Evaluation of anti‐bacterial activity of silver nanoparticles synthesised by coprophilous fungus PM0651419

Abstract

The study explored biological synthesis of metallic silver nanoparticles (AgNPs) from the less explored non‐pathogenic coprophilous fungus, sterile mycelium, PM0651419 and evaluates the antimicrobial efficacy of biosynthesised AgNPs when impregnated in wound fabrics and in combination with six antimicrobial agents. AgNPs alone proved to be potent antibacterial agents and in combination they enhanced the antibacterial activity and spectrum of antibacterials used in the study against a microbiologically diverse battery of Gram positive, Gram negative and multidrug‐resistant bacteria. AgNPs impregnated on the wound dressings established their antibacterial activity by significantly reducing the bacterial load of pathogenic bacteria like Staphylococcus aureus and Bacillus subtilis e stablishing potential as effective antimicrobial wound dressings for treatment of polymicrobial wound infections. This study presents the first report on the potential of biosynthesis of AgNPs from the under explored class of coprophilous fungi. Their promise to be used in wound dressings and as potent antibacterials alone and in combination is evaluated

1 Introduction

Metallic nanoparticles have amassed interest in the field of nanotechnology owing to their distinct properties‐physical, chemical and biological. Nanoparticles have been extensively investigated for biomedical applications in therapeutic areas of oncology, infectious diseases, drug delivery, imaging and biosensors [1, 2, 3]. Amongst metallic nanoparticles, the nanoparticles of noble metals have provoked particular interest due to their distinguishing surface plasmon resonance (SPR). SPR relates to the physical property of elements generated due to the cumulative oscillation of the free electrons caused by incident light. In case of metallic nanoparticles, this resonance is pronounced in the visible and UV parts of the spectrum allowing metallic nanoparticles to be optically differentiated from the surrounding tissues making them ideal candidates for imaging and guided chemotherapy [3]. Gold nanoparticles (AuNPs) conjugated with tumour necrosis factor‐α (TNFα) have demonstrated higher photo thermal efficacy when compared with the use of laser monotherapy or combination of laser therapy with conventional TNF formulation in murine carcinoma model. This foundational study has been substantiated by progression of TNFα conjugated gold nanoparticles to phase II clinical trials, thereby opening up new avenues for TNFα therapy in oncology [4]. Surface functionalised platinum nanoparticles (PtNPs) are being developed to augment their innate anticancer potential [5]. PtNPs are also under exploratory research for treatment of inflammatory conditions such as chronic obstructive pulmonary disease and UV‐induced inflammation on account of their antioxidant property [6, 7].

Silver nanoparticles (AgNPs) have generated a great deal of interest as antimicrobial agents due to their broad antibacterial spectrum and multiple mechanisms of antibacterial action. Emergence of multidrug‐resistant microorganisms has led to increased morbidity as well as mortality in the area of infectious diseases or debilitating conditions like diabetes. Newer antimicrobial approaches are also required to address rising number of clinical cases of poly microbial infections associated with chronic diabetic ulcers, burn wounds, nosocomial wound infections and chronic non‐healing ulcers of the foot. Although associated with different etiopathologies, all the aforementioned conditions are associated with compromised immune system, impaired circulation and the presence of antimicrobial inactivating mechanisms such as exudates and biofilms. Thus, antimicrobial agents which are effective in controlling infection of the above‐mentioned types and are competent enough to prevent development of multidrug resistance are the need of the hour. In the current clinical scenario, AgNPs are emerging as an effective alternate strategy to overcome drug resistance in microorganisms. Synergistic activity of AgNPs with several anti‐infective agents has been reported [8, 9]. AgNPs are used not only to coat medical devices like prostheses and catheters but are also incorporated in commercially available wound dressings like Acticoat and Allevyn Ag for chronic wound conditions. AgNP incorporation on hydrogel dressings such as chitin membranes [10] and poly(vinyl alcohol)/poly(N ‐vinyl pyrrolidone) [11] have demonstrated efficient activity against wound pathogens like Pseudomonas and Staphylococcus aureus. AgNPS have demonstrated antimicrobial activity in the exudate rich micro‐environments well as have biofilm disrupting capabilities [12]. In addition, due to its multimodal antimicrobial effects, development of resistance to Ag is difficult with limited literature reports to that effect. Additional biological activities of AgNPs such as their anti‐angiogenic and anti‐inflammatory potential in a mouse model of postoperative hypertrophic scarring could be beneficial in several clinical settings like burn wounds and post‐surgical infections which are associated with morbid scarring contractures [13].

AgNPs can be synthesised by numerous chemical and biological means. Alternatives to chemical synthesis are being explored as it is expensive and involves the usage of hazardous chemicals such as sodium borohydride, hydroxyl amines, organic solvents like chloroform and harsh treatment conditions such as high pressure and temperature [14]. In order to achieve economy of scale up and limit the use of toxic solvents microbial biosynthesis of AgNPs is gaining importance as an environmentally friendly method of AgNP synthesis [15]. Biological synthesis has been reported from plants such as geranium leaf extract [16], alfalfa sprouts [17], aloevera plant extract [18], actinomycetes [19], bacteria such as Klebsiella pneumoniae [20], S. aureus [21], Lactic acid bacteria [22], and fungi such as Penicillium sp. [23], and Fusarium sp. [24]. Amongst the options available for biosynthesis of AgNPs, fungi are increasingly being sought after owing to the ease of separating the mycelia thereby achieving cell‐free synthesis of nanoparticles [25, 26]. Biological synthesis could be either extracellular or intracellular depending upon the nature of the fungi. Extracellular method of synthesis is favoured over the latter due to convenience of separation of nanoparticles and conducive downstream processing [27, 28]. Extracellular synthesis of AgNPs by Trichophyton species of dermatophytes [29], Trichoderma reesei [26], Penicillium sp. [23], Aspergillus flavus [30] and Fusarium oxysporum [31] have been reported previously. However, some of these fungi are known to be pathogenic to humans making their large‐scale production and applications a matter of medical concern [32, 33].

Coprophilous fungi as the name suggests are ‘dung loving’ fungi and play a major contribution in the breakdown of animal dung. The dung environment is complex with several interactions between the different organisms such as bacteria, mites, flies and the weather conditions that influence the germination and fruiting of these fungi. Literature reports on coprophilous fungi have been relatively sparse leaving a vast scope of research on this lineage. Known to be non‐pathogenic with thicker cell walls, newer taxons such as Thielavia gigaspora sp. nov. have been reported in this class to be Thermotolerant species [34]

Our study has used the coprophilous fungus PM0651419 which has been identified to be Lepidosphaeria species which produces an anti‐inflammatory compound – mutolide. AgNP synthesis from this species and its potential application as an antimicrobial agent is being attempted which has never been explored previously. This study could add to the explorative research on this unique lineage of fungi [35].

2 Experimental methods

2.1 Isolation and identification of fungus PM0651419

PM0651419 is a fungus isolated from Rajkot, a state in India, in the month of February 2006 from horse dung samples collected by the method as described by Krug et al. Potato dextrose agar (PDA) medium fortified with 50 mg/L of chloramphenicol was used for culture isolation [36, 37]. The culture was perpetuated on PDA slant tubes for further identification and production of nanoparticles. The fungus, a sterile mycelium identified based on partial sequence analysis of the internal transcribed spacer (ITS) region of rDNA using ITS‐1 and ITS‐4 primers [38]. A nucleotide to nucleotide BLAST query of the gene bank database (http://www.ncbi.nlm.nih.gov/BLAST) recovered GQ203760.1, Lepidosphaeria nicotiae as the closest match to the ITS rDNA of PM0651419 (92%) [39]. The 92% similarity score does not provide confident species‐level identification in the genus Lepidosphaeria, hence it was designated simply as a Lepidosphaeria sp.

2.2 Production of fungal biomass

The fungal isolate ofPM0651419 was aerobically cultivated in 100 mL of malt extract peptone medium comprising of malt extract (0.3 g), glucose (1 g), yeast extract (0.3 g), peptone (0.5 g) and distilled water (100 mL). The Erlenmeyer flasks were inoculated with fungal mycelia and further incubated at 25 ± 1°C under shaking at 200 rpm. Post 3 days of incubation, mycelia were removed from the culture broth by filtering contents through sterile Whatman filter paper no. 1. The biomass harvested on the filter paper was washed thrice with sterile demineralised water making the mycelia free of any media components [31]. Washed fungal biomass was maintained in 100 mL sterile demineralised water for 72 h at 25°C and170 rpm on a rotary shaker (News Brunswick Scientific Innova 44 Incubator Shaker series). After incubation, the cell‐free filtrate was collected by filtering the mycelia containing solution through sterile Whatman filter paper no. 1 for the extracellular synthesis of AgNPs. Experiments were performed in triplicates with freshly grown culture of PM0651419 for each cycle.

2.3 Synthesis of AgNPs by extracellular filtrate

The cell filtrate obtained by the above‐mentioned method was then used for bio reduction. 0.5 mM silver nitrate (AgNO₃) (Himedia Laboratories Pvt Ltd, Mumbai, India) was added to 50 mL of the cell filtrate. Formation of AgNPs in solution was characterised by a visual inspection of colour change in solution over a period of 72 h [40]. Controls consisting of cell filtrate without AgNO₃ and a negative control containing only AgNO₃ solution were also maintained. All the flasks were agitated at 170 rpm at 25°C [18]. The AgNPs thus obtained were separated by centrifugation (Remi Cooling Centrifuge C30BL) at 13,000 rpm for 10 min and the concentrated nanoparticles were washed with MilliQ water (three times). Re‐dispersion of the purified AgNPs was done by ultrasonication (Branson 3510 Ultrasonic Cleaner).

2.4 Characterisation of silver nanoparticles

AgNPs were characterised using UV–Visible Spectroscopy (UV–Vis Spectroscopy), dynamic light scattering (DLS) technique, Fourier transformed infrared spectroscopy (FTIR), X‐ray diffraction (XRD), particle size, transmission electron microscopy (TEM). Atomic absorption spectroscopy (AAS) was used to identify the presence of Ag + ions and characterise changes to the Ag + ion concentration in the cell‐free extract with respect to time.

2.4.1 UV–Vis spectroscopy

Sample was withdrawn after colour change at 72 h for recording of the UV–Vis spectra of the nanoparticles produced. The UV–Vis spectrum was recorded in the 200–600 nm range at regular intervals to monitor the change in absorption (Jasco V‐630 spectrophotometer)

2.4.2 DLS

Hydrodynamic particle size measurements were carried out by DLS technique. The particle size of a 2 mL sample placed in a cuvette was estimated by laser diffraction (Delsa Nano C Particle Analyzer, Beckman Coulter, USA). Polydispersity index was studied for evaluating the narrow size distribution of the particles. The particle size reported is an average of three independent experiments and is expressed as mean ± SD.

2.4.3 FTIR spectroscopy

FTIR spectroscopy analysis was performed on the aqueous solution of AgNPs and the spectra were recorded in the 4000–600 cm⁻¹ range using an Infrared Spectrophotometer (Perkin Elmer Fourier Transform Infrared Spectrophotometer).

2.4.4 XRD

The vacuum dried AgNPs were subjected to powder XRD analysis (Bruker D8 ADVANCE). For XRD analysis, the AgNPs obtained from cell‐free filtrate were subjected to centrifugation, washed and then resuspended in ethanol. The ethanol‐washed AgNPs were dried at 45°C on a rotary evaporator. The dried mixture thus collected was used for the determination of formation of AgNPs.

2.4.5 TEM

TEM analysis was performed on FEI Technai G ² 12 BioTwin (FEI Company, Eindhoven, the Netherlands). For TEM, the sample preparation was done by drop‐coating the AgNP solution on a carbon‐coated grid and dried under infrared radiation.

2.4.6 AAS

AAS was also performed to determine the concentration of AgNPs (GBC Scientific Equipment Pty Ltd).

2.5 Effect of the combination of AgNPs with antibiotics on bacterial susceptibility

2.5.1 Bacterial strains

The bacterial strains Pseudomonas aeruginosa ATCC 9027, Pseudomonas aeruginosa M‐35, Escherichia coli ATCC 8736, Staphylococcus aureus 209 P, Staphylococcus aureus ATCC 6338, MRSA E‐710, VRE 323, Staphylococcus epidermidis 32965, Proteus vulgaris spr 6 and Klebsiella pneumoniae 1522E were utilised for evaluating bacterial susceptibility. These bacteria were cultivated in tryptic soya broth (TSB) media (Himedia Laboratories Pvt Ltd, Mumbai, India) for 24 h before the experiment and by using the pour plate technique were seeded in agar plates.

2.5.2 Antibiotics

Antibiotics were selected based on their different modes of action and their widespread use in the anti‐infective therapeutic area. Vancomycin (glycopeptides), Gentamycin (aminoglycoside) (Himedia), Amikacin (aminoglycoside and less susceptible to inactivating enzymes), Amoxicillin (β‐lactam antibiotic), Levofloxacin (fluoroquinolone) (Sigma‐Aldrich, St. Louis, MO, USA) and Linezolid (oxazolidinone) (Symed Labs Limited, Andhra Pradesh, India) were evaluated in the bacterial susceptibility assay.

2.5.3 Antibiotic preparation for assay

The antibiotics were prepared (in a mixture of water and methanol) to obtain the following concentrations‐:Vancomycin – 30 µg/mL, Amikacin – 30 µg/mL, Gentamycin ‐ 100 µg/mL, Linezolid – 30 µg/mLAmoxicillin – 30 µg/mL, Levofloxacin – 10 µg/mL

2.5.4 Antibacterial assay

The antibacterial activity was assessed against ten test cultures by well‐diffusion method. The test cultures were grown overnight in tryptic soy broth (TSB) with incubation at 37°C and OD₅₆₀ was adjusted to 1 for the assay. The Tryptic soya agar plates were prepared by seeding 15 µL of culture in 40 mL of the agar. Parallel cavities were made at equal distances on the plate using a cork borer (6 mm diameter). Wells were filled with 50 µL/well of the antibiotic solutions and allowed to diffuse. Subsequently, the AgNPs solution was filled in one set of wells, allowed to diffuse and incubated at 37°C for 24 h.

Antibacterial activity was recorded as the diameter of the zone of inhibition measured in millimetres (Kirby Bauer method) [41]. The assays were performed in triplicates. Data was obtained from three independent biological experiments and reported as mean ± SD.

2.6 Statistical analysis

Statistical differences were determined using t ‐test. A P ‐value of <0.05 was considered statistically significant.

2.6.1 Textile impregnation of AgNPs

Gauze fabrics were cut (5 cm × 5 cm), sterilised and dried before impregnation. They were then impregnated with AgNPs by incubating them in 50 mL of 17.27 ppm of AgNP solution in an Erlenmeyer flask (250 mL capacity), shaking at 170 rpm for 72 h. Post AgNP impregnation, the gauze fabrics were dried at 55°C and the percentage of AgNPs incorporated in them was measured by SEM (JSM‐6360; JEOL, Tokyo, Japan).

2.6.2 Antibacterial effects exhibited by the AgNP impregnated fabrics

Antimicrobial activity was investigated using small gauze (about 1 cm) pieces prepared in aseptic manner [42]. Each of the gauze pieces was subjected to pretreatment with 800 µL distilled water in sterile glass tubes for 10 min before 2.2 mL of TSB was added to them. Eight microliters of S. aureus and B. subtilis suspensions (about 10⁷ CFU/mL) were then added to the tubes and placed on shaker at 200 rpm. Experimental controls consisted of broths with and without bacteria. Ten microliters of broth was drawn at the end of 24 h incubation and serially diluted to spread on to the plates in duplicates. The plates were incubated at 37°C and bacterial counts were performed after 24 h. The bacteriostatic activity was calculated by estimating the percentage reduction of bacterial count by the following equation [40]:

$$R\left(\text{\%}\right)=\frac{\left(A-B\right)}{A}\times 100$$

where R is the the reduction rate, A is the the number of bacterial colonies from untreated fabrics and B is the the numbers of bacterial colonies from treated fabrics [40].

3 Results

3.1 Synthesis and characterisation of AgNPs using PM0651419 culture supernatant

After addition of aqueous AgNO₃ (0.5 mM), the mycelia‐free cell filtrate demonstrated a gradual colour change with time at room temperature, from yellow to light red and finally turned reddish brown on incubation at room temperature for 72 h (Fig. 1). The presentation of a colour change in solution from colourless to reddish brown is suggestive of the formation of AgNPs on account of the extracellular reduction facilitated by reducing agents released in the cell‐free filtrate by the fungus [43].

Fig. 1.

Extracellular synthesis of AgNPs. cell‐free filtrate of PM0651419

(a) Without AgNO₃, and (b) With 0.5 mM AgNO₃

The control sets of cell‐free filtrate without AgNO₃ and AgNO₃ only in MilliQ water demonstrated no colour change under the same experimental conditions.

3.2 UV–Vis spectrophotometry

Spectral analysis of the suspension of AgNPs was conducted with UV–Vis spectrophotometer at 24, 72, 96 h post addition of AgNO₃. The suspension exhibited a single absorbance peak and maximal OD in the range of 410–422 nm at the end of 72 h. This range is characteristic for AgNPs and is accredited to the SPR of AgNPs (Fig. 2) [44].

Fig. 2.

UV–vis spectroscopy. Absorption spectrum of cell‐free filtrate with AgNO₃ (0.5 mM) with SPR peak in the 410–422 nm range

The observation of a single SPR band is suggestive of generation of spherical metal nanoparticles. A gradual increase in O.D. with time indicates time dependent increase in concentration of AgNPs formed [45]. After 72 h absorbance showed no increase which could be due to saturation of solution with the nanoparticles thus formed [46]. Absence of secondary peaks accompanied by an increase in peak intensity is indicative of formation of a homogenous suspension devoid of particle aggregation [28]. These results have been substantiated with the DLS results which demonstrated a unimodal and narrow particle size distribution curve for the biosynthesised AgNPs. The spherical nature of the particles was also substantiated by the TEM analysis of the sample.

3.3 DLS analysis

The biosynthesised AgNPs had an average particle size of 72.6 ± 0.47 nm with >90% of particles having size <85 nm as measured by DLS. The AgNPs formed by the optimised process parameters exhibited unimodal distribution with polydispersity index of <0.2 indicating generation of homogeneous suspension.

3.4 FTIR

The FTIR measurements of the liquid sample of AgNPs exhibited eight peaks at 3662.76, 3362.62, 3339.55, 3252.64, 1856.20, 1134.74, 614 and 530 cm⁻¹ (Fig. 3). Of these, the peaks at 3339.55 and 3362.62 cm⁻¹ are attributed to primary amines and the peak at 1134.74 cm⁻¹ can be assigned to the C–N stretching vibrations of aliphatic amines suggestive of the presence of proteins associated with nanoparticles [42]. Other peaks at 614 and 530 cm⁻¹ are indicative of C–Cl and C–Br stretch. The strong broad peak at 3662.76 and 3252.64 cm⁻¹ correspond to the –OH stretch and C–H stretching vibrations, respectively. The protein coatings or proteins associated with nanoparticles have been previously reported and hypothesised to result in stabilisation of AgNPs in suspension and could be responsible for the peaks recorded in FTIR [47].

Fig. 3.

FTIR profile of AgNPs synthesised by mycelia‐free cell filtrate

3.5 XRD

The AgNPs synthesised exhibited four peaks in the XRD spectrum at 2θ values of 38.17, 44.3, 64.5 and 77.4 corresponding to (111), (200), (220) and (311) in the 2θ values spectrum ranging from 3° to 80° as seen in Fig. 4. In comparison with the standard (JCPDS file no 04‐0783), crystalline AgNPs formed was confirmed [48].

Fig. 4.

XRD profile of AgNPs synthesised by mycelia‐free cell filtrate 2θ values 38.17, 44.3, 64.5 and 77.4 corresponding to (111), (200), (220) and (311)

3.6 TEM

TEM analysis was performed to confirm homogeneity and shape of AgNPs in suspension which revealed the presence of spherical particles in the size range of 60–80 nm as seen in Figs. 5 a and b. The particles were dispersed in solution and exhibited little agglomeration which is in correlation with the UV‐Visible and particle size data.

Fig. 5.

TEM images of the AgNPs

(a,b) TEM images of AgNPs (c) Energy dispersive spectroscopy spectrum

3.7 Energy dispersive spectroscopy (EDS) analysis

EDS displayed sharp signals which confirmed the presence of elemental silver (Fig. 5 c). The peaks observed in the range of 3–4 keV is typical for metallic AgNP [49].

3.8 Atomic absorption spectroscopy

AAS was performed for quantitative analysis of Ag content in the AgNP solution. AAS analysis indicated the presence of 17.27 µg/mL of elemental silver in AgNP suspension. The calibration curve for AAS was established using 3, 4, 5, 6, 7 and 10 ppm of AgNO₃ [47].

3.9 Antibacterial activity of AgNPs and enhancement in activity seen in conjunction with antibiotics

The synergism of AgNPs with antimicrobials was dependent on the gram nature of the bacteria and the class of antibiotics used (Table 1). While an increase in fold area of the zones of inhibition was observed for most antibiotics in combination with AgNPs, the highest fold change was observed with Linezolid + AgNPs against Proteus vulgaris (nine fold increase) followed by eight fold increase with Vancomycin + AgNPs and Amikacin + AgNPs against Proteus vulgaris followed by a seven fold increase by Gentamycin + AgNPs against S. aureus 209P. Amikacin + AgNPs and Gentamycin + AgNPs demonstrated six fold increase against ESS 2231 and S. aureus 209P. Vancomycin + AgNPs and Linezolid + AgNPs demonstrated five fold increase in inhibition against K. pneumoniae while Gentamycin + AgNPs demonstrated five fold increase in inhibition against P. aeruginosa M‐35. A four fold increase in activity was seen with Gentamycin + AgNPs against MRSA, VRE, Amikacin + AgNPs against S. aureus ATCC6338, MRSA, K. pneumoniae, P. aeruginosa M‐35 and Levofloxacin + AgNPs also demonstrated a four fold increase against Proteus vulgaris. A three fold increase in activity was demonstrated by several antibiotic + AgNP combinations such as Vancomycin + AgNPs against S. aureus ATCC 6338, S. epidermidis, Amikacin + AgNPs against P. aeruginosa ATCC 9027, VRE, Gentamycin + AgNPs against Proteus vulgaris, Amoxicillin + AgNPs and Linezolid + AgNPs against P. aeruginosa ATCC9027. The most pronounced increase in antimicrobial activity of AgNPs was observed in combination with Vancomycin, Amikacin and Gentamycin as recorded by the increase in fold area of inhibition and spectrum of antimicrobial activity.

Table 1.

Antibacterial efficacy of antibiotics alone and in combination with AgNPs. Instances where bacterial inhibition zone is absent, the cork borer diameter (6 mm) is used to calculate the fold increase; increase in fold area = (b ² –a ²)/a ². Significance of data in each group (antibiotic versus antibiotic +AgNPs) has been analysed using t ‐test.

Sample details	S. aureus 209P	S. aureus ATCC 6338	ESS 2231	E. coli ATCC 8739	P. aeruginosa ATCC 9027	P. aeruginosa M‐35	K. pneumoniae 1522E	S. epidermitis 32965	P. vulgaris spr‐6	MRSA E‐710	VRE 323	Average	P value
Zone of inhibition(mm) of various antibiotic combinations with AgNPs (average ± standard deviation)
Vancomycin(a)	13 ± 0.57	7 ± 0	12 ± 0.70	9 ± 0	—	14 ± 0.57	—	—	—	12 ± 0.57	11 ± 0	11.14	P ***
AgNPs	15 ± 0.57	15 ± 0.57	15 ± 0.57	16 ± 0.57	12 ± 0.57	15 ± 0.57	15 ± 0.57	10 ± 0.57	17 ± 0.57	13 ± 0.57	12 ± 0.57	14.09
Vancomycin + AgNPs(b)	14 ± 0.57	15 ± 0.57	14 ± 0.70	16 ± 0	12 ± 0.57	15 ± 0.57	15 ± 0.57	12 ± 1.5	18 ± 0.57	14 ± 0.57	12 ± 0	14.27
increase in fold area	0.16	3.6	0.36	2.16	3	0.14	5.25	3	8	0.36	0.19
Amikacin	—	7 ± 0	—	9 ± 0.7	—	—	7	—	—	—	—	7.66	P ***
AgNPs	15 ± 0.57	15 ± 0.57	15 ± 0.57	16 ± 0.57	12 ± 0.57	15 ± 0.57	15 ± 0.57	10 ± 0.57	17 ± 0.57	13 ± 0.57	12 ± 0.57	14.09
Amikacin + AgNPs	16 ± 1.5	17 ± 1	16 ± 1.4	16 ± 0.57	13 ± 0	14 ± 0.57	16 ± 0.57	10 ± 0	18 ± 1	14 ± 0.57	12 ± 0.57	14.73
increase in fold area	6.11	4.89	6.11	2.16	3.69	4.44	4.22	1.77	8	4.44	3
Gentamycin	—	10 ± 0.57	—	10 ± 0.57	9 ± 0.57	—	11 ± 0.57	18 ± 0	9 ± 0.7	—	—	11.16	P ***
AgNPs	15 ± 0.57	15 ± 0.57	15 ± 0.57	16 ± 0.57	12 ± 0.57	15 ± 0.57	15 ± 0.57	10 ± 0.57	17 ± 0.57	13 ± 0.57	12 ± 0.57	14.09
Gentamycin + AgNPs	17 ± 1.5	16 ± 0	16 ± 0.7	16 ± 0	15 ± 0.57	15 ± 0	17 ± 0.57	18 ± 0.57	18 ± 0.57	14 ± 0.57	13 ± 0.57	15.9
increase in fold area	7.02	1.56	6.11	1.56	1.77	5.25	1.38	0	3	4.44	4.52
Amoxicillin	22 ± 0.57	15 ± 0.57	21 ± 0.7	14 ± 1	—	24 ± 0.57	15 ± 0.57	15 ± 1	11 ± 0.57	20 ± 1.15	25 ± 0.57	18.2	ns
AgNPs	15 ± 0.57	15 ± 0.57	15 ± 0.57	16 ± 0.57	12 ± 0.57	15 ± 0.57	15 ± 0.57	10 ± 0.57	17 ± 0.57	13 ± 0.57	12 ± 0.57	14.09
Amoxicillin + AgNPs	22 ± 0.57	17 ± 1	21 ± 0.7	17 ± 0.57	12 ± 0	25 ± 0	18 ± 0.57	15 ± 0.57	18 ± 0.57	21 ± 0	26 ± 0.57	19.27
Increase in fold area	0	0.28	0	0.47	3	0.08	0.44	0	1.67	0.1	0.08
Levofloxacin	15 ± 0	25 ± 1.15	15 ± 0	24 ± 0.57	23 ± 0.57	15 ± 0.57	22 ± 0	17 ± 0.57	13 ± 0.57	14 ± 0.57	17 ± 1	18.18	ns
AgNPs	15 ± 0.57	15 ± 0.57	15 ± 0.57	16 ± 0.57	12 ± 0.57	15 ± 0.57	15 ± 0.57	10 ± 0.57	17 ± 0.57	13 ± 0.57	12 ± 0.57	14.09
Levofloxacin + AgNPs	16 ± 0.57	27 ± 0.57	15 ± 0	26 ± 0.57	24 ± 1.15	16 ± 0	24 ± 0.57	18 ± 0	30 ± 0.57	15 ± 0.57	19 ± 1.15	20.9
increase in fold area	0.13	0.166	0	0.17	0.08	0.13	0.19	0.08	4.32	0.14	0.25
Linezolid	23 ± 1.15	10 ± 0	23 ± 0.7	10 ± 0	—	25 ± 2.88	—	30 ± 0	—	21 ± 0.57	25 ± 1.15	20.87	ns
AgNPs	15 ± 0.57	15 ± 0.57	15 ± 0.57	16 ± 0.57	12 ± 0.57	15 ± 0.57	15 ± 0.57	10 ± 0.57	17 ± 0.57	13 ± 0.57	12 ± 0.57	14.09
Linezolid + AgNPs	23 ± 0.57	17 ± 1	25 ± 0	17 ± 0.57	12 ± 0	30 ± 0	16 ± 2.08	30 ± 0	19 ± 0.57	22 ± 0.57	25 ± 0	21.45
increase in fold area	0	1.89	0.18	1.89	3	0.44	5.25	0	9.02	0.08	0

AgNPs, silver nanoparticles; P ***, significance; ns, no significance.

Singularly, Amikacin, Vancomycin and Gentamycin displayed limited spectrum of bacterial inhibition with zone sizes <10 mm diameter and were inactive against S. aureus 209P, ESS 2231, P. aeruginosa M‐35, MRSA, VRE (amikacin and gentamycin), P. aeruginosa ATCC 9027, S. epidermidis, P.vulgaris (vancomycin and amikacin) and K. pneumoniae (vancomycin). However, the combination of AgNPs with amikacin, vancomycin and gentamycin demonstrated significant improvement in broadening the spectrum of antimicrobial action and in increasing the zones of inhibition to 10 mm and above (Table 1). Among all the test cultures, significant improvement in antimicrobial activity profile was seen for the combination of Vancomycin with AgNPs, Amikacin + AgNPs and gentamycin + AgNPs. The antimicrobial activity of amoxicillin demonstrated marginal improvement in combination with AgNPs. Antimicrobial activity of Levofloxacin was not significantly enhanced against any of the test cultures when combined with AgNPs. Broadening of antimicrobial spectrum was obtained on combining AgNPs with Linezolid as observed in Fig. 6.

Fig. 6.

Efficacy of antibiotics alone and in combination with AgNPs. VA, Vancomycin; AK, Amikacin; GM, Gentamycin; AM, Amoxicillin; LE, Levofloxacin; LI, Linezolid

(a) E. coli ATCC 8739, (b) K. pneumoniae, (c) MRSA, (d) P. vulgaris, (e) P. aeruginosa, (f) S. aureus.

AgNPs alone displayed activity against all test cultures with zone sizes of 10 mm and above indicating their inherent potential as an antibacterial agent. With cultures like S. epidermidis, P. aeruginosa and VRE, which are difficult to inhibit by most antibiotics, AgNPs have shown promise in not only inhibiting them alone but in combination, have proven to improve the activity of the conventional antibiotics. This finding could open new channels in the therapeutic approaches and research against such resistant organisms.

3.10 Textile impregnation of AgNPs

The textile (gauze) impregnated with AgNPs was analysed using SEM to determine incorporation of AgNPs onto the fabric (Fig. 7).

Fig. 7.

SEM images of the gauze surface. SEM, scanning electron microscopy

(a) Untreated fabric, (b) Silver nanoparticle impregnated fabric

3.11 Antibacterial activity of the AgNPs impregnated fabrics

The AgNP impregnated fabric pieces exhibited bacteriostatic activity against Bacillus subtilis and S. aureus cultures and were able to reduce the bacterial load substantially when compared with the untreated gauze pieces (10⁷ CFU/mL). The gauze pieces impregnated with AgNPs demonstrated inhibition of 87.14% against B. subtilis and 87.3% inhibition against S. aureus when compared with the untreated controls.

These results further prove that the AgNPs generated could be used in wound dressings to prevent development of recalcitrant wound infections.

4 Discussion

The present study was devised to evaluate the potential of PM0651419, a fungus belonging to an unexplored group of fungi called coprophilous fungi to biosynthesize AgNPs using a cell‐free extract. Coprophilous fungi have been known in the past to be producers of active metabolites such as Ovalicin [50], Tulasnein [51], Flutimide [52], Australifungin [53] and Sordarins [54] and are able to perpetuate in the hostile environment of the mammalian digestive system as well as dung in the presence of competing bacteria and protists. Even though their non‐pathogenic nature makes them promising candidates in terms of scale up feasibility, their potential has not been exploited for the biosynthesis of AgNPs. In addition to exploring biosynthesis with PM0651419, the present study has investigated the effectiveness of the synthesised AgNPs to improve antibacterial spectrum and activity of commonly used antibiotics. Further, this study has evaluated the efficiency of AgNPs as antimicrobial agents when incorporated in wound dressings.

AgNP biosynthesis was optimised by varying concentrations of AgNO₃ from 1 to 0.5 mM. AgNPs in the size range of 60–80 nm were generated at 0.5 mM AgNO₃ indicated by colour change of the cell‐free filtrate from yellow to reddish brown and the UV–Vis peak at 410–422 nm. The single UV–Vis peak formed suggested formation of spherical metallic nanoparticles formed in the range of 2–100 nm and correlates with the specific SPR seen with AgNPs. These findings were confirmed by DLS and TEM which demonstrated the presence of spherical particles in the size range of 60–80 nm.

The synthesis of AgNPs made of Ag^o from Ag⁺ (AgNO₃) in the cell‐free filtrate has been attributed to presence of metabolically active enzymes such as nitrate reductase and phytochelatin synthase in the extracellular cell‐free filtrate [55]. Critical to the secondary metabolite production in fungi is the abundance of the enzymatic machinery in the cell which would be specific for fungal species under evaluation and the experimental conditions. This will also have a bearing on the rate and amount of synthesis of nanoparticles making some fast AgNP producers while some slow AgNP producers. In this study, the fast production of AgNPs could be attributed to the fact that secondary metabolite production is found with filamentous fungi exclusively like the basidiomycetes which are found in abundant members in the coprophilous community of fungi. In this study, the fungal machinery could carry out the biotransformation at 0.5 mM of AgNO₃, thus strengthening our belief on their biotransformation potential [35]

The crystalline nature of biosynthesised AgNPs in this study using coprophilous PM0651419 fungus is comparable to reported literature on extracellular biosynthesis of AgNPs like the ones obtained from Fusarium oxysporum [54] and Penicillium brevicompactum [46]. Silver has been used since historical times as silver nitrate and silver sulfadiazine in the treatment of burns and infections [55]. Ag‐based antibiotics have demonstrated broad spectrum of antibacterial as well as anti‐fungal activity. Due to the multiple mechanisms of antibacterial activity and their inhibitory effects on biofilms often responsible for recalcitrant wound infections, AgNPs are known to circumvent development of multi drug resistance (MDR) in comparison to conventional chemical antimicrobials.

Several reports suggest that AgNPs exert their antimicrobial action in several ways by lodging themselves onto microbial cell membranes and then penetrating the cell, thus making the cell leaky incapable of repairing [56]. Currently on account of their broad spectrum antimicrobial activity, silver salts like silver nitrate and silver sulfadiazine are used for treatment of several wound infections; however, the superior antimicrobial activity of Ag is limited by insufficient release of silver and larger particle size lowering surface area to volume ratio, thus decreasing the overall exposure of Ag to the wound bed.

Unlike Ag salts, AgNPs are not associated with interfering effects of salts and owing to their nanosize, they offer higher surface area to volume ratio and continued release in the form of Ag⁺ ions in the wound microenvironment at concentrations that are toxic to the microorganisms but not the host tissue.

In the current study, it is observed that the AgNPs exhibited broad spectrum antibacterial activity against Gram negative as well as Gram positive test cultures. However, the most significant improvement in antimicrobial activity was observed on combining AgNPs with protein‐synthesis inhibitors such as gentamycin, amikacin (aminoglycosides) and vancomycin (glycopeptide) where an enhancement in the fold area of inhibition was seen against all test cultures evaluated. Similar findings have also been reported with AgNPs synthesised from Acinetobacter calcoaceticus wherein maximum inhibition against E. aerogenes with 3.8 fold increase was observed due to the synergistic effect of combining vancomycin with AgNPs [28].

Enhancement of antimicrobial efficacies of vancomycin, gentamycin, kanamycin, streptomycin with AgNPs synthesised from Fusarium oxysporum has also been demonstrated [31]. The major difference between the current study and the reported literature has been the greater fold area change observed in the current study. This difference in fold area could be explained by differences in the evaluating assays. The literature reports have evaluated the antimicrobial efficacy using the disc diffusion method while the current study utilised the well‐diffusion assay. In contrast to the filter paper disc method, the particulate matter present if any in the sample could be expected to interfere less with the diffusion of antimicrobial substance into the agar. The filter paper disc contains many free hydroxyl groups which render the surface of the disc hydrophilic, which could result in adsorption of cationic agents like Ag + ions to the exterior of the disc limiting free diffusion into the medium efficiently [57].

Apart from synergistic activity, the AgNPs expanded the spectrum of antimicrobial activity for Amikacin, Vancomycin, Gentamycin, Linezolid for Gram positive organisms such as S. aureus 209P, S. aureus ATCC 6338, MRSA, S.epidermidis and Gram negative organisms like Escherichia coli super sensitive strain (ESS 2231), E. coli, P. aeruginosa M‐35, P. aeruginosa ATCC 9027, K. pneumoniae, P. vulgaris and VRE.

Amongst all the drugs evaluated, AgNPs had most pronounced effect on aminoglycosides group of antibiotics. This could be due to ability of AgNPs to introduce permeable pits in the bacterial membrane improving penetration of these compounds most of which are protein‐synthesis inhibitors requiring access to their intracellular target on the ribosomes for efficient action.

Amongst the panel of microorganisms, AgNPs demonstrated maximal inhibitory effect on Proteus vulgaris either alone or in combination with other antimicrobial agents evaluated. Proteus vulgaris is an opportunistic pathogen to humans often implicated in urinary tract infections post instrumentation as well as chronic wound infections. One application of AgNPs from the current study would be to find use in bladder wash either alone or in combination with another antimicrobial as a prophylactic measure in case of patients requiring frequent urethral instrumentation.

Silver as an antimicrobial agent is an important component of wound dressings. Wound infections are often polymicrobial in nature. Chronic wound infections like diabetic ulcers are predominantly infected with gram positive cocci such as the biofilm forming S. aureus while burn wounds and post‐surgical wounds are dominated by gram negative rods like Pseudomonas aeruginosa. Hence to address the diversity of microbial infections, antimicrobials with wide spectrum of activity are desired such as AgNPs. Towards that effort, in this study we assessed the antimicrobial ability of AgNP impregnated fabric against S. aureus and B. subtilis. B. subtilis is a common soil bacterium and a gut commensal but causes diseases in severely immunocompromised conditions such as burns, HIV and diabetes.

This study has successfully demonstrated that gauze fabric impregnated with AgNPs was capable of reducing the bacterial load of about 10⁷ CFU/mL by about 87% in case of S. aureus and B. subtilis elaborating the antibacterial potential exhibited by the AgNPs impregnated onto the fabric gauze. This could prove to be of great application and importance in therapeutic areas like burn wounds which are often confounded by other complications like constant exudate release and infections leading to sepsis. This study reports AgNPs of 60–80 nm which are of great significance in the treatment of such wounds as this size facilitates activity on the skin and is able to counter the superinfecting pathogens without entering any systemic circulation that could lead to toxicity thereby ensuring specificity in action and sustained activity. Moreover, the AgNPs could damage the bacterial cell wall increasing the uptake of antibiotics thereby decreasing the development of resistance and accelerating the recovery process. In addition to these studies, several studies reported in literature have demonstrated the ability of AgNPs to limit biofilm formations and enhance wound healing due to their anti‐inflammatory action in animal models of wound infections [24].

Biosynthesis of AgNPs using coprophilous fungi is a novel and innovative attempt to tap the unexplored potential of the vast microbial nanofactories in nature. AgNPs generated from this repository were not only potent antibacterials alone but could enhance the action of other antibacterials in combination. They also proved to be promising as wound dressings that could be potential additions to the current standards of wound care thus accelerating recovery and due to their potent activity against P. vulgaris, AgNPs can be constituted in bladder washes to improve local microbial load in debilitated individuals requiring long‐term medical interventions. Their antimicrobial potential also makes them promising to be used with medical devices and implants like catheters as coatings to prevent infections. Most wound conditions are vulnerable to infections caused by drug resistant and bacteria and those capable of forming biofilms. Such infections have a significant impact on recovery and can contribute to extended stay of the patients at medical facilities. The use of AgNP‐coated devices could offer superior care in such conditions accelerating recovery and possible prevention of super infections [58, 59].

5 Conclusion

In this report, it is demonstrated that the coprophilous fungus PM0651419 is amenable to extracellular biosynthesis of AgNPs. The monodisperse and crystalline AgNPs thus synthesised demonstrated particle size in the range of 60–80 nm. The AgNPs produced displayed synergistic activity with microbial protein‐synthesis inhibitors like Vancomycin, Gentamycin, Amikacin, Linezolid and had most pronounced effect on P. vulgaris. The AgNPs were amenable to fabric impregnation and demonstrated significant antimicrobial activity against S. aureus and B. subtilis. The usage of AgNP coated wound dressings opens up an entirely new area for wound management. Several steps in that direction including use of several moisturising membranes such as AgNP impregnated chitosan membranes, dehydrated amniotic membranes are ongoing. Exploration of using AgNPs with such membranes to promote wound healing and recovery could lay the path towards improved wound management.

The ability of the biosynthesised AgNPs from PM0651419 to improve and widen the spectrum of antimicrobial activity offers immense promise and potential for their use in AgNP impregnated wound bandages for conditions such as chronic infected ulcers, burn wounds and diabetic foot infections.