Anticandidal Efficacy of Green Synthesized Silver Nanoparticles Using Trans-Himalayan Plant Extracts Against Drug Resistant Clinical Isolates of Candida auris

Abstract

Candida auris is the most common, globally detected nosocomial fungal pathogen with multi-drug resistance. The high prevalence of C. auris infections has raised concern about drug resistance and adverse effects, compounded by a lack of effective alternative drugs. Bioengineered nanomaterials play a significant role in combating nosocomial infections. Silver nanoparticles (AgNPs) have emerged as an extensively used nanomaterial due to their prominent antimicrobial properties. One of the most promising approaches is to incorporate herbal extracts that contain a range of phytoconstituents, being used for curing various chronic illnesses. This study aimed to produce eco-friendly, cost-effective green synthesized AgNPs with trans-Himalayan medicinal plant extracts (Trillium govanianum & Bergenia ligulata) and assess their anticandidal and antibiofilm potential. The green-synthesized AgNPs formation and crystalline nature were confirmed by UV–visible spectroscopy, dynamic light scattering and X-ray diffraction analysis. The UV–Vis spectra of the AgNPs revealed bands in the range of 415–430 nm. Phytoconstituents as reducing agents were involved in the stabilization of AgNPs as identified by FTIR spectra. HR-TEM of AgNPs’ displayed a spherical shape with size in the range of 10–100 nm. Results of activity tests performed using various C. auris clinical strains showed half maximum growth inhibition (IC₅₀) at 8.02 µg/mL, which inhibited 65% of biofilm for T. govanianum extract. The free radical scavenging activity evaluated for green synthesized AgNPs using DPPH showed more than 90% antioxidant activity. Green synthesized AgNPs displayed potent growth inhibition (IC₅₀) at 4.01 µg/mL with 87.0% biofilm inhibition. Green synthesized AgNPs coated bandages and catheters inhibited the growth of C. auris. This study concluded that green synthesized AgNPs formulation in conjunction with antifungal agents exhibits potential biomedical application and also could be used as alternative therapeutics.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01277-8.

Introduction

Candida species are the most prevalent, nosocomial fungal pathogens and they are the fourth leading cause of transmitted infections in hospitals [1–3]. There are 0.4 million candidemia infections caused by Candida species around the world, with fatality rates exceeding 40% [3, 4]. Thus, they are on WHO’s priority list of pathogens and essentially required to work on the Antifungal Surveillance Program; SENTRY [5]. Though Candida albicans is the common cause of candidemia infections, several Candida species, including C. auris, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei, are also responsible for destructive candidiasis and invasive infections with increasing drug resistance [6–8]. Among the various species, C. auris is a growing nosocomial pathogen with multi-drug resistance and is involved in the spread of invasive infections [7]. It has become a problem in clinical treatment due to a high death rate (30- 72%), multi-drug resistance, environmental resilience, difficulties in identification, and horizontal transmission [6]. The levels of multi-drug resistance are rising at an alarming rate, if there are no advancements in the management of ailments carried out by multi-drug resistant pathogens there will be ten million annual fatalities worldwide due to these infections by the year 2050 [9].

C. auris has the ability to develop biofilms on medical devices, which act as the main route to cause infections in hospital care settings [10]. Several virulence traits of C. albicans are also exhibited by C. auris particularly pathways involved in cell wall modelling, siderophore-based iron acquisition, enzyme secretion and nutrient acquisition [11]. Genetic analysis of C. auris has revealed an upsurge of genes related to multidrug efflux and drug resistance [12]. Further, mutations in the genes encoding the lanosterol 1,4-alpha-demethylase (ERG11) gene and drug target 1,3-beta-glucan synthase (FSK1), lead to resistance [6]. Also, efflux pumps such as the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) play a major role in azole resistance resulting in higher mortality in C. auris invasive infections [13]. The Centre for Disease Control and Prevention (CDC), in 2019 published a report on increasing C. auris infections and warned of an “urgent antimicrobial resistance threat” that C. auris poses [14]. C. auris was listed on the WHO’s first-ever Fungal Priority Pathogen List (FPPL) in 2022, under the critical priority pathogen category [15]. The Indian Council of Medical Research (ICMR) issued an advisory to healthcare providers in India to stop the spread of a difficult-to-manage infection [16].

Nanotechnology is currently among the most vibrant research fields in modern science with specialized applications in the formation of nanoparticles (NPs) using herbal plant products. Silver nanoparticles (AgNPs) have displayed better ionic conductivity, nontoxicity, catalytic activity and potential medicinal features such as tissue repair ointments, antimicrobial, surgical equipment, food packaging, fabric coatings, and cosmetics [17, 18]. The antimicrobial (antibacterial, antifungal, and antiviral ability of AgNPs is a principal cause for their prospective utility in combating microbial pathogens in the medical, industrial, and agricultural fields [19–21]. It is commonly acknowledged that fungal pathogens have a significant impact on the health sector [4]. Various new approaches like herbal extracts have shown versatile activity against almost all microorganisms and other biomedical conditions in humans. Specifically, in addition to their anti-inflammatory, anti-ageing and antibacterial activities, some medicinal plants have also reported notable antifungal activity [22]. Often plants found in the trans-Himalayan region have gained interest for their medicinal properties, almost curing various critical ailments because of their high value and robust antioxidant phytoconstituents. Trillium govanianum of the family, Melanthiaceae and Bergenia ligulate belonging to the family, Saxifragaceae are among the recently identified trans-Himalayan herbs with effective medicinal properties including the healing of burn wounds, antifungal, anti-inflammatory and immunomodulatory properties [23–28]. Plant species of the genus Trillium have frequently been documented with a high steroidal saponin content which contributes to antifungal properties in addition to anti-inflammatory and immune adjuvant activities [27, 28]. Similarly, the root extract of Bergenia genus plants has been considered a promising treatment against urinary stones due to its high polyphenolic content [24]. Further, the successful application of bio-nanotechnology can enhance the antimicrobial properties of these medicinal plants as the amalgam of medicinal plant extract and metal salts yielded metal nanoparticles with robust activity [29]. Hence, the current study was undertaken to evaluate the antifungal potential of green synthesized AgNPs using the plant extracts of T. govanianum and B. ligulata against clinical strains of C. auris. This highlights the ethnopharmacological importance of trans-Himalayan herbs against the clinically emerging strains of C. auris.

Material and Methods

Fungal Strains and Growth Media

In the current study C. auris clinical strains viz., NCCPF 470151, NCCPF 470153, NCCPF 470197, and NCCPF 470200 were procured from the National Culture Collection of Pathogenic Fungi (NCCPF), Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. The purity of all strains was checked and biochemically characterized using a standard test procedure. Sabouraud dextrose agar (SDA) and silver nitrate (AgNO₃) were procured from HiMedia (India). Other reagents like glacial acetic acid (Fisher Scientific, USA), and fluconazole (Cipla, USA) were also purchased and used in this study.

Preparation of Trans-Himalayan Medicinal Plant Extracts

The preparation of organic extracts of T.govanianum and B.ligulata plants was carried out according to the method described by Redfern et al. 2014 with minor modifications. The fresh roots and rhizomes of medicinal plants were procured from the Chamba district of the trans-Himalayan region. The roots and rhizomes of plants were washed with 3% hydrogen peroxide, and double deionized water (ddH₂O), and were allowed to dry in the oven at 50 °C for 4 h. Dried plant material was crushed and chopped into fine pieces in each cycle of dry pulverization using a blender for 10 min to generate fine powder for higher surface area to be extracted with a solvent. Using the Soxhlet extraction process for 24 h, the plant extracts were prepared with ethanol solvent. For Soxhlet extraction, the pulverized plant material was tightly wrapped in a cellulose thimble of Whatman filter paper no 1, in the amounts of 50 g for each plant material to be extracted. The thimble was subsequently placed in the Soxhlet extractor and added 300 mL of organic solvent to the round bottom flask, connected with the Soxhlet extractor and condenser on an isomantle. A rig was built using stands and clamps to support the extraction apparatus. The solvent in the round bottom flask was heated on isomantle at 70 ⁰C for 24 h. Evaporated solvent passes through the condenser, and condensate is poured on the thimble inside the Soxhlet extractor. Once the level of condensate reaches the siphon, it is poured back into the round bottom flask, and the cycle begins again. After 24 h of extraction, the plant material was removed from the thimble. The cycle was repeated to remove the excess solvent. The remaining extracts were stored at -20 ºC until frozen and concentrated by lyophilizing at -50 ºC until the complete solvent was evaporated. The extracted yield was calculated (Supplementary Table S1), and the extract was kept in the refrigerator until used to prepare Ag nanoparticles from an AgNO₃ precursor solution. The stock was prepared (1 and 5 mg/ml) in double distilled water for T. govanianum and B. ligulata, respectively.

Source and Preparation of Silver Nitrate

Silver nitrate (CAS No. 7761–88-8,10 g) was procured from Himedia, India. The stock solution of 1 mM AgNO₃ was prepared by dissolving 108 mg of silver nitrate powder in 1 L of distilled water and used as a master stock. From this a succession of 0.1 mM, 0.2 mM, and 0.5 mM as working stocks.

Cell Surface Hydrophobicity

The hydrophobic/hydrophilic surface characteristics of different strains of C. auris were determined using microbial adhesion to solvents (MATS), with slight modifications [30]. The growing cells were processed by centrifugation at 8000 rpm for 10–15 min. Further, 10⁸ cells were collected and washed twice with 0.1 M KNO₃ and OD was measured at 600 nm (A₀). Then 3 mL each of the same number of suspended cells were mixed with different solvents, hexane (a hydrocarbon), chloroform (polar acidic solvent), and xylene (nonpolar solvent) and incubated at room temperature for 10 min. After vortexing, phases were allowed to separate by incubating for 2 h at room temperature. The aqueous phase was removed, and OD was measured at 600 nm (A₁). Finally, the hydrophobicity of the cell surface was measured by using the formula Hydrophobicity (%) ¼ (1-A₁/ A₀) 100.

Qualitative Analysis of Phytoconstituents in the Medicinal Plant Extracts

The phytoconstituents of the test plant extracts were evaluated using previously established protocols in crude extracts qualitatively as described [31]. Briefly, tannins were identified using 0.5 mL of test solution that was added with 1 mL of sterile distilled water and a few drops of 0.1% ferric chloride solution. Terpenoids were detected by adding 2 mL chloroform and 3 mL of Sulfuric acid to 5 mL of test solution. Flavonoids were detected by adding a few drops of 1% ammonia solution to 1 mL of the sample. Similarly, glycosides and alkaloids were identified by adding Anthrone and Mayer’s reagents, respectively. For detection of saponins sample dissolved in water was shaken vigorously to obtain persistent froth and simultaneously 3 drops of olive oil were added to the test solution to observe emulsion formation.

Green Synthesis and Optimization of AgNPs Using Medicinal Plant Extracts

AgNPs were green-synthesized using T. govanianum and B. ligulata as reducing agents, the plant extracts were added to flasks containing 1 mM AgNO₃ solution, covered with aluminium foil, and incubated on a rotary shaker (150 rpm, 37 ºC) for 48 h. Another flask containing AgNO₃ solution was also incubated under identical conditions and used as a control. The silver ion reduction was evaluated using a UV-VIZ Spectrophotometer (Neumann & Miller Co. USA), considering the entire bio-reduction of Ag⁺. AgNPs were collected by ultracentrifugation at 10,000 rpm for 30 min and washed using distilled water. The samples were then lyophilized (SCANVAC, CoolSafe) at -50 ºC and stored for subsequent analyses.

Effect of pH, Silver Nitrate and Extract Concentration

Various factors influence the biogenesis, size, and shape of nanoparticles. By varying one variable at a time, environmental and cultural factors, the impact of parameters (pH, AgNO₃ concentration, and extract concentration) on AgNP biosynthesis was examined using UV-VIZ Spectroscopy. To observe the effect of pH on AgNP production, a pH ranging from 6.5 to 9.0 pH was set and incubated. The production and analysis of AgNPs were observed. Flasks with different concentrations of silver nitrate (0.1–1.5 mM) were incubated for 24–48 h, and the production of AgNPs was observed. The effect of extract concentration was evaluated using different concentrations of extracts (100–500 µg/mL) of T. govanianum and for B. ligulata (1–2.5 mg/mL) and incubating them along with the control.

Characterization of Green-Synthesized AgNPs

Initially, the bioconversion of Ag⁺ to Ag° in an aqueous solution was confirmed using an ultraviolet–visible spectrophotometer ranging from 300 to 700 nm. The hydrodynamic diameter (size), surface charge and polydispersity index (PDI) of colloidal AgNPs were investigated using dynamic light scattering (DLS) at 25 °C using a Zeta potential/particles sizer. Finally, the chemical composition and the functionalization of AgNPs were detected by FTIR analysis. For FTIR analysis, 2 mg of the material was combined with KBr of 200 mg and pressed into a pellet. The sample pellet was transferred into the sample holder, and FTIR spectra were captured with a resolution of 4 cm⁻¹ in the range 4000–450 cm⁻¹ at Panjab University Chandigarh's CIL (Central instrumentation laboratory). The primary purpose of FTIR was to identify the chemical functional groups in the material. The crystalline structure of green synthesized AgNPs was examined using an X-ray diffractometer (XRD). AgNPs were centrifuged at 10,000 rpm for 20 min, and the formed pellets were dispersed again in double distilled water. The filtered pellets were oven-dried at 50 °C and were analysed by an X-ray diffractometer which was operated at a 45 kV voltage and 40 mA current by using Cu-Kα radiation source in 2-degree scattering range, 1-degree divergence slit, 2θ ordinary mode. To analyse the results Origin Pro and High Score Plus software were used. High-resolution Transmission electron microscopy (HRTEM) was analysed to confirm the size of AgNPs. For HRTEM analysis, extract samples containing Ag nanoparticles were spread on a copper grid followed by solvent evaporation and dried at room temperature. HRTEM operating at 200 kV was used to collect micrographs. The size distributions of the generated nanoparticles were observed on the ground of HRTEM micrographs. The images were captured at CIL (Central Instrumentation Laboratory), Panjab University, Chandigarh.

Antimicrobial Assay

The anticandidal activity of green-synthesized AgNPs was evaluated by comparing the growth inhibition zones of AgNPs with the inhibition zones of plant extracts by Agar well diffusion assay. The inhibitory impact of extracts and their AgNPs against various fungal strains of C. auris in accordance with the method described [32]. To obtain the log phase culture of the test strain, a fresh inoculum was prepared from overnight culture and incubated for 4–5 h. About 100 µl of inoculum (10⁶ CFU/mL) was dispersed on the SDA plate and wells (8 mm) were punched, and loaded with extracts and AgNPs. Distilled water was used as a control. After the incubation period of 24 h at 30 °C, the zone of inhibition was measured. The antifungal agent (Posaconazole) was used as a positive control in this study, which is known to inhibit C. auris with a minimum inhibitory concentration (MIC) of 125–250 µg/ml varied for all the strains according to NCCPF.

Minimum Inhibitory Concentration (MIC) of Extracts and their AgNPs

The MIC of extracts and AgNPs was determined using both the micro and the macro dilution methods performed following CLSI guidelines [33]. In SDB, two-fold serial dilutions of the agents (extracts and AgNPs) were made volumetrically. Dilutions up to 1:256 were made. 1 × 10⁶ CFU/mL inoculum was added to each tube/well and incubated for 24 h at 30 ºC. The results were interpreted by comparing them to a blank and a positive control. The MIC was determined visually as the lowest dose that fully suppressed fungal growth. It was validated further by streaking from tubes/wells containing MIC and a greater concentration of extract or AgNPs on SDA plates and incubating at 30 ºC for 24 h.

Antioxidant Activity Determination

The antioxidant activity of the plant extract AgNPs was examined on the basis of the scavenging effect on the stable DPPH free radical activity. Ethanolic solution of DPPH free radical (0.5 mM) 300 µl was added to 40 µl of extract solution with different concentrations (0.25, 0.125, 0.5, 0.75, and 1 mg/mL). DPPH solution was prepared freshly and kept at dark at 4 °C. 96% Ethanol (2.7 ml) was added and the mixture was shaken vigorously. The mixture was left to stand for 4 min and absorbance was checked at 517 nm. The radical scavenging activity was calculated using the formula i.e., % inhibition = AB- AA/AB X 100, where AB = Blank Absorbance, AA = Test Absorbance [34]. Ascorbic acid was used as a standard.

Biofilm Quantification and Inhibition by Crystal Violet (CV) Staining Assay

Quantification and inhibition of biofilm formation was performed on 96 well, flat-bottomed polystyrene plates in accordance with the method described [35]. Agents were introduced at sub-MIC doses to each well in the first column containing sterile SDB and serially diluted volumetrically except for the blank and control wells. 1 × 10⁶ CFU/mL inoculum was added to each well. Plates were incubated statically at 30 ºC for 24 h. After incubation, the media was removed from each well without disrupting the biofilms. To eliminate planktonic cells, wells were washed two or three times with PBS and air-dried. The fixing of biofilms was done by adding methanol to each well for 15 min. After decanting methanol, 0.1% (w/v) CV solution was added and incubated at room temperature for 5 min. An excessive amount of CV was removed, and plates were rinsed twice with distilled water and allowed to dry. Crystal violet bound to the cells was destained using 33% glacial acetic acid. The aforementioned solution was then transferred to a new reading plate. Biofilm formation was assessed in terms of OD at 595 nm using a microplate reader.

Field Emission Scanning Electron Microscope (FE-SEM)

C. auris NCCPF 470153 biofilm was developed on sterile coverslips in 6 well plates. Fungal cell suspension (10⁶ cells/ml) was inoculated onto each coverslip and treated with TG-AgNPs and BL-AgNPs at MIC/2 concentrations. Wells without treatment were taken as control. The plate was incubated for 24 h at 30 ºC under static conditions. Post incubation the culture media was discarded and the wells were gently washed with PBS to remove planktonic cells. For FE-SEM analysis the biofilm cultures were fixed with 4% paraformaldehyde for 1 h. The biofilm was then subjected to gradient ethanol dehydration (40–100%). The biofilm-coated coverslips were then mounted onto an aluminium stub, air-dried and coated with gold for 30 s for observation. The samples and control were examined at 5 kV using an FE-SEM (Hi-Tachi, Japan) at various magnifications at the Central Instrumentation Laboratory (CIL), Panjab University, Chandigarh.

Determination of Adhesive Ability of AgNPs Used as a Coating Agent

The TG-AgNPs (0.5 mg/mL) and BL-AgNPs (1 mg/mL) were coated onto Whatman filter paper No. 1 under laminar flow, and dried for two days in an oven at 45 °C. For preparing AgNPs coated catheters, Foley urinary catheters were used and proceeded by peristaltic pump method which was operated through rotary motion. The AgNPs with concentration TG-AgNPs (1 mg/ml) and BL-AgNPs (1.5 mg/ml) were deposited on one side of the tube and the suspension was allowed to flow from one end of the tube to the other end. The peristaltic pump was operated for 48 h and then catheters were cut into pieces and oven-dried for 2 days. Further, in-vitro analysis of its antifungal and antibiofilm potential was checked.

In Vitro Analysis of Coated AgNPs

The inhibitory antifungal impact of coated nanoparticles on bandages against various fungal strains of C. auris was in accordance with the method described [36]. To obtain the log phase culture of the test strain, a fresh inoculum was prepared from overnight culture and incubated for 4–5 h. About 100 µl of inoculum (10⁶ CFU/mL) was dispersed on the MHA plate, coated bandages were placed with forceps. After the incubation period of 24 h at 30 °C, the zone of inhibition was observed that confirmed antifungal properties.

Biofilm Formation in the Presence of AgNPs Coated Bandages and Catheters

To assess the biofilm formation on coated bandages and catheters, SDB media was added to each well of the plate and in the presence of Coated filter paper and catheters, 4 µl of fresh inoculum was added. Resazurin dye test which is a dye reduction test, was performed to observe the growth of microorganisms and in the presence of viable cells, the dye turns its colour from bluish to pinkish colour. 100 µl of resazurin dye was added to the formed biofilm and media.

Results

Qualitative Analysis of Phytochemicals in Plant Extracts

Trans-Himalayan medicinal plants T. govanianum and B. ligulata are widely used as traditional medicinal plants to treat pain, inflammation, burns and wounds. The root extract obtained by Soxhlet extraction from T. govanianum and B. ligulata yielded 21% and 24% respectively (Supplementary Table S1), by ethanol extractant procedure. The available phytoconstituents in these plant extracts were determined qualitatively based on the observation of colour changes, emulsion formation, and precipitation (Supplementary Fig S1). The observed bioactive compounds in both extracts were found to be tannins, terpenoids, saponins and flavonoids. However, alkaloids and glycosides were detected only in T. govanianum but were not found in B. Ligulata (Table 1).

Table 1.

Preliminary phytoconstituent analysis of the plant extracts

Phytochemicals	T. govanianum	B. ligulata
Tannins	+	+
Terpenoids	+	+
Flavonoids	+	+
Saponins	+	+
Glycosides	+	−
Alkaloids	+	−

Cell Surface Hydrophobicity

The clinical strains of C. auris displayed a hydrophobic nature as we analyzed cell surface hydrophobicity using different solvents. Results revealed that strains had varied affinity to hexane, chloroform and xylene. The stronger and the maximum affinity was observed for hexane, xylene and chloroform as 69%, 62% and 51%, respectively (Supplementary Fig S2). The different clinical strains of C. auris affinity for all solvents implies that its cell surface is complicated.

Green-Synthesis of Silver Nanoparticles (AgNPs) Using Medicinal Plants Extract

In the present study, aqueous extracts of these plants were added to 1 mM AgNO₃ at different dilutions. However, the plant extracts, when extracted through the Soxhlet extraction process using ethanol as a solvent revealed 25 to 100 times higher yield in comparison to extraction with distilled water. Preliminary confirmation of nanoparticle formation by a progressive shift in the color of the colloidal solution from translucent to brown in T. govanianum and to reddish brown in B. ligulata is clear indicator of silver nanoparticles (AgNPs) formation (Supplementary Fig S3). Increase in the incubation period over 48 h changed the hue to a darker tone and color stabilized after 72 h. AgNPs suspended in the solvent with no signs of precipitation even after 6 months of duration indicates stability of the nanoparticles.

Optimization of Green-Synthesized AgNPs Using Plant Extract

The green synthesis of AgNPs using medicinal extracts of T. govanianum and B. ligulata was optimized by standardizing various process parameters including AgNO₃ concentration, reducing agent concentration (plants extract), and pH using the UV–visible spectroscopy method. For the maximum synthesis of AgNPs, silver nitrate concentration was optimized. A gradual shift in absorption peak to higher wavelengths was observed with increasing AgNO₃ concentrations and highest value of peak intensity equivalent to + 2.0 was measured at 430 nm with 1 mM AgNO₃ (Fig. 1a). Increasing AgNO₃ concentration to 1.5 mM resulted in peak symmetry distortion and reduced peak intensity. Likewise optimum concentration for the green-synthesis of AgNPs was determined to be 1 mM of AgNO₃ for B. ligulata, which showed maximum peak value of + 2.02 at 420 nm. Increasing concentration resulted in agglomeration and precipitation of green synthesized AgNPs at the bottom of flask. Optimization of reducing agent concentration for maximal biosynthesis of AgNPs for both the plant extracts was determined and with increasing extract concentration, nanoparticle production increased in a linear manner. The greatest value of peak intensity for T. govanianum was observed to be 2.24 at 417 nm for 500 µg/ml and 1.94 at 421 nm for 200 µg/ml extract (Fig. 1b). At 420 nm and 419 nm, respectively, for 1.5 mg/ml and 1 mg/ml extract, the highest peak intensities of 2.02 and 1.90 for B. ligulata were observed, indicating the highest production of AgNPs. (Fig. 1d). At neutral or acidic pH values, no particle formation was seen. However, as the pH of the solution reaction mixture was raised, the reaction mixture's colour rapidly changed from pale to dark brown. Additionally, the absorbance peaks grew sharper and narrower, signifying the development of smaller, spherical nanoparticles. Peaks at pH 8.0 and 9.0 were discovered to be very symmetrical, supporting the consistent production of AgNPs. T. govanianum had maximum peak intensities of 2.23 and 1.66 at 415 and 419 nm, respectively (Fig. 1c). The peak was found to be very symmetrical, thinner, and sharper at pH 8.0, indicating evenly dispersed, spherical, and smaller-sized AgNPs. In case of B. ligulata, the maximum peak intensity corresponding to 1.90 was observed at 419 nm at pH 9.0, indicating that the amount of AgNPs produced was highest in the reaction medium (Fig. 1e). Further, with an increase in pH, the peak shifted towards a lower wavelength, which indicates a reduction in the size of AgNPs.

Fig. 1.

UV–vis absorption spectra of green-synthesized AgNPs using extracts of T. govanianum and B. ligulata a variable AgNO₃ concentration (0.5 mM—2 mM); b effect of reducing agent concentration (100-500 µg/mL); c various pH values (pH 8–9.5) and d reducing agent concentration (1–2.5 mg/mL); e variation of pH (pH 8–9.5) respectively

Characterization of Green Synthesized Silver Nanoparticles

UV- visible spectrometry was used to analyze the formation of colloidal AgNPs by the reduction of Ag⁺ ions using T. govanianum and B. ligulata plant extract. The solution's UV–vis absorption spectra revealed the surface plasmon resonance originating from the silver nanoparticles around 400–450 nm. The transition of silver ions into AgNPs was measured with a peak between 410–450 nm, indicating AgNPs have a strong surface plasmon resonance (SPR) in the visible wavelength range that increased over time. It was analyzed for all the different samples as mentioned above in the figures (Fig. 1a–e). After the AgNPs formation confirmation, dynamic light scattering (DLS)was used for determining average particle size, charge in aqueous solutions of AgNPs, and polydispersity index of the synthesized AgNPs. The study found that the average particle size of green synthesized AgNPs of plant extract T. govanianum and B. ligulata were found to be 80.75 and 85.87 nm, respectively (Fig. 2a, c). Another important element of nanoparticles is their surface charge or zeta potential, which impacts their capacity to connect with or compound with macromolecules found on the surface or inside cells. The results indicated that AgNPs formed from extracts T. govanianum and B. ligulata had charges of −25.4 and −56.7 mV, respectively (Fig. 2b, d).

Fig. 2.

Size determination by DLS analysis of silver nanoparticles synthesized using plants extract of a T. govanianum b B. ligulata c zeta potential analysis of T. govanianum and d B. ligulata

The particle size distribution within the sample is reflected by poly-dispersive index (PDI). AgNPs, which was found to be 0.516 and 0.43 for T. govanianum and B. ligulata, respectively, thus indicating the stability and mono dispersity of AgNPs. HRTEM was used to examine the shape, size, and morphology of green synthesized AgNPs. HRTEM images of drop-coated films of AgNPs were produced by treating AgNO₃ solution with a 500 µg/ml extract of T. govanianum. The silver nanoparticles' shape was spherical and well separated, with no aggregation seen in the micrograph. The analysis confirmed that the green synthesized AgNPs were 10 to 100 nm in size (Fig. 3). The nanoparticles had a crystalline structure as revealed by SAED (Selected area electron diffraction) pattern (Supplementary Figure S4). The multiple functional groups in biomolecules that are involved in the bio-reduction of Ag + and the capping/stabilization of AgNPs have been identified by FTIR spectroscopy analysis. To analyze the functional group present in extracts, FTIR spectrum captured multiple absorption peaks from T. govanianum and B. ligulata green-synthesized AgNPs. The observed N–H extension vibrations indicate the presence of strong hydrogen bonds with broad peaks between 3650 and 3250 cm⁻¹_, specifically at 3413.90 cm⁻¹_. Peaks found at 1600–1300, 1200–1000, 800–600 cm⁻¹confirmed hydroxyl compound. The narrow bands below 3000 cm⁻¹ were found specifically at 2928.23 cm-1 revealed long chain aliphatic compounds. The double bond region (1500–2000) cm ⁻¹, sharp peak was observed at 1743, 1715 and 1641 cm⁻¹ conforming carbonyl group (C = C). This indicates the presence of carbonyl double bond from aldehydes, ketones, carboxyl or esters. The peaks below 1700 cm⁻¹indicated amides carboxylate functional group, specific peak at 1641 cm⁻¹. The peaks at 900 cm⁻¹ and 990 cm⁻¹ depicted that agent had vinyl terminals (CH = CH₂). At 890 cm⁻¹, peaks were observed confirming double olefinic bonds in single vinyl (C = CH₂)_. The FTIR analysis of green-synthesized AgNPs also displayed similar spectra. Peaks found in the range of 3650–3250 cm⁻¹were indicative of hydroxyl group stretching vibration. The narrow bands below 3000 cm⁻¹ were found specifically at 2926.05 and 2856.16 cm⁻¹, revealing long-chain aliphatic compounds. Triple bond region (2000–2500) cm⁻¹ was detected by observing a specific peak at 2100 cm⁻¹. In context to double bond region (1500–2000) cm⁻¹, sharp peak was observed at 1740.39, and 1681.26 cm⁻¹ conforming carbonyl group (C = C) stretching of aldehyde and ester. The peaks below 1700 cm⁻¹ confirmed amides carboxylate functional group. The similarity in spectra obtained for plant extract and green-synthesized AgNPs indicates capping of plant extracts from aldehydes, ketones, carboxyl, or esters (Fig. 4). Further, determination of crystalline structure of the green-synthesized AgNPs was carried out by XRD analysis showed diffraction peaks at 32.4°, 38.1°, 46.2°, 55.4°, 58.3°, 64.3°, 76.9°, and 32.5°, 38.2°, 46.5°, 57.5°, 64.5°, 76.9° for TG-AgNPs and BL-AgNPs, respectively (Fig. 5). When compared with the standard, the obtained XRD spectrum confirmed that the synthesized AgNPs were in nanocrystal form and crystalline in nature. The face-centred cubic silver was examined and matched to those existing in the Joint Committee on Powder Diffraction Standard, USA (JCPDS No. 89–3722) database in order to verify that green synthesised AgNPs are pure crystalline in nature. The peaks can be assigned to the planes (122), (111), (200), (220), and (311) facets of silver crystal, respectively.

Fig. 3.

Visualization ofgreen-synthesized AgNPs using HRTEM micrograph T. govanianum &B. ligulata, with different magnifications showing spherical shape. [The scalar bar represents as a 200 nm b 100 nm c 20 nm d 10 nm of TG-AgNPs and e 200 nm f 100 nm g 20 nm h 10 nm of BL-AgNPs]

Fig. 4.

FTIR spectra of freeze-dried plant extracts of T. govanianum and B. ligulata and their green-synthesized AgNPs a T. govanianum b TG-AgNPs c B. ligulata d BL-AgNPs

Fig. 5.

XRD pattern of green-synthesized AgNPs a TG-AgNPs b BL-AgNPs

Antimicrobial Assay

The anticandidal activity of plant extracts and their green-synthesized AgNPs was evaluated in triplicates by testing against various clinical strains of C. auris. Sensitivity response against clinical strains of C. auris was varied with concentrations and a strong inhibition zone was observed at a concentration of 1 mg/mL of T. govanianum plant extract and for the green synthesized-AgNPs of this extract it was 0.5 mg/mL. In the case of B. ligulata plant extract and its BL-AgNPs, the concentration used was 10 and 1 mg/mL, respectively. It was observed that in contrast to the prepared AgNPs, C. auris strains demonstrated greater sensitivity to TG-AgNPs and BL-AgNPs (Fig. 6). The green-synthesized BL-AgNPs showed a strong sensitivity response in comparison to the extract as the inhibition zone diameter increased from 13 to 28 mm with the increase in concentration (Fig. 6). The antifungal agent posaconazole with a concentration of 31.25 µg/mL was used in the study as a positive control which showed an inhibition zone of 10–15 mm (Fig. 6).

Fig. 6.

Determination of antimicrobial activity of plant extracts and their green-synthesized AgNPs against different clinical strains of C. auris by agar well diffusion (Inset showing inhibition zone by extracts and their green-synthesized AgNPs against C. auris)

Determination of Minimum Inhibitory Concentration (MIC)

The plants extract and their green-synthesized AgNPs showed remarkable antimicrobial activity when compared to the control after 24 h of incubation against the four strains of C. auris used as test strains. The antimicrobial activity was varied with different concentrations of extracts and their AgNPs (Table 2). The minimum inhibitory concentration (IC₅₀) of T. govanianum extract was in the range of 8–32 µg/mL, and in the case of green synthesized TG-AgNPs the MIC (IC₅₀) was 4–16 µg/mL (Table 2). The MIC (IC₉₀) of T. govanianum extract ranged between 32 and 125 µg/mL. Whereas, in the case of TG-AgNPs it ranged between 64 -250 µg/mL. Similarly, the MIC (IC₅₀) of B. ligulata varied between 32 -64 µg/mL and 16–32 µg/mL in the case of BL-AgNPs (Table 2). The MIC (IC₉₀) ranges from 125 to 250 µg/mL in BL-AgNPs against all four strains of C. auris (Table 2).

Table 2.

MIC(IC₅₀)/MIC(IC₉₀) of plant extracts and their green synthesized silver nanoparticles against different strains of C. auris using broth-micro dilution method

Candida strains	MIC(IC50)/MIC(IC90) (Trillium govanianum)	MIC(IC50)/MIC(IC90) (Bergenia ligulata)
T. govanianum		B. ligulata
C. auris NCCPF 470151	32.25/125	64.5/125
C. auris NCCPF 470153	32.25/125	32.25/125
C. auris NCCPF 470197	16.12/125	32.25/125
C. auris NCCPF 470200	8.02/32	64.5/250
	TG-AgNPs	BL-AgNPs
C. auris NCCPF 470151	16.12/250	32.25/250
C. auris NCCPF 470153	16.12/250	16.12/250
C. auris NCCPF 470197	8.02/250	16.12/250
C. auris NCCPF 470200	4.01/64.5	32.25/125

*All MIC results were expressed in µg/mL

Antioxidant Activity Determination

The considerable antioxidant activity of green synthesized AgNPs was observed by DPPH free radical scavenging assay. The free radical scavenging activity of plant extracts was obtained in order as for TG-AgNPs were more for 0.25, 0.125, 0.5, 0.75 and 1 mg/ml, and the same in case of BL-AgNPs. TG-AgNPs showed higher DPPH scavenging activity than BL-AgNPs (Supplementary Figure S5).

Biofilm Quantification and Inhibition

Quantification of biofilm formation by the clinical strains of C. auris was performed by CV assay, which showed a gradual increase in biofilm biomass in all four strains (C. auris strains NCCPF 470151, NCCPF 470153, NCCPF 470197, NCCPF 470200) up to 72 h (Supplementary Figure S6). The highest peak was obtained at 72 h most notably in the case of C. auris strain NCCPF 470200. The inclusion of different extracts and their AgNPs caused inhibition in the biofilm compared to their test contr2ols. In the current investigation, the in-vitro study was evaluated in a dose-dependent manner against the biofilm-forming organism C. auris strains. Results of the experiment revealed that plant extracts of T. govanianum and B. ligulata inhibited biofilm formation in the range of 62–68% against different strains of C. auris (Fig. 7a, c). Similarly, in case of green-synthesized AgNPs derived from T. govanianum and B. ligulata showed inhibition of biofilm formation in the range of 83–87% (Fig. 7b, d) as observed in 96-well microtiter plate analysis (inset, Fig. 7).

Fig. 7.

Determination of antibiofilm activity of plant extracts and their green-synthesized AgNPs. a T. govanianum, b TG-AgNPs c B. ligulata and d BL-AgNPs against four clinical strains of C. auris. ODs measured in triplicate and bars represents the standard deviation with p value (< 0.001). (Inset shows biofilm inhibition images of 96-well microtiter plate)

Field Emission Scanning Electron Microscope (FE-SEM) Analysis

Sub-MIC concentrations of TG-AgNPs and BL-AgNPs were found to inhibit the C. auris biofilm and cause cellular morphological disruption (Fig. 8). The control group's C. auris biofilm maintains its architectural integrity, while the treated samples show evidence of cell aggregation with slightly changed morphology. Complete damage to the cell membrane as well as intracellular component leakage due to plasmolysis were also observed in treated cells, along with distortion in cell morphology caused by cell shrinkage.

Fig. 8.

FE-SEM micrograph of Candida auris NCCPF 470153 biofilm in treated and untreated cells (a, b, c) Control at magnification 50.0, 20.0 and 10 µm resepctively. (d, e, f) TG-AgNPs treated cells at magnification 50.0, 20.0 and 10 µm resepctively. (g, h, i) BL-AgNPs treated cells at magnification 50.0, 20.0 and 10 µm resepctively

Determination of Adhesive Ability of AgNPs

Biofilm deposition on skin surfaces and hospital equipment is persistent and had a negative impact on human health. Therefore, AgNPs adhesive ability was tested for use as a coating agent by in-vitro analysis. The bandages were coated with green synthesized AgNPs and upon subsequent processing, they were tested for inhibiting activity by spread plate method. Results showed no growth on the surface and periphery of the bandages. The narrow inhibiting zone in bandage surroundings clearly indicates the irreversible binding of green-synthesized AgNPs to the bandages (Fig. 9a and b). The inhibition zone varied between 11 and 12 mm for different test strains (Fig. 9a and b). Likewise, AgNPs coated bandages and catheters were also tested for antibiofilm ability by allowing formation in the presence of coated bandages and catheters using resazurin assay. In the presence of TG-AgNPs and BL-AgNPs wells showed purple colour indicating the absence of biofilm on green-synthesized AgNPs coated bandages, whereas, pink colour observed in control wells without coated bandages indicated the formation of an active biofilm. Similarly, biofilm formation in the presence of TG-AgNPs and BL-AgNPs coated catheters was also assessed by resazurin assay. No biofilm was formed in the presence of coated catheters indicating intact coating of TG-AgNPs and BL-AgNPs on catheters that prevented their diffusion into media in comparison to uncoated catheters (Fig. 9c- 9f).

Fig. 9.

Determination of adhesive ability of AgNPs. a, b AgNP coated bandages showing zone of inhibition. c, d, e, f Biofilm inhibition ability of AgNP coated bandages and catheters using resazurin assay. (Inset shows in vitro biofilm adhesive and biofilm inhibition ability of AgNPs)

Discussion

The emergence of multi-drug resistant clinical strains resulted in an urgent need to look for a new regime of antifungal drugs at the point of care settings. C. auris is such an emerging opportunistic pathogenic fungus that causes outbreaks in hospital care settings. It shows nosocomial spread and only a few drugs have been approved for treatment, whose efficacy is still questionable. Various new antifungal medicines are being explored with fewer side effects and lower cost, amongst which medicinal plants are found to be most efficient due to the bioactive elements present in them conferring specific properties [36–38]. In light of this problem, the present study was designed to evaluate the efficacy of green synthesized AgNPs against multi-drug resistance clinical isolates of C. auris. The study was focused on clinical isolates as they are taken from candidemia patients.

It was reported that medicinal plants, T. govanianum and B. ligulata were employed as traditional medicinal plants to treat various ailments [23, 25]. In fact, Rahman et al. reported the antifungal activity of the hydro-methanolic extract of T. govanianum against fungal strains but,ethanolic extract of T. govanianum offered higher antifungal activity [39]. It wasalso identified that govanoside A and borassoside E compounds belonging to saponin class were present in T. govanianum extract, which are responsible for the antifungal activity [40, 41]. Other reports have also shown the antifungal potential of T. govanianum plant extract [23]. The antimicrobial property was reputed to be associated with certain secondary metabolites that plants produce in self-defence such as saponins, terpenoids, tannins and flavonoids [39]. Likewise, secondary metabolites including tannins, terpenoids, glycosides, and flavonoids present in B. ligulata were reported to be involved in antifungal activity [42]. Thus, we have proceeded with the preliminary testing to detect phytochemicals present in T. govanianum and B. ligulata plant extracts, which identified the presence of tannins, terpenoids, flavonoids, saponins, glycosides and alkaloids in both the plants extract. C. auris being a multi-drug resistant strain, both medicinal plant extracts were tested to find antimicrobial activity. Both the tested medicinal plants exhibited strong inhibitory activity against clinical isolates of C. auris (Fig. 6). Further, a remarkable difference in the MIC values of all the strains was observed, which can be co- related to the strain variance (Table 2). Furthermore, a significant difference in the MIC values of both the plant extracts was observed, which might be attributed to the different bioactive compounds’ composition in the extracts. The composition was analyzed and confirmed with the assignment of functional groups using FTIR analysis. Upon confirmation that the selected plants extract showed good antifungal activity, green synthesis of silver nanoparticles using both the plant extracts were performed to enhance the activity. Plant extracts are frequently used for the green-synthesis of metal nanoparticles as preferred method compared to other chemical methods, especially AgNPs, because they are easily available, cost-effective, and are not harmful to the environment. The findings by UV–Vis spectroscopy of the AgNPs were corelated with predictions and earlier experiments where the strong SPR bands occur around 420 nm when AgNPs dispersed in other polymeric matrices. AgNPs produced in this study showed smaller size with uniform dispersion, the narrower and more symmetrical the peaks. This is directly tied to the nanoparticles' stability and antifungal activity. DLS confirmed that with increasing plant extract concentration, the particles’ hydrodynamic size (diameter) was increased. The zeta potential of TG-AgNPs was highly negative. A high value of negative or positive represents lower chances of aggregation, while a low zeta potential level is indicative of aggregation of synthesized nanoparticles [43–45]. The high peaks in the XRD analysis indicated the active silver composition with the indexing. This indicates that the AgNPs face-centred, cubic and crystalline in nature [46]. HRTEM images of green synthesized silver nanoparticles revealed that most of the particles are spherical in shape and crystalline in character, with the existence of a lattice fringe corresponding to the Ag plane, which is an important factor in bio-organic compounds nanoparticle formation as reported using TEM and SEM analysis [47–50]. Though both plants extract showed antifungal activity, remarkably, the antifungal and antibiofilm potential of plants extract was enhanced by more than two-fold increase with silver nanoparticles formulation. The activity enhancement could be due to the capping of the particular bioactive compound of the extracts that coated silver nanoparticles. The functional groups present in AgNPs were identified by FTIR. In line with earlier reports, the FTIR spectrum analysis in this study detected several absorption peaks that are attributed to compounds present in extracts that are linked to silver ion reduction leading to nanoparticle stabilization [49–52]. AgNPs synthesized using environmentally benign solvents showed better inhibition in C. albicans [53]. The anti-biofilm activity of AgNPs against infectious agents responsible for microbial keratitis has also been studied in the literature [54]. The majority of biofilm related ailments are connected to metal equipment like respirators, urinary catheters, fixed orthopaedic devices, dental supplies, central lines, prosthetics, etc. As a result, targeted treatments and cost-effective wound treatments are necessary for the development of alternative strategies. Therefore, novel nanotechnology including the green synthesized AgNPs can present another platform for the development of therapeutic agent for wound infection caused by clinical strains of C. auris. Additionally, the antioxidant activity observed by these extract-bound AgNPs indicates their radical scavenging ability, which is essentially required for human applications of AgNPs [55].

C. auris is an opportunistic fungal pathogen with multidrug resistance which is severely involved in spread of infections. These findings suggest the selective use of plants extract and their green-synthesized silver nanoparticles as an adjuvant to standard anticandidal treatments against biofilm-forming microorganisms such as C. auris. The most important limitation of using these plants extract is that crude extract/ raw material contains some impurities which can show some adverse effects on the host. Characterization of different compounds present in crude plant extracts is further being carried out in our laboratory. Future research could see an increase in the production of AgNPs using green technology, which could have applications in various fields, including plant-derived phytochemicals. Though phytochemicals pose complexity they modulate the cytotoxicity of nanoparticles. Biological activity of AgNPs is driven by surface charge, size, and shape, which are not easy to control during the preparation. Considering their toxicity and distribution, AgNPs may release into the environment and requires thorough investigation [56]. Furthermore, toxicity of AgNPs must be investigated in living cells, specifically considering their application to treat diseases [57]. Further combinatorial therapy of AgNPs against microbes using antibiotics or tumours using anticancer agents requires to study synergistic effect and toxicity issues. Such combinations with low or no toxicity could be considered for efficient treatment and may further develop AgNPs based efficient products. It is pertaining to mention that research studies have demonstrated non-toxic nature of AgNPs if they are used in appropriate and controlled environments suggesting their potential external applications involving below toxicity levels [58]. Further, antimicrobial AgNPs are increasingly used in textiles and laundry, cosmetic products, and food packaging material as per regulatory norms [59]. AgNPs were non cytotoxic to human fibroblast cells at low concentrations in in-vitro and in-vivo experiments using mice with significant adverse effects [60]. Furthermore, clinical trials of wound dressings, without adverse effects confirmed non-toxic nature in human subjects [61]. Current evidence indicates that AgNPs can be safely integrated into a range of applications without creating significant hazardous dangers even though continuing research continues to refine our understanding.

Conclusion

The green-synthesis approach used in this study for formulating silver nanoparticles is eco-friendly and non-toxic. The inhibitory and killing activities against clinical strains of C. auris supported the premise that medicinal herbs and their green-synthesized silver nanoparticles are potent antifungal and antibiofilm agents. After being validated in vivo, the encouraging findings of this study may contribute to the relief of symptoms and treatment of fungal diseases. Because of their effective antifungal action, green-synthesized AgNPs might greatly benefit in the medical sector. This can further help to develop a strain-specific novel antifungal drug.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.

Declarations

Conflict of interest

The authors report no declarations of interest.