRD results (Fig. 3), in agreement with the previous reports in the literature (Han et al. 2011; Rajasekharreddy and Rani 2014; Ansari et al. 2018). Zhang and coworkers also reported a 6.1 eV splitting of 3d doublet to characterize metallic Ag, and this splitting was observed for all nanoparticles in Fig. 6, suggesting that metallic silver (AgNPs) were successfully synthesized through all different plant and fungus extracts (Zhang et al. 2011).

Fig. 6
figure 6

High-resolution Ag 3d spectra of synthesized AgNPs-GT (a), AgNPs-D (b) and AgNPs-F (c) obtained by XPS measurements

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3.3.6 Dynamic light scattering (DLS)

The DLS measurements revealed that all synthesized AgNPs are at nanoscale, with hydrodynamic average size less than 60 nm, and with a monomodal size distribution (Fig. 7a–c). As can be seen in Fig. 7, the values of average hydrodynamic size distribution for the nanoparticles are higher compared with the nanoparticle size obtained from AFM analysis (at solid state) (Fig. 5). As expected, higher values of nanoparticle size are obtained from DLS measurements related to the size values obtained at solid state (Rolim et al. 2019a). The presence of extra hydrate layers, along with ions or molecules attached to the nanoparticle surface in an aqueous environment, was responsible for the higher hydrodynamic sizes (Rolim et al. 2019a).

Fig. 7
figure 7

Hydrodynamic size distribution of AgNPs-GT (a), AgNPs-D (b) and AgNPs-F (c)

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The PDI values for AgNPs-GT, AgNPs-D and AgNPs-F were found to be 0.28 ± 0.01, 0.37 ± 0.01 and 0.52 ± 0.04, respectively. These values indicate a moderate polydispersity. Zeta potential values of − 35.5 ± 1.18 mV, − 23.5 ± 0.40 mV and − 35.93 ± 3.51 mV were obtained for AgNPs-GT, AgNPs-D and AgNPs-F, respectively. The negative zeta potential value is expected due to the presence of polyphenols and proteins attached on nanoparticles surface (Fernández et al. 2016; de Oliveira Silva and Seabra 2016). In addition, the magnitude of the obtained zeta potential values indicates high stability of synthesized AgNPs in aqueous suspension. Our results obtained by DLS are in accordance with similar green synthesized AgNPs (Rajasekharreddy and Rani 2014; de Oliveira Silva and Seabra 2016; Seabra et al. 2017; Singh et al. 2018).

3.4 Antioxidant activity of green synthesized AgNPs

The use of DPPH protocol provides a rapid and easy method to evaluate the antioxidant activity of the synthesized AgNPs. Plant phytochemicals and fungus enzymes act as reducing and capping agents of the nanoparticles. This capping layer on the nanoparticle surface is responsible for antioxidant activity of AgNPs (Mittal et al. 2012). Figure 8 shows the percentage of DPPH radical inhibition upon incubation with different concentrations of AgNPs-GT, AgNPs-D and AgNPs-F. The results demonstrate a concentration-dependent free radical scavenging action (antioxidant activity) for all tested nanoparticles. Overall, AgNPs-F and AgNPs-GT showed superior antioxidant activity. This behavior can be associated with the high content of reducing enzyme in fungus extract (Durán et al. 2005; Cuevas et al. 2015) and high phenolic content in green tea extract (Masek et al. 2017). At nanoparticle concentration of 80 µg mL−1, the percentages of free radical scavenging activity of AgNPs-GT, AgNPs-D and AgNPs-F were found to be 62.41, 46.74 and 79.73%, respectively.

Fig. 8
figure 8

DPPH radical scavenging activity of AgNPs-GT (black bar), AgNPs-D (red bar) and AgNPs-F (blue bar)

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These results demonstrated that all synthesized AgNPs have antioxidant activity, highlighting the potent antioxidant activity of AgNPs-F and AgNPs-GT. Our results are in agreement with the previous published work based on AgNPs synthesized by green routes (Niraimathi et al. 2013; Mittal et al. 2014; Sudha et al. 2017). These results indicate that all AgNPs prepared in this work might be used as potent antioxidant agents.

3.5 Antibacterial activity

The antibacterial activities of AgNPs-GT, AgNPs-D and AgNPs-F were evaluated against Gram-positive S. aureus (ATCC 25923) and Enterococcus faecalis (ATCC 3229), and against Gram-negative Klebsiella pneumoniae (ATCC 13803), Escherichia coli (ATCC 25922) and the multidrug-resistant P. aeruginosa (KPC 37) bacterial strains at different nanoparticle concentrations. MIC and MBC values are shown in Tables 1 and 2, respectively. The results presented on Tables 1 and 2 show a potent antibacterial activity of all synthesized AgNPs against different bacterial strains. Interestingly, by comparing the MIC and MBC values, it is possible to observe that the P. aeruginosa was more sensitive for all synthesized AgNPs. In contrast, the bacterial strain Klebsiella pneumoniae demonstrated to be less susceptive to AgNPs-GT and AgNPs-F with MIC and MBC values of 25 µg mL−1. Even though, all green synthesized AgNPs demonstrated relatively low MIC and MBC values, suggesting their potent antibacterial activity. Recently, Rolim and coworkers reported MIC and MBC values in the range of 7–500 µg mL−1 and 15–500 µg mL−1 for AgNPs-GT against different Gram-positive and Gram-negative bacterial strains (Rolim et al. 2019a). MIC values in the range of 20–25 μg mL−1 were reported for AgNPs synthesized by using bark extract of the medicinal plant Holarrhena pubescens Wall ex G. Don (HP) against clinical isolates of P. aeruginosa (Ali et al. 2018). A MIC value of 6.25 µg mL−1was reported for AgNPs synthesized by using spice blend extract against Streptococcus faecalis, Bacillus cereus, Escherichia coli and Shigella flexneri (Ali et al. 2018). Overall, our results are in accordance with the previous reports (de Lima et al. 2012).

Table 1 MIC values (μg mL−1) for synthesized AgNPs evaluated against different bacterial strains
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Table 2 MBC values (μg mL−1) for synthesized AgNPs evaluated against different bacterial strains
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Furthermore, the tolerance level (ratio between MBC and MIC values) of all bacterial strains used in this work was estimated. The tolerance level equal or greater than 16 indicate a bacteriostatic effect, while tolerance level equal or less than 4, suggest a bactericidal effect (Neethu et al. 2018). According to Tables 1 and 2, all synthesized AgNPs have tolerance level values equal or less than 2, for all tested bacterial strains. Therefore, our results demonstrate that all synthesized AgNPs are bactericidal in nature with a potent antibacterial activity against both Gram-positive and Gram-negative bacteria.

The mechanism of antibacterial activity of AgNPs is reported to involve the following steps: (i) attachment of the nanoparticles to bacterial cell membrane through interactions with sulfur-containing proteins, (ii) disturbance of membrane permeability leaking the intracellular content and impairment of cell respiration, (iii) DNA damage, (iv) generation of reactive oxygen species, (v) regulation of phosphotyrosine profiles of proteins, (vi) cell cycle progression, (vii) synthesis of capsular polysaccharides, (viii) impairment of protein synthesis and functions and (ix) cell death (Durán et al. 2016a; Alsammarraie et al. 2018). In addition, the biological effects of nanoparticles are dependent on nanoparticle size, nanoparticle morphology, degree of agglomeration, surface chemistry and surface charge, among others (de Lima et al. 2012). In this sense, small size nanoparticles might increase the nanoparticle interaction and penetration in membrane cell wall. As the surface nature of nanoparticles is important for particle biological activity, the presence of phytochemicals from plant extracts or proteins from fungus extract might enhance the antibacterial activity of the nanoparticle. The phytochemicals derived from green tea and dill have antimicrobial activities. For instance, the phytochemical catechin gallate might undergo intercalation into phospholipid membrane bilayers of bacteria membrane, disturbing functions of key processes related to the bacterial cytoplasmatic membrane (Durán et al. 2016a). Pure plant extracts are known to have antimicrobial effects, which might be enhanced upon coupled with AgNPs.

3.6 In vitro biofilm formation assay

AgNPs are well known to have antibiofilm effects (Gurunathan et al. 2014). A biofilm allows bacterial persistence by antibiotic resistance and by evading host immune responses. Biofilms are highly impermeable leading to antibiotic resistant (Parrino et al. 2019). In the current study, the ability to inhibit biofilm formation of AgNPs-GT, AgNPs-D and AgNPs-F was evaluated against S. aureus, E. coli and the P. aeruginosa (Fig. 9a–c). Figure 9 shows the potent antibiofilm activity of all synthesized AgNPs against different bacteria strains, at different nanoparticle concentrations.

Fig. 9
figure 9

Effect of biosynthesized AgNPs-GT (black bars), AgNPs-D (red bars) and AgNPs-F (blue bars) on S. aureus ATCC 25923 (a), E. coli ATCC 25922 (b) and Pseudomonas aeruginosa KPC 37 (c) biofilm formation

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In the case of S. aureus (Fig. 9a), it is possible to observe that at nanoparticle concentration of 50 µg mL−1, all tested AgNPs were able to inhibit ca. 90% of biofilm formation, and ca. 80% of biofilm inhibition was observed for nanoparticle concentration of 12.5 µg mL−1, for all tested AgNPs. Even the lowest tested concentration (1.56 µg mL−1) of AgNP-F inhibited ca. 80% of S. aureus biofilm (Fig. 9a). In the case of E. coli (Fig. 9b), all tested AgNPs at concentration of 12.5 µg mL−1 inhibited ca. 95% of biofilm. Interestingly, at the lowest tested concentration (1.56 µg mL−1), AgNPs-F inhibited 90% of E. coli biofilm. In the case of P. aeruginosa (Fig. 9c), all tested nanoparticles at concentration 12.5 µg mL−1 showed potent antibiofilm activity (ca. 97% of biofilm inhibition). In addition, P. aeruginosa was found to be very sensitive to AgNPs, with a strong inhibitory effect (almost 91%) at nanoparticle concentration of 1.56 µg mL−1. AgNPs-GT and AgNPs-D presented a biofilm inhibition of more than 90% at nanoparticle concentration of 25 µg mL−1 (Fig. 9c). Thus, in this study, a clinical P. aeruginosa strain, which is resistant to a variety of antibiotics, and has a strong capacity to form biofilms, was very susceptible to biogenically synthesized AgNPs-F.

Our results are in accordance with the previous published papers (Lotha et al. 2018). For instance, Kalishwaralal et al. synthesized AgNPs using a green route and demonstrated an antibiofilm activity against P. aeruginosa and S. epidermidis biofilms with an inhibition of 95% to 98% at nanoparticle concentration of 100 nmol L−1 of AgNPs (Kalishwaralal et al. 2010). In another work, Singh and collaborators (2018) produced AgNPs by using Cannabis sativa and demonstrated a potent antibiofilm efficacy of almost 100% against P. aeruginosa, E. coli and S. epidermidis (Singh et al. 2018). Studies report that bacteria within biofilms are more resistant to antibiotics than planktonic bacteria, making difficult to control the infections (Velázquez-velázquez et al. 2015). Our results demonstrated that AgNPs synthesized by green routes might be effectively used against biofilm formation, at low nanoparticle concentration.

3.7 Imaging of MDR P. aeruginosa KPC 37 biofilm using scanning confocal laser microscopy

Biofilms represent one of the major issues in bacterial infection, being ten thousand times more resistant in comparison with planktonic bacteria (Sonesson et al. 2017). In vitro tests indicated that biofilms were able to survive to common antibiotics treatment even at a concentration one thousand times higher than the necessary to inhibit free-floating bacteria (Stewart and Costerton 2001). P. aeruginosa biofilms commonly occur in various hospital settings, mostly in medical devices, such as catheters and ventilators and chronic lung infections (Taylor et al. 2014). Despite causing severe and chronic infections, P. aeruginosa biofilm rapidly develops antibiotic resistance leading to difficulty in antibiotic-based treatments (Folkesson et al. 2012; Jeukens et al. 2014). In contrast, AgNPs present a promising alternative in the combat of P. aeruginosa biofilms.

To quantify the percentage of live bacteria in the biofilm formed, live–dead staining was carried for MDR P. aeruginosa KPC 37 strain. The formation of P. aeruginosa KPC 37 biofilm was carried out on the sample holder for confocal microscopy. After 24 h of incubation, the medium was removed and a fresh medium containing AgNPs-GT, at concentrations of 50 or 100 µg mL−1, was added. The biofilm viability was analyzed using confocal laser scanning microscopy. This technique allows the evaluation of the efficacy of nanoparticles in the elimination of formed P. aeruginosa KPC 37 biofilm. Results are shown in Fig. 10. Figure 10a shows the confocal microscope image of P. aeruginosa KPC 37 biofilm without treatment (control group). Figure 10b, c shows the images of viable biofilm after the biofilm treatment with AgNPs-GT (50 or 100 µg mL−1, respectively). In this work, AgNPs-GT was selected to demonstrate the efficacy of biogenic synthesized nanoparticles against biofilm, since AgNPs-GT showed lower MBC value, compared with AgNPs-F, and superior phenolic content and antioxidant activity compared with AgNPs-D. Moreover, the previous results from our group demonstrated potent antibacterial activity of AgNPs-GT (Rolim et al 2019a, Rolim et al. 2019b). Live bacteria appeared green in the images after the treatment. Fluorescence intensity of positive control (control—Fig. 10a) was taken as 100%, and fluorescence of other images was quantified relative to the control. Figure 10d shows the fluorescence intensities of the biofilms. It is possible to observe higher fluorescence intensity for the lowest tested nanoparticle concentration (50 µg mL−1). The treatment of P. aeruginosa KPC37 biofilm with 100 µg mL−1of AgNPs-GT caused bacteria death of more than 75%.

Fig. 10
figure 10

Confocal micrograph images of MDR Pseudomonas aeruginosa KPC 37 biofilms. a Positive control (untreated biofilm); b biofilm incubated with AgNPs-GT at 50 µg mL−1 and c biofilm incubated with AgNPs-GT at 100 µg mL−1. Green cells are viable. d Efficacy of MDR P. aeruginosa KPC 37 biofilm destruction, upon incubation with AgNPs-GT at (ii) 50 µg mL−1 and (iii) 100 µg mL−1. (i) Negative control

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By comparing results in Fig. 9 with Fig. 10d, it is possible to observe that nanoparticle concentrations that are able to avoid almost 100% of biofilm formation (Fig. 9) were not sufficient to kill biofilm cells after the establishment of the biofilm (Fig. 10d). Radzig and coauthors (2013) observed the same behavior for AgNPs against biofilms of E. coli AB1157 (Radzig et al. 2013). In other work, Oliver and coauthors observed that catechin, the major constituent of green tea, can exert antibiofilm activity of P. aeruginosa by attenuation of quorum sensing (Oliver et al. 2018). Thus, Fig. 10d shows that AgNPs-GT is effective in killing bacteria in established MDR P. aeruginosa KCP 37 biofilms. Our results are in accordance with the previous papers (Lotha et al. 2018). It should be noted that biofilms of P. aeruginosa KPC 37 are very resistant. Even though, green synthesized AgNPs were able to kill bacteria in established and resistant biofilms. This result suggests the potent applications of green synthesized AgNPs in the combat of resistant bacterial infections.

4 Conclusions

This study describes the green synthesis of AgNPs by using plant extracts of green tea and dill and by using the mycelium-free extract produced by the white-root S. hirsutum, a white-root Chilean fungus. All plant extracts used in the green synthesis of AgNPs demonstrated a high phenolic content and thus a potent antioxidant activity. Several physicochemical and morphological characterizations of synthesized AgNPs were performed demonstrating the formation of small spherical particles at the nanoscale, coated with plant or fungus extracts. The prepared nanoparticles showed potent antioxidant and antibacterial activity, with relative low values of MIC and MBC (in the range of 1.56–25 µg mL−1) against Gram-positive and Gram-negative bacteria. Overall, AgNPs-D showed lower values of MIC and MBC, compared to other nanoparticles. Furthermore, the synthesized AgNPs demonstrated a potent antibiofilm activity, at low nanoparticle concentration. AgNPs-F demonstrated higher percentage of biofilm inhibition, in comparison with the other nanoparticles, at the lowest tested nanoparticle concentration (1.56 µg mL−1), against all tested bacterial strains. AgNPs-GT was efficiently applied in the disruption of MDR P. aeruginosa KPC37 biofilms. Thus, all synthesized nanoparticles reported here might find important biomedical applications, especially in the treatment of multiresistant bacteria in clinical settings.