Chapter 3. Antimicrobial activity of the silver nanoparticles. biosynthesized using the characterized. fungi

Transcription

1 Chapter 3 Antimicrobial activity of the silver nanoparticles biosynthesized using the characterized fungi

2 INTRODUCTION Excessive use of the conventional antibiotics has led to the emergence of microbial resistance to antimicrobial drugs which has become a major public health concern particularly in developing countries (Isturiz and Carbon, 2000). This has become the biggest challenge in the treatment of infectious diseases. Resistance is most often based on evolutionary processes taking place during, for example, antibiotic therapy, and leads to inheritable resistance. In addition, horizontal gene transfer by conjugation, transduction or transformation can be a possible way for resistance to build up (Mazela and Davies, 1999). This has prompted the development of alternative strategies to overcome the microbial resistance. Metal nanoparticles have emerged as novel antimicrobial agents. Several classes of antimicrobial nanoparticles and nanosized carriers for antibiotics delivery have proven their effectiveness for treating infectious diseases, including antibiotic-resistant ones. The use of nanoparticles is gaining impetus in the present century as they posses defined chemical, optical and mechanical properties. Metallic nanoparticles also have a large contact area with a microorganism owing to their small sizes and higher surface area to volume ratio. This feature enhances biological and chemical activity, and therefore, renders nanoparticles higher antibacterial activity as compared to their bulk counterparts. The metallic nanoparticles are promising antimicrobial alternatives as they show enhanced antimicrobial properties, which is generated interest in the researchers due to the growing microbial resistance against antibiotics and the development of resistant strains (Gong et al., 2007). Various nanomaterials like copper, zinc, titanium, magnesium, gold, alginate and silver have been developed (Gu et al., 2003; Ahmad et al., 2005; Schabes-Retchkiman

3 et al., 2006) but silver nanoparticles have proved to be most effective as they exhibit potent antimicrobial efficacy against different bacteria, viruses and fungi (Gong et al., 2007). Silver nanoparticles are known to target a broad spectrum of Gram-negative and Gram-positive bacteria including antibiotic-resistant strains and are able to stay without microbial resistance. Most importantly silver nanoparticles are also non-toxic to the human body at low concentrations (Baker et al., 2005). Nanoparticles can disturb functions of cell membranes such as permeability and respiration. The silver nanoparticles get attached to the cell membrane and also penetrate inside the bacteria. Inside the bacterial cells silver nanoparticles can disturb the functions of sulfurcontaining proteins and phosphorus-containing compounds such as DNA by effectively reacting with them leading to the inhibition of enzyme functions (Singh et al., 2008; Gordon et al., 2010). The nanoparticles bind to proteins and DNA and damage them by inhibiting replication. They attack the respiratory chain, cell division finally leading to cell death. The nanoparticles release silver ions in the bacterial cells, which enhance their bactericidal activity (Feng et al., 2000; Sondi and Salopek-Sondi, 2004; Song et al., 2006). In addition, complex action mechanisms of metals decrease the probability of bacteria developing resistance to them (Chopra, 2007). Thus, one of the promising approaches for overcoming antibiotic resistance of microorganisms is the use of silver nanoparticles. The association of metal nanoparticles and antibiotics is a very promising area of research. Silver nonoparticles are interesting when compared with silver ions due to their larger size, which in turn, improve the capacity to react with several molecules (Li et al., 2005). This chapter presents the study on the antimicrobial

4 properties of the biosynthesized silver nanoparticles which were synthesized using selected fungal isolates obtained from the soil as well as fungi endophytic on plants. MATERIALS AND METHODS Antimicrobial assays of the biosynthesized silver nanoparticles The antimicrobial activity of the silver nanoparticles synthesized using the fungal isolates was assessed against Gram positive as well as Gram negative bacteria namely, Staphylococcus aureus MTCC96, Salmonella enterica MTCC735, Escherichia coli MTCC730 and Enterococcus faecalis MTCC2729 and also against the pathogenic fungus Candida albicans MTCC183. The assay was carried out on Mueller Hinton agar (MHA) medium by plating the test organism on the medium plates. Wells were cut on the plates using sterile cork borer and 50μL of Silver nanoparticles solution was dispensed in each well. The mycelia-free water extract alone was used as control. The plates were incubated overnight at 37 C for 24 hr and observed for the presence of zones of inhibition. Combination study on activity of silver nanoparticles with antibiotic The combined effect of extracellularly synthesized silver nanoparticles with commonly used broad spectrum antibiotic chloramphenicol (HiMedia, India) was studied to test the antimicrobial efficacy of these nanoparticles alone and in combination with the antibiotic. To determine the synergistic effect, antibiotic disk alone and disk impregnated with 20 μl of freshly prepared silver nanoparticles were separately placed onto the Muller-Hinton agar (MHA) medium inoculated with test

5 organisms. Fungal cell-free filtrate was used as negative control. After incubating at 25 C for 24 hr the plates were observed for the antimicrobial activity (zones of growth inhibition) by the antibiotic alone and in combination with silver nanoparticles. All the assays were performed in triplicates. The increase in fold area was assessed by calculating the mean surface area of the inhibition zone generated by an antibiotic alone and in combination with Silver nanoparticles. The fold area increase was calculated by the equation: (b 2 a 2 )/a 2 where a and b refer to the zones of inhibition for antibiotic alone and antibiotic with silver nanoparticles respectively (Birla et al., 2009). Concentration of the mycosynthesized silver nanoparticles The concentration of silver nanoparticles was determined by the method of Liu et al. (2007). To determine the average number of atoms per nanoparticle, the following relation was used: Where, N is the number of atoms per nanoparticles, π = 3.14, ρ is the density of face centered cubic (fcc) silver (=10.5 g/cm3), D is the average diameter of nanoparticles, M is the atomic mass of silver (= g), N A is the number of atoms per mole (Avogadro s number= ). The molar concentration of the nanoparticles solution was determined using the following formula:

6 where, C is the molar concentration of nanoparticle solution, N T is the total number of silver atoms added as AgNO 3 =1M, N is the number of atoms per nanoparticle, V is the volume of the reaction solution in L, N A is the Avogadro s number. Minimum inhibitory concentration and minimum bactericidal concentration Minimum inhibitory concentration (MIC) of biosynthesized silver nanoparticles was also measured using tube dilution method (Mayr-Harting et al., 1972). Bacterial inoculum of Staphylococcus aureus MTCC96, Salmonella enterica MTCC735, Escherichia coli MTCC730 and Enterococcus faecalis MTCC2729 as well as the fungal inoculum of Candida albicans MTCC183 were prepared by growing a single colony in nutrient broth (NB)/Potato dextrose broth (PDB) until it achieved the turbidity of the 0.5 McFarland standards resulting in approximately ~10 8 CFU/mL suspension. The concentration of the silver nanoparticles was determined using the formula described above (Liu et al., 2007) and the growth media was supplemented with different concentration of nanoparticles (1, 5, 10, 15 and 20 µm) and inoculated with standardized inoculum. Control tubes were maintained without silver nanoparticles. The MIC was determined after 24 hr of incubation at 37 C by observing the visible turbidity and measuring the optical density of these culture broths at 600 nm (Panacek et al., 2006). The Minimum bactericidal concentration (MBC) was also determined by plating the treated cells with different concentration of silver nanoparticles on fresh agar

7 plates. Standardized bacterial as well as the fungal inocula were inoculated in the growth media supplemented with different concentration of nanoparticles (1, 5, 10, 15 and 20 µm) and incubated for 24 hr at 37 C. 100 μl of the culture was then spread onto Muller-Hinton agar plates and incubated at 37 C for 24 hr. After incubation, the agar plates were observed for the growth of the test organisms. Growth kinetics of indicator organisms in presence of silver nanoparticles To study the microbial growth kinetics in presence of silver nanoparticles, indicator bacteria were grown in 100 ml of nutrient broth and the fungus was grown in potato dextrose broth medium. Growth was allowed till the optical density reached 0.1 at 600 nm (OD of 0.1 corresponds to a concentration of 10 8 CFU ml 1 of medium). Subsequently, CFU from above were inoculated to 100 ml of respective liquid media supplemented with different concentrations of silver nanoparticles (5, 10, 15, 20 µm). Control broths were used without nanoparticles and incubated at 37 C and 100 rpm. The growth rate was determined by recording the absorbance at 600 nm for 30 hr at regular time interval. Scanning Electron Microscopy analysis In order to find out and elucidate the antibacterial mechanism of silver nanoparticles, Scanning Electron Microscopy technique was used. Cells of test strains before and after treatment with nanoparticles were fixed overnight with 2.5% glutaraldehyde and drop coated on cover glass. Air dried samples were sputter coated with gold and observed under Scanning Electron Microscope, Leo 1430vp instrument.

8 RESULTS Antimicrobial activity The antimicrobial efficacy of the biosynthesized silver nanoparticles was assessed against Gram positive and Gram negative bacteria viz., Staphylococcus aureus MTCC96, Enterococcus faecalis MTCC2729, Salmonella enterica MTCC735 and Escherichia coli MTCC730 as well as the pathogenic fungus Candida albicans MTCC183 (Fig 3.1). Silver nanoparticles showed remarkable antimicrobial activity against the test strains as indicated by the inhibition zones against the test pathogenic strains (Table 3.1). The silver nanoparticles synthesized using twelve selected fungal isolates namely, Fusarium oxysporum MP5, Aspergillus niger NH6, Paecilomyces lilacinus SF1, Arthrinium sp KL1, Aspergillus fumigatus SP5, Cladosporium cladosporioides RS1, Aspergillus tamarii PFL2, Aspergillus niger PFR6, Penicillium ochrochloron PFR8, Cryptosporiopsis ericae PS4, Alternaria solani GS1 and Penicillium funiculosum GS2 showed considerable antibacterial as well as antifungal activity with the inhibition zones ranging from 10mm-19mm diameters. Among the twelve samples, the silver nanoparticles synthesized using Cryptosporiopsis ericae PS4 with the average particle size 5.5 ± 3.1nm gave the maximum antimicrobial activity against all the tested pathogenic strains viz., Staphylococcus aureus MTCC96, Enterococcus faecalis MTCC2729, Salmonella enterica MTCC735, Escherichia coli MTCC730 and Candida albicans MTCC183 with inhibition zone diameters of 14mm, 14mm, 15mm, 17mm and 19mm respectively.

9 The combined effect of the antimicrobial agent with the nanoparticles was also investigated using disc diffusion method (Fig 3.2). The diameter of inhibition zones was measured and increase in fold area for all the test bacteria and the fungus were also determined. The antibacterial activity of the broad spectrum antibiotic chloramphenicol against the tested bacterial strains increased significantly in presence of silver nanoparticles (Table 3.2 & 3.3). And also the antifungal activity of antifungal agent fluconazole was also found to have increased when combined with the biosynthesized silver nanoparticles. Therefore, such silver nanoparticles can be considered as excellent broad-spectrum antimicrobial agent and used as antimicrobial agent alone or in combination with antibiotics after further trials on experimental animals.

10 Candida albicans Escherichia coli s Enterococcus faecalis Salmonella enterica Staphylococcus aureus Fig 3.1: Antimicrobial activity (zone of inhibitions) of the biosynthesized silver nanoparticles against test organisms A A+NP A A Control A+NP NP A+NP A+NP NP A Control Fig 3.2: Antimicrobial activity (zone of inhibitions) of the biosynthesized silver nanoparticles and their combined effect with antibiotics against test organisms

11 Table 3.1: Mean zone of inhibition (in mm) produced by mycosynthesized silver nanoparticles Fungal isolates used for biosynthesis of nanoparticles Aspergillus fumigates SP5 Aspergillus niger NH6 Paecilomyces lilacinus SF1 Arthrinium sp KL1 Cladosporium cladosporioides RS1 Fusarium oxysporum MP5 Aspergillus tamarii PFL2 Aspergillus niger PFR6 Penicillium ochrochloron PFR8 Cryptosporiopsis ericae PS4 Alternaria solani GS1 Penicillium funiculosum GS2 Silver nitrate (1mM) Mycelium-free filtrate (-ve control) Escherichi a coli MTCC730 Salmonella enterica MTCC735 Zone of inhibition (mm) Enterococcu s faecalis MTCC2729 Staphylococcus aureus MTCC96 Candida albicans MTCC

12 Table 3.2: Mean zone of inhibition (in mm) produced by antimicrobial agents alone or in combination with silver nanoparticles (AgNPs) biosynthesized using soil fungi Mean zone of inhibition (in mm) Escherichia Salmonella Enterococcus Staphylococcus Candida coli enterica faecalis aureus Albicans MTCC730 MTCC735 MTCC2729 MTCC96 MTCC183 Antimicrobial agent* Antimicrobial agent* + AgNPs (MP5) Increase in fold area Antimicrobial agent* + AgNPs (SP5) Increase in fold area Antimicrobial agent * + AgNPs (SF1) Increase in fold area Antimicrobial agent * + AgNPs (KL1) Increase in fold area Antimicrobial agent* + AgNPs (NH6) Increase in fold area Antimicrobial agent* + AgNPs (RS1) Increase in fold area *Chloramphenicol is used in case of bacterial strains and fluconazole is used in case of fungal strain

13 Table 3.3: Mean zone of inhibition (in mm) produced by antimicrobial agents alone or in combination with silver nanoparticles (AgNPs) biosynthesized using endophytic fungi Mean zone of inhibition (in mm) Escherichia Salmonella Enterococcus Staphylococcus Candida coli enterica faecalis aureus Albicans MTCC730 MTCC735 MTCC2729 MTCC96 MTCC183 Anticrobial agent* Antimicrobial agent * + AgNPs (PFL2) Increase in fold area Antimicrobial agent * + AgNPs (PFR6) Increase in fold area Antimicrobial agent * + AgNPs (PFR8) Increase in fold area Antimicrobial agent * + AgNPs (PS4) Increase in fold area Antimicrobial agent * + AgNPs (GS1) Increase in fold area Antimicrobial agent* + AgNPs (GS2) Increase in fold area *Chloramphenicol is used in case of bacterial strains and fluconazole is used in case of fungal strain

14 Minimum inhibitory concentration and minimum bactericidal concentration Further antimicrobial assays were performed using the silver nanoparticles synthesized by Cryptosporiopsis ericae PS4 since it showed maximum antimicrobial activity against the test microbial strains. The MIC of the biosynthesized silver nanoparticles using Cryptosporiopsis ericae PS4 was determined using tube dilution method (Fig 3.3). MIC of the silver nanoparticles was obtained at 15µM for the Gram negative bacterial strains Escherichia coli MTCC730 and Salmonella enterica MTCC735 whereas for the Gram positive strains Staphylococcus aureus MTCC96 and Enterococcus faecalis MTCC2729, higher inhibitory concentration of 20 µm was observed. In case of the fungal strain Candida albicans MTCC183, MIC was observed at much lower concentration (10 µm). MBC of the silver nanoparticles was also determined by plating the test strains treated with different concentration of silver nanoparticles. MBC was obtained at 20µM concentration for the Gram negative bacterial strains Escherichia coli MTCC730 and Salmonella enterica MTCC735 (Fig 3.4a & b). For the Gram positive strains Staphylococcus aureus MTCC96 and Enterococcus faecalis MTCC2729, MBC were obtained at 25 µm concentrations of silver nanoparticles respectively (Fig 3.4c & 3.4d). In case of MBC of silver nanoparticles against Candida albicans MTCC183, it was found to be 20 µm (Fig 3.4e).

15 Fig 3.3: MIC of the biosynthesized silver nanoparticles against test strains Fig 3.4: MBC of the biosynthesized silver nanoparticles against a) Escherichia coli MTCC730, b) Salmonella enterica MTCC735, c) Staphylococcus aureus MTCC96, d) Enterococcus faecalis MTCC2729 and e) Candida albicans MTCC183

16 Growth kinetics of indicator strains in presence of silver nanoparticles The microbial growth curve was monitored in liquid media supplemented with different concentrations of nanoparticles. The growth curves of bacterial cells as well as the yeast cells treated with silver nanoparticles indicated inhibition of the growth and reproduction of microbial cells by silver nanoparticles. Culture without nanoparticles did not show any growth inhibition and reached stationary phase at the end of 30 hr. The bacterial culture as well as the fungal culture when treated with increasing concentration of silver nanoparticles, the exponential phase was significantly delayed compared to untreated samples leading to complete arrest of growth (Fig ). The lowest concentration of silver nanoparticles that totally arrested the growth of all four tests bacterial strains was observed at 15µM concentration (Fig ). However, upon comparison of the bacterial growth curves, the growth curves of the silver nanoparticles treated bacteria indicated a faster growth inhibition of Gram negative bacteria Escherichia coli MTCC730 and Salmonella enterica MTCC735 than of Gram positive bacteria Staphylococcus aureus MTCC96 and Enterococcus faecalis MTCC2729. While complete inhibition in the growth of the fungal strain Candida albicans MTCC183 was found at lower concentration of the silver nanoparticles i.e. 10 µm (Fig 3.9).

17 Fig 3.5: Effect of the silver nanoparticles on growth of Escherichia coli MTCC730 (values are average of three observations ± SEM) Fig 3.6: Effect of the silver nanoparticles on growth of Salmonella enterica MTCC735 (values are average of three observations ± SEM)

18 Fig 3.7: Effect of the silver nanoparticles on growth of Staphylococcus aureus MTCC96 (values are average of three observations ± SEM) Fig 3.8: Effect of the silver nanoparticles on growth of Enterococcus faecalis MTCC2729 (values are average of three observations ± SEM)

19 Fig 3.9: Effect of the silver nanoparticles on growth of Candida albicans MTCC183 (values are average of three observations ± SEM)

20 Scanning electron microscopy analysis SEM microscopy was used to evaluate the surface morphology of the untreated microbial cells and the effect of silver nanoparticles on the surface morphology of the treated cells. The cell surface of the untreated cells was intact and damage was not seen as shown in the Fig 3.10a-3.14a. However, after treatment with nanoparticles, structural changes and major damages in the morphology of cells were clearly observed in the scanning electron micrographs as the surfaces of the cells were damaged (Fig 3.10b- 3.14b). In case of the Gram negative bacteria Escherichia coli MTCC730 and Salmonella enterica MTCC735, very clear deformation and fragmentation of the cell membrane was observed. The morphological membrane disruption of Gram positive bacteria Staphylococcus aureus MTCC96 and Enterococcus faecalis MTCC2729 was much lesser as compared to Gram negative bacteria Escherichia coli MTCC730 and Salmonella enterica MTCC735. This difference could be attributed to the difference of the peptidoglycan layer of the bacterial cell between Gram positive and Gram negative bacteria. SEM analysis also revealed the ability of silver nanoparticles to disrupt the fungal envelope structure. The result showed that the treated fungal cells of Candida albicans MTCC183 showed significant damage, which was characterized by the rupture of cell membrane (Fig 3.14b).

21 a b Fig 3.10: SEM image of cells of Escherichia coli MTCC730 a) before and b) after treatment with silver nanoparticles

22 a b b Fig 3.11: SEM image of cells of Salmonella enterica MTCC735 a) before and b) after treatment with silver nanoparticles

23 a b Fig 3.12: SEM image of cells of Staphylococcus aureus MTCC96 a) before b) after treatment with silver nanoparticles

24 a b Fig 3.13: SEM image of cells of Enterococcus faecalis MTCC2729 a) before and b) after treatment with silver nanoparticles

25 a b Fig 3.14: SEM image of cells of Candida albicans MTCC183 a) before and b) after treatment with silver nanoparticles

26 DISCUSSION Antibiotic resistance developed by microorganisms constitutes an increasing problem in medical care facilities worldwide (Ho et al., 2010). Silver is known to have broad-spectrum antimicrobial activity against bacteria, viruses and eukaryotic microorganisms and could be explored as an alternative to the conventional antibiotics (Feng et al., 2005; Morones et al., 2005). Smaller particles with a larger surface area possess higher antibacterial effects compared to the larger particles (Panacek et al., 2006). It is known that silver nanoparticles exhibit a high antimicrobial activity due to their well-developed surface which provides the maximum contact with the environment. Furthermore, toxicity is presumed to be size and shape dependent (Choi et al., 2008) because small size nanoparticles may pass through cell membranes. For example, inside a bacterium, nanoparticles can interact with DNA, thus making the microbe unable to replicate leading to the cell death. Kim et al. (2007) reported effective antimicrobial activity of silver nanoparticles against Escherichia coli and Staphylococcus aureus. In vitro antimicrobial activity of silver nanoparticles obtained using soil and endophytic fungi Fusarium oxysporum MP5, Aspergillus niger NH6, Paecilomyces lilacinus SF1, Arthrinium sp KL1, Aspergillus fumigatus SP5, Cladosporium cladosporioides RS1, Aspergillus tamarii PFL2, Aspergillus niger PFR6, Penicillium ochrochloron PFR8, Cryptosporiopsis ericae PS4, Alternaria solani GS1 and Penicillium funiculosum GS2 was carried out which indicated that the biosynthesized silver nanoparticles are promising antimicrobial agents. The mycosynthesized silver nanoparticles showed potent antimicrobial activity against both Gram positive and Gram negative pathogenic strains as well as the pathogenic fungal

27 strain. However, the biosynthesized nanoparticles were found to have maximum antimicrobial activity against the fungal strain Candida albicans MTCC183 followed by Gram negative bacterial strains Escherichia coli MTCC730 and Salmonella enterica MTCC735. Silver nanoparticles had a minimal microbicidal activity on Gram positive bacteria. This is due to the high lipopolysaccharide and thick peptidoglycan layer of the microorganisms (Feng et al., 2000; Jung et al., 2008). The negatively charged silver nanoparticles can bind to Gram negative cell wall better. The Gram positive bacteria are made up of rigid peptidoglycan layer and thus are more stable with minimal binding sites for silver nanoparticles (Fayaz et al., 2010). Combined use of silver nanoparticles with antibiotics, such as penicillin G, amoxicillin, and vancomycin have been reported to enhance their activity through synergistic antimicrobial effects against various Gram-positive and Gram-negative bacteria (Shahverdi et al., 2007; Rai et al., 2009b; Fayaz et al., 2010). Present study on combined effect of antimicrobial activity of the biosynthesized silver nanoparticles with commonly used antibiotic chloramphenicol against the test bacterial strains viz., Staphylococcus aureus MTCC96, Enterococcus faecalis MTCC2729, Salmonella enterica MTCC735 and Escherichia coli MTCC730 showed significant enhancement of antibacterial activity of the antibiotics in presence of the biosynthesized silver nanoparticles. These results corroborate with the findings reported by Birla et al. (2009) who reported that in vitro activity of commercially available antibiotics was significantly increased in the presence of silver nanoparticles produced from Phoma glomerata. Similarly, our findings of combined effects of silver nanoparticles with antimicrobial agents corroborate with the report of Shahverdi et al. (2007) who

28 demonstrated the increase in antibacterial activities of penicillin G, amoxicillin, erythromycin, clindamycin, and vancomycin in combination with the mycosynthesized silver nanoparticles against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. It is observed from the present study that the biosynthesized silver nanoparticles inhibit the growth of Gram-negative and Gram-positive bacteria as well as the fungal pathogen. Further, the silver nanoparticles in combination with antimicrobial agents proved to have enhanced antimicrobial activity against broad range of microorganisms. Thus, these findings suggest that the biosynthesized silver nanoparticles singly or their formulations in combination with conventional antimicrobial agents could be used as excellent broad-spectrum antimicrobial agent. The growth curves of bacterial cells as well as the yeast cells of the indicator strains treated with different concentrations of nanoparticles were monitored. The bacterial culture as well as the fungal culture treated with increasing concentration of silver nanoparticles shoed the exponential phase significantly delayed compared to untreated samples leading to complete arrest of growth. Biosynthesized silver nanoparticles were found to have less significant effect on the growth of Gram positive than the Gram negative bacteria. And also the fungal strain Candida abicans MTCC183 was effectively inhibited at much lower concentration as compared to the bacterial strains. The mechanism by which the nanoparticles are able to inhibit bacterial growth is not well understood, but it is suggested that the silver nanoparticles affects the membrane of both Gram negative and Gram positive bacterial strains. It may lead to significant increase in the permeability and affect membrane transport (Sondi and Salopek-Sondi, 2004). The morphological changes of bacterial cells after treatment with

29 silver nanoparticles were evaluated using SEM. The morphological damage of the five tested microorganism were observed which could result in cell lysis. The damage of the cell membrane was much intense in case of the fungal strain Candida albicans MTCC183 and the Gram positive bacterial strains Staphylococcus aureus MTCC96, Enterococcus faecalis MTCC2729 as compared to the Gram negative bacterial strains Salmonella enterica MTCC735 and Escherichia coli MTCC730. This is attributed to the thick peptidoglycan layer of the Gram positive bacteria. An essential function of the peptidoglycan layer is to protect against antibacterial agents such as antibiotics, toxins, chemicals, and degradative enzymes (Silhavy et al., 2010). From the findings of the present study, it can be concluded that the silver nanoparticles synthesized using the endophytic fungus Cryptosporiopsis ericae PS4 was found to be an excellent antimicrobial agent against a broad range of microorganisms such as Gram positive bacteria, Gram negative bacteria as well as the fungal pathogen with potential scope to bioprospect for biomedical and clinical applications.