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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1 ´ anticandidosique de nanoparticules d’argent synthe ´ tise ´ es Activite `a l’aide de Streptomyces sp.VITPK1 P. Sanjenbam, J.V. Gopal, K. Kannabiran * Division of Biomolecules and Genetics, School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India Received 1 July 2013; received in revised form 6 March 2014; accepted 31 March 2014
KEYWORDS Streptomyces sp.VITPK1; Silver nanoparticles; Anticandidal activity
Summary Objectives. — The aim of the present study was to evaluate the anticandidal activity of biologically synthesized silver nanoparticles using the culture filtrate of Streptomyces sp.VITPK1. Materials and methods. — Silver nanoparticles were synthesized using the culture filtrate of Streptomyces species isolated from brine spring located at Thoubal District, Manipur, India. The isolate was identified by molecular taxonomic characterization and designated as Streptomyces sp.VITPK1. The synthesized silver nanoparticles (AgNPs) were characterized by UV-visible spectra, X-ray diffraction (XRD) patterns, Energy Dispersive Analysis of X-rays (EDAX) and Fourier Transform Infrared (FTIR) analysis. The antifungal activity of the synthesized silver nanoparticles was evaluated against selected Candida species. Results. — The synthesized AgNPs showed a surface plasmon resonance peak at 425 nm. XRD patterns showed the crystalline peaks at 38.158 (111), 44.358 (200), 64.528 (220) and 77.498 (311) matching with the diffraction facets of silver. The size of the AgNPs was in the range of 20— 45 nm. The EDAX analysis revealed the presence of silver as the major metal in the sample. The synthesized AgNPs showed anticandidal activity against Candida albicans, Candida tropicalis and Candida krusei with a maximum zone of inhibition of 20 mm against C. albicans. Conclusions. — The results of this study suggest that the green synthesis of silver nanoparticles using Streptomyces sp.VITPK1 have the ability to act against pathogenic Candida strains. # 2014 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (K. Kannabiran). http://dx.doi.org/10.1016/j.mycmed.2014.03.004 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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MOTS CLÉS Streptomyces sp.VITPK1 ; Nanoparticules d’argent ; Activité anticandidosique
Re ´sume ´ Objectifs. — L’objectif de la présente étude était d’évaluer l’activité anticandidosique des nanoparticules d’argent biologiquement synthétisées en utilisant le filtrat de culture de Streptomyces sp.VITPK1. Mate´riel et me´thodes. — Les nanoparticules d’argent ont été synthétisées en utilisant le filtrat de culture de Streptomyces sp. isolés d’une source d’eau salée située à Thoubal District, Manipur, Inde. Les isolats ont été identifiés et caractérisés par taxonomie moléculaire et désignés comme Streptomyces sp.VITPK1. Les nanoparticules synthétiques d’argent (AgNPs) étaient caractérisées par leur spectre UV-visible, la diffraction des rayons X, et par l’analyse EDAX et FTIR. L’activité antifongique nanoparticules a été évaluée contre divers Candida sp. Re ´sultats. — Les AgNPS synthétisés ont montré un pic de résonance de plasmon de surface à 425 nm. L’analyse XRD schémas ont montré des pics cristallins à 38,158 (111), 44,358 (200), 64,528 (220) et 77,498 (311) correspondant avec les facettes de diffraction de l’argent. La taille de l’AgNPs était dans la gamme des 20—45 nm. L’analyse EDXA a révélé la présence d’argent comme principal métal dans l’échantillon. Les particules de AgNPs ont montré une activité contre Candida albicans, Candida tropicalis et Candida krusei avec une zone d’inhibition maximale de 20 mm contre C. albicans. Conclusions. — Les résultats de l’étude suggèrent que les nanoparticules d’argent synthétisées via Streptomyces sp.VITPK1 ont la capacité d’agir contre des souches pathogènes de Candida sp. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Candida species cause 6.2% of the human infections, ranking the 4th most prevalent infectious agent [5]. Epidemiological data from the Indian subcontinent showed that 67—90% of nosocomial candidaemia cases were due to Candida and Candida-non-albicans species [17]. Vulvovaginal candidiasis caused by Candida was more prevalent in reproductive-age Indian women living in rural India [27]. Candida albicans can colonize skin and mucosal surfaces of healthy people and thus occurs commensally in the gastrointestinal tract, oral cavity and vagina, often causing superficial infections [23]. Moreover, C. albicans can enter the bloodstream by direct penetration from the epithelium after tissue damage, or by dissemination from biofilms formed on medical devices introduced into the patient’s organs, e.g. catheters, dental implants, endoprostheses, artificial joints or central nervous system shunts [23]. Yeast cells disseminate with the blood flow and infect almost all inner organs, including lungs, kidney, heart, liver, spleen and brain, causing fungaemia and lifethreatening septicemia. Amphotericin B and fluconazole are the antibiotics used for the treatment candidiasis. Candida infections are more frequently reported in both HIV and cancer patients. Recent reports have shown that the association of Candida and its role in malignant transformation of oral fibrosis [28]. Fungi being eukaryotes, it is very difficult to find new leads without causing further toxicity to humans [8]. Drug-resistant pathogens have increased tremendously in the recent past and searching for an effective drug is of great interest to the researchers [22]. Reports suggest that C. albicans pathogenesis is complex and multifactorial [13]. The rise in resistant strain of Candida has increased tremendously becoming a serious concern [4]. The formulation of nanosciences and microbiological sciences is emerging as an effective tool for the discovery of new drugs [33]. There are lots of nanomaterials available currently but biologically synthesized nanomaterials are very few. Biologically synthesized nanomaterials are cost-effective and
eco-friendly [11,14]. Among the numerous nanomaterials reported till date, silver nanoparticles are most effective as silver exhibits the highest plasmon excitation [6]. It was reported that Aspergillus formed silver nanoparticles modulate the cytokines involved in wound healing [37], being an excellent producer of bioactive metabolites and reports shows that these secondary metabolites produced by Streptomyces have been widely used for synthesis of nanoparticles which are proved to be effective antimicrobial agents [15]. Anticandidal activity of AgNPs synthesized using Gracilaria corticata against Candida species has already been reported [26]. Manipur, India, being an Indo-Burma hotspot serves as a treasure island for exploration of actinomycetes diversity and their secondary metabolites [24,25]. In this investigation, we synthesized silver nanoparticles using Streptomyces sp.VITPK1 and evaluated the antifungal activity of AgNPs against drug-resistant C. albicans and other Candida species.
Materials and methods Sample collection Soil sediments were collected using sterile polythene bags from the brine spring located at Thoubal District, Manipur. It lies between 238450 N and 248450 N latitude and 938450 E and 948150 E longitude. Collected soil samples were transported to the laboratory and stored at 4 8C.
Isolation of actinomycetes Actinomycetes were isolated using Actinomycetes Isolation Agar (AIA) medium by serial dilution method [21]. The plates were incubated for 14 days at 27 8C. The isolated colonies were purified and stored at 4 8C. Systematic screening of isolates for anticandidal activity resulted in the selection of VITPK1 for further studies.
Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Characterization of the isolate
Characterization of AgNPs
The isolate was identified by molecular taxonomic characterization [34]. The morphology of the isolate was determined by scanning electron microscopic analysis (Hitachi, 10 mm). Later 16S rDNA sequencing was carried out as reported earlier [39]. The sequence was submitted in GenBank, NCBI, USA. The sequence similarity estimated by CLUSTAL W software tool available online at http://www.genome.jp/ tools/clustalw/.
The reduction of silver ions was monitored by visual observation of the solution. The absorption spectra of this solution were recorded using a UV-Visible spectrophotometer (ELICO, India) from 200—800 nm at regular intervals [31]. The synthesized silver nanoparticles were separated by centrifugation and used for XRD analysis. XRD patterns were collected in the range of 20—70 8C (2u) operated at 40 kV and 30 mA (Advance Powder X-ray diffractometer, Germany). The synthesized silver nanoparticles were imaged by AFM (Nano-Surf Easy Scan2, Switzerland) and SEM to determine the exact configuration. In addition, the presence of silver metals in the sample was confirmed by energy dispersive X-ray analysis (EDXA). Fourier Transform Infra-red (FTIR) analysis was also carried out to know the presence of functional groups.
Biosynthesis of AgNPs The isolate VITPK1 was grown on starch casein agar for 7 days at 278 C. Inoculum was prepared by transferring 10 ml of 1 McFarland standard of bacterial suspension prepared from the culture grown on starch casein agar, into 250 ml flasks containing 100 ml ISP 1 media [7]. The culture was grown for seven days at 150 rpm and 27 8C in rotary shaker [36]. The cell-free supernatant was collected by centrifuging at 4000 rpm for 15 min. The extracellular supernatant (100 ml) was mixed with 100 ml of 1 mM silver nitrate solution in a 250 ml flask and incubated in a rotary shaker. The formation of silver nanoparticles was observed by color change and the reduction of silver confirmed by UV-visible spectra observed between the range of 400—500 nm [12,31].
Anticandidal assay Anticandidal assay was carried out using the Kirby-Bauer method [3]. Three candidal strains were chosen: C. albicans (MTCC 227), Candida tropicalis (MTCC 184) and Candida krusei (MTCC 9215). A fresh culture of the Candida strains were prepared using Sabouraud’s dextrose broth and incubated for 48 h. Sabouraud’s dextrose agar plates were prepared and swabbed with fresh culture of Candida. Then the wells were loaded with 50 mg/ml of the synthesized AgNPs in triplicates and the zone of inhibition (mm) was measured against C. albicans, C. tropicalis and C. krusei.
Results The isolate VITPK1 grew well in all the media tested and formed a white color aerial mycelium in Actinomycetes Isolation Agar (AIA). Arrangement of spores was observed under SEM (Fig. 1). Cultural and biochemical characteristics of the isolate are listed in Table 1. The isolate used starch as sole carbon source and potassium nitrate as sole nitrogen source for its growth. The blast search of 16S DNA sequence (1032 bases) with sequences available in the GenBank (NCBI) database revealed that the isolate showed 98% similarity with Streptomyces variabilis (NR043840). The phylogeny and molecular taxonomy of the isolate showed that it belongs to the genus Streptomyces and designated as Streptomyces sp.VITPK1 (JX139609). Cultural characteristics of Streptomyces sp.VITPK1 was compared to the closest neighbour S. variabilis are given in Table 2. The phylogenetic tree of the isolate is shown in Fig. 2. AgNPs were formed within 48 h of incubation evidenced by change in pale yellow color to dark brown color indicating the reduction of silver nitrate and formation of silver nanoparticles. The synthesized nanoparticles were stable evidenced by stable color even after nine days. The synthesized
Figure 1 A. SEM image showing the arrangement of spores of Streptomyces sp.VITPK1. B. Growth of Streptomyces sp.VITPK1 on Actinomycetes Isolation Agar. A. Image SEM montrant l’arrangement des spores de Streptomyces sp.VITPK1. B. Croissance de Streptomyces sp.VITPK1 sur une ´ lose d’isolement pour Streptomyces. ge
Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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P. Sanjenbam et al. Table 1 Cultural and biochemical characteristics of Streptomyces sp.VITPK1. ´ ristiques culturelles et biochimiques de StreptomyCaracte ces sp.VITPK1.
Table 2 Cultural characteristics of Streptomyces sp.VITPK1 with reference to the closest neighbour. ´ ristiques culturelles de Streptomyces sp.VITPK1 avec Caracte ´ fe ´ rence `a son voisin le plus proche. re
Media
VITPK1
Biochemical test
VITPK1
Characteristics
VITPK1
AIA ISP 1 ISP 2 ISP 3 ISP 4 ISP 5 ISP6 ISP7
Good growth Good growth Good growth Poor growth Good growth Good growth No melanin Good growth
Catalase Oxidase Indole Methyl red Voges proskauer Citrate utilization Starch hydrolysis Gelatin liquefaction
+ + + + +
Streptomyces variabilis
Morphological characterization Gram staining Motility Spore mass white Diffusible pigment Spore surface
+ + Smooth
+ Smooth
Physiological characterization Growth at 45 8C Growth at NaCl 7% (w/v)
+ +
Biochemical characterization Hypoxanthine Starch Urea Oxidase H2S production Casein Gelatin liquefaction Sucrose 1% (w/v) Mannitol 1% (w/v) Rhamnose 1% (w/v)
+ + + + + + + +
+ + + + + + + + + +
+: positive; : negative.
AgNPs showed a prominent activity against Candida strains. The anticandidal activity of cell-free supernatant and synthesized AgNPs are given in Table 3. The culture supernatant (50 mg/ml) of the isolate showed high inhibition zone against C. tropicalis (15 0.06) followed by C. krusei (13.5 1.0) and C. albicans (13 0.21). The synthesized AgNPs (50 mg/ ml) showed maximum zone of inhibition against C. albicans (20 0.067) followed by C. tropicalis (18 0.23) and C. krusei (16 1.07). The observed result indicated that synthesized AgNPs showed higher zone of inhibition when compared to the cell-free supernatant of the isolate (Fig. 3). Silver nanoparticles produced a strong and broad UVvisible spectra observed at 425 nm, indicating the presence of AgNPs. The SPR bands of AgNPs solution remain close to 420—450 nm throughout the reaction period (Fig. 4). XRD patterns showed the crystalline peaks at 2u values of 38.158, 44.358, 64.528 and 77.498 which can be indexed as 111, 200, 220, 311 planes of FCC silver (Fig. 5). The size of the nanoparticles was found to be in the range of 21—45 nm.
+: positive; : negative.
The surface morphology and topographical structure of the synthesized AgNPs were measured by AFM (Fig. 6). The surface morphology of silver nanoparticles was also observed under SEM imaging (Fig. 7A). The EDX spectrum obtained is shown in Fig. 7B. The purity of the biosynthesized AgNPs was
Figure 2 Phylogenetic tree analysis of the Streptomyces sp.VITPK1. ´ ne ´ tique de Streptomyces sp.VITPK1. Arbre phyloge Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1 Table 3 Anticandidal activity of synthesized silver nanoparticles from Streptomyces sp.VITPK1. ´ anticandidosique de nanoparticules d’argent synActivite ´ tise ´ es de Streptomyces sp.VITPK1. the Pathogen
Cell-free supernatant
AgNPs
Candida albicans Candida tropicalis Candida krusei
13 0.21 15 0.06 13.5 1.0
20 0.067 18 0.23 16 1.07
Values are mean S.D. (n = 3).
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examined and the EDX spectrum obtained revealed a strong signal for silver. The FTIR analysis revealed the presence of functional groups like amines or proteins leading to the formation of silver nanoparticles from Streptomyces sp.VITPK1 (Fig. 8). The peak at 3662.82 cm1and 3410.15 cm1 due to stretching of C = O, 2956.87 cm1 and 2926.1 cm1 due to stretching of anti-symmetric CH3, 1674.21 cm1 stretching of C = C, 1616.35 cm1 scissoring of —NH2, 1454.33 cm1 aromatic ring, 1402.25 cm 1 and 1274.95 cm1 due to —OH and —COOH groups present in the cell-free supernatant of Streptomyces sp.VITPK1 (Fig. 8A). The functional groups which leads to the reduction of Ag+ ions where observed at peaks 3695.61 cm1, 1585.49 cm1,
Figure 3 A. Antifungal activity of cell-free supernatant of Streptomyces sp.VITPK1 against Candida albicans (MTCC227). B. Activity shown by AgNPs. ´ antifongique du surnageant acellulaire de Streptomyces sp.VITPK1 contre Candida albicans (MTCC227). B. Activite ´ illustre ´ A. Activite par AgNPs.
Figure 4 UV-visible spectra of synthesized silver nanoparticles at regular intervals. ´ guliers. Spectre dans l’UV-visible des nanoparticules d’argent `a intervalles re Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Figure 5 XRD pattern of synthesized silver nanoparticles confirming the formation of silver nanoparticles. ´ tise ´ es confirAspect XRD des nanoparticules d’argent synthe mant la formation de nanoparticules d’argent.
1398.39 cm1, 1151.50 cm1 and 1068.56 cm1 due to the stretching of a C-N groups (Fig. 8B).
Discussion Currently, nanosciences are developing drastically because of its application in various fields of microelectronics and
biomedical stream [18,38]. Biologically synthesized nanoparticles are simpler and eco-friendly as compared to those produced by physical and chemical methods [19]. The nanoparticulate system proved to be of great potential to convert the labile bioactive compounds to promising leads [1]. Both bacteria and fungi have the ability to produce nanoparticles as they can easily reduce metal ions to metallic nanoparticles [2]. In this context, Streptomyces is known for its ability to produce numerous bioactive metabolites [10] and silver is well known for its antimicrobial properties [35] which could be used to produce new leads. It is advantageous to combine both the antimicrobial property and nanoparticle synthesizing ability of the compound to be considered as one of the powerful antimicrobial agent [16]. There are few reports on the extracellular biosynthesis of silver nanoparticles using cell filtrate of Streptomyces sp. [7,40]. In our study, AgNPs were synthesized successfully from the cell-free supernatant of Streptomyces sp.VITPK1. The secondary metabolites produced by the Streptomyces sp.VITPK1 reduce silver nitrate leading to the formation of nanoparticles (NPs). The peak at 450 nm may be due to the excitation of longitudinal plasmon vibrations and formation of quasi-linear superstructures of nanoparticles [32]. The intensity gradually increased in the range of 350—600 nm as the incubation time increased [19]. The functional groups involved in interaction with silver was analysed by FTIR. The FTIR spectra showed the presence of proteins in the secondary metabolites which interact with AgNPs by acting as capping agent as reported earlier [2]. Our results support the observation that the
Figure 6 Atomic force micrographs of synthesized silver nanoparticles from aqueous filtrate of Streptomyces sp.VITPK1. ` se `a partir de la phase aqueuse de filtrat de Streptomyces Micrographes de la force atomique des nanoparticules d’argent de synthe sp.VITPK1. Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Figure 7 A. SEM imaging of silver nanoparticles. B. EDAX spectrum of silver nanoparticles from Streptomyces sp.VITPK1. A. Images SEM des nanoparticules d’argent. B. Spectre EDX des nanoparticules d’argent de Streptomyces sp.VITPK1.
AgNPs can interact with the amine residues [9]. Interaction of amines with the AgNPs was already reported [37]. It was also reported that Streptomyces produces proteins as extracellular metabolites which easily interact with the AgNPs as a capping agent [26].
Thus exploration of actinomycetes for the formation of NPs could be beneficial for screening their ability against various pathogens and antibiotic resistant microorganism [29]. Candida being a polymorphic organism, it is very difficult to control its infection in immunocompromised patients.
Figure 8 FTIR spectra of (A) cell-free supernatant of Streptomyces sp.VITPK1 and (B) synthesized silver nanoparticles. ´ tise ´ es. Spectre FTIR de (A) surnageant acellulaire de Streptomyces sp.VITPK1 et (B) nanoparticules d’argent synthe Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Several reports are available on existence of drug-resistant Candida species. It is more significant to have AgNPs with profound inhibition over drug-resistant C. albicans. In our study, the synthesized AgNPs showed high anticandidal activity when compared with the activity shown by the cell-free supernatant. The fungi static effect exhibited by the synthesized AgNPs against Candida species highlights the significance of this work. The mode of action of AgNPs against the fungal pathogens such as Candida species may be by destructing the integrity of cell membrane and stopping the process of budding [20]. The results suggest that green synthesis of nanoparticles finds extensive pharmaceutical and biomedical applications, but the mechanism of action needs to be explored.
Conclusion Based on the results, it can be concluded that the actinomycetes isolate Streptomyces sp.VITPK1 is an excellent microbial resource for the extracellular synthesis of AgNPs. Inhibition of pathogenic Candida strains by biologically synthesized AgNPs from VITPK1 isolate needs to be studied further.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgement The authors thank the management of VIT University for providing facilities to carry out this study.
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Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1 ´ anticandidosique de nanoparticules d’argent synthe ´ tise ´ es Activite `a l’aide de Streptomyces sp.VITPK1 P. Sanjenbam, J.V. Gopal, K. Kannabiran * Division of Biomolecules and Genetics, School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India Received 1 July 2013; received in revised form 6 March 2014; accepted 31 March 2014
KEYWORDS Streptomyces sp.VITPK1; Silver nanoparticles; Anticandidal activity
Summary Objectives. — The aim of the present study was to evaluate the anticandidal activity of biologically synthesized silver nanoparticles using the culture filtrate of Streptomyces sp.VITPK1. Materials and methods. — Silver nanoparticles were synthesized using the culture filtrate of Streptomyces species isolated from brine spring located at Thoubal District, Manipur, India. The isolate was identified by molecular taxonomic characterization and designated as Streptomyces sp.VITPK1. The synthesized silver nanoparticles (AgNPs) were characterized by UV-visible spectra, X-ray diffraction (XRD) patterns, Energy Dispersive Analysis of X-rays (EDAX) and Fourier Transform Infrared (FTIR) analysis. The antifungal activity of the synthesized silver nanoparticles was evaluated against selected Candida species. Results. — The synthesized AgNPs showed a surface plasmon resonance peak at 425 nm. XRD patterns showed the crystalline peaks at 38.158 (111), 44.358 (200), 64.528 (220) and 77.498 (311) matching with the diffraction facets of silver. The size of the AgNPs was in the range of 20— 45 nm. The EDAX analysis revealed the presence of silver as the major metal in the sample. The synthesized AgNPs showed anticandidal activity against Candida albicans, Candida tropicalis and Candida krusei with a maximum zone of inhibition of 20 mm against C. albicans. Conclusions. — The results of this study suggest that the green synthesis of silver nanoparticles using Streptomyces sp.VITPK1 have the ability to act against pathogenic Candida strains. # 2014 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (K. Kannabiran). http://dx.doi.org/10.1016/j.mycmed.2014.03.004 1156-5233/# 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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MOTS CLÉS Streptomyces sp.VITPK1 ; Nanoparticules d’argent ; Activité anticandidosique
Re ´sume ´ Objectifs. — L’objectif de la présente étude était d’évaluer l’activité anticandidosique des nanoparticules d’argent biologiquement synthétisées en utilisant le filtrat de culture de Streptomyces sp.VITPK1. Mate´riel et me´thodes. — Les nanoparticules d’argent ont été synthétisées en utilisant le filtrat de culture de Streptomyces sp. isolés d’une source d’eau salée située à Thoubal District, Manipur, Inde. Les isolats ont été identifiés et caractérisés par taxonomie moléculaire et désignés comme Streptomyces sp.VITPK1. Les nanoparticules synthétiques d’argent (AgNPs) étaient caractérisées par leur spectre UV-visible, la diffraction des rayons X, et par l’analyse EDAX et FTIR. L’activité antifongique nanoparticules a été évaluée contre divers Candida sp. Re ´sultats. — Les AgNPS synthétisés ont montré un pic de résonance de plasmon de surface à 425 nm. L’analyse XRD schémas ont montré des pics cristallins à 38,158 (111), 44,358 (200), 64,528 (220) et 77,498 (311) correspondant avec les facettes de diffraction de l’argent. La taille de l’AgNPs était dans la gamme des 20—45 nm. L’analyse EDXA a révélé la présence d’argent comme principal métal dans l’échantillon. Les particules de AgNPs ont montré une activité contre Candida albicans, Candida tropicalis et Candida krusei avec une zone d’inhibition maximale de 20 mm contre C. albicans. Conclusions. — Les résultats de l’étude suggèrent que les nanoparticules d’argent synthétisées via Streptomyces sp.VITPK1 ont la capacité d’agir contre des souches pathogènes de Candida sp. # 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction Candida species cause 6.2% of the human infections, ranking the 4th most prevalent infectious agent [5]. Epidemiological data from the Indian subcontinent showed that 67—90% of nosocomial candidaemia cases were due to Candida and Candida-non-albicans species [17]. Vulvovaginal candidiasis caused by Candida was more prevalent in reproductive-age Indian women living in rural India [27]. Candida albicans can colonize skin and mucosal surfaces of healthy people and thus occurs commensally in the gastrointestinal tract, oral cavity and vagina, often causing superficial infections [23]. Moreover, C. albicans can enter the bloodstream by direct penetration from the epithelium after tissue damage, or by dissemination from biofilms formed on medical devices introduced into the patient’s organs, e.g. catheters, dental implants, endoprostheses, artificial joints or central nervous system shunts [23]. Yeast cells disseminate with the blood flow and infect almost all inner organs, including lungs, kidney, heart, liver, spleen and brain, causing fungaemia and lifethreatening septicemia. Amphotericin B and fluconazole are the antibiotics used for the treatment candidiasis. Candida infections are more frequently reported in both HIV and cancer patients. Recent reports have shown that the association of Candida and its role in malignant transformation of oral fibrosis [28]. Fungi being eukaryotes, it is very difficult to find new leads without causing further toxicity to humans [8]. Drug-resistant pathogens have increased tremendously in the recent past and searching for an effective drug is of great interest to the researchers [22]. Reports suggest that C. albicans pathogenesis is complex and multifactorial [13]. The rise in resistant strain of Candida has increased tremendously becoming a serious concern [4]. The formulation of nanosciences and microbiological sciences is emerging as an effective tool for the discovery of new drugs [33]. There are lots of nanomaterials available currently but biologically synthesized nanomaterials are very few. Biologically synthesized nanomaterials are cost-effective and
eco-friendly [11,14]. Among the numerous nanomaterials reported till date, silver nanoparticles are most effective as silver exhibits the highest plasmon excitation [6]. It was reported that Aspergillus formed silver nanoparticles modulate the cytokines involved in wound healing [37], being an excellent producer of bioactive metabolites and reports shows that these secondary metabolites produced by Streptomyces have been widely used for synthesis of nanoparticles which are proved to be effective antimicrobial agents [15]. Anticandidal activity of AgNPs synthesized using Gracilaria corticata against Candida species has already been reported [26]. Manipur, India, being an Indo-Burma hotspot serves as a treasure island for exploration of actinomycetes diversity and their secondary metabolites [24,25]. In this investigation, we synthesized silver nanoparticles using Streptomyces sp.VITPK1 and evaluated the antifungal activity of AgNPs against drug-resistant C. albicans and other Candida species.
Materials and methods Sample collection Soil sediments were collected using sterile polythene bags from the brine spring located at Thoubal District, Manipur. It lies between 238450 N and 248450 N latitude and 938450 E and 948150 E longitude. Collected soil samples were transported to the laboratory and stored at 4 8C.
Isolation of actinomycetes Actinomycetes were isolated using Actinomycetes Isolation Agar (AIA) medium by serial dilution method [21]. The plates were incubated for 14 days at 27 8C. The isolated colonies were purified and stored at 4 8C. Systematic screening of isolates for anticandidal activity resulted in the selection of VITPK1 for further studies.
Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Characterization of the isolate
Characterization of AgNPs
The isolate was identified by molecular taxonomic characterization [34]. The morphology of the isolate was determined by scanning electron microscopic analysis (Hitachi, 10 mm). Later 16S rDNA sequencing was carried out as reported earlier [39]. The sequence was submitted in GenBank, NCBI, USA. The sequence similarity estimated by CLUSTAL W software tool available online at http://www.genome.jp/ tools/clustalw/.
The reduction of silver ions was monitored by visual observation of the solution. The absorption spectra of this solution were recorded using a UV-Visible spectrophotometer (ELICO, India) from 200—800 nm at regular intervals [31]. The synthesized silver nanoparticles were separated by centrifugation and used for XRD analysis. XRD patterns were collected in the range of 20—70 8C (2u) operated at 40 kV and 30 mA (Advance Powder X-ray diffractometer, Germany). The synthesized silver nanoparticles were imaged by AFM (Nano-Surf Easy Scan2, Switzerland) and SEM to determine the exact configuration. In addition, the presence of silver metals in the sample was confirmed by energy dispersive X-ray analysis (EDXA). Fourier Transform Infra-red (FTIR) analysis was also carried out to know the presence of functional groups.
Biosynthesis of AgNPs The isolate VITPK1 was grown on starch casein agar for 7 days at 278 C. Inoculum was prepared by transferring 10 ml of 1 McFarland standard of bacterial suspension prepared from the culture grown on starch casein agar, into 250 ml flasks containing 100 ml ISP 1 media [7]. The culture was grown for seven days at 150 rpm and 27 8C in rotary shaker [36]. The cell-free supernatant was collected by centrifuging at 4000 rpm for 15 min. The extracellular supernatant (100 ml) was mixed with 100 ml of 1 mM silver nitrate solution in a 250 ml flask and incubated in a rotary shaker. The formation of silver nanoparticles was observed by color change and the reduction of silver confirmed by UV-visible spectra observed between the range of 400—500 nm [12,31].
Anticandidal assay Anticandidal assay was carried out using the Kirby-Bauer method [3]. Three candidal strains were chosen: C. albicans (MTCC 227), Candida tropicalis (MTCC 184) and Candida krusei (MTCC 9215). A fresh culture of the Candida strains were prepared using Sabouraud’s dextrose broth and incubated for 48 h. Sabouraud’s dextrose agar plates were prepared and swabbed with fresh culture of Candida. Then the wells were loaded with 50 mg/ml of the synthesized AgNPs in triplicates and the zone of inhibition (mm) was measured against C. albicans, C. tropicalis and C. krusei.
Results The isolate VITPK1 grew well in all the media tested and formed a white color aerial mycelium in Actinomycetes Isolation Agar (AIA). Arrangement of spores was observed under SEM (Fig. 1). Cultural and biochemical characteristics of the isolate are listed in Table 1. The isolate used starch as sole carbon source and potassium nitrate as sole nitrogen source for its growth. The blast search of 16S DNA sequence (1032 bases) with sequences available in the GenBank (NCBI) database revealed that the isolate showed 98% similarity with Streptomyces variabilis (NR043840). The phylogeny and molecular taxonomy of the isolate showed that it belongs to the genus Streptomyces and designated as Streptomyces sp.VITPK1 (JX139609). Cultural characteristics of Streptomyces sp.VITPK1 was compared to the closest neighbour S. variabilis are given in Table 2. The phylogenetic tree of the isolate is shown in Fig. 2. AgNPs were formed within 48 h of incubation evidenced by change in pale yellow color to dark brown color indicating the reduction of silver nitrate and formation of silver nanoparticles. The synthesized nanoparticles were stable evidenced by stable color even after nine days. The synthesized
Figure 1 A. SEM image showing the arrangement of spores of Streptomyces sp.VITPK1. B. Growth of Streptomyces sp.VITPK1 on Actinomycetes Isolation Agar. A. Image SEM montrant l’arrangement des spores de Streptomyces sp.VITPK1. B. Croissance de Streptomyces sp.VITPK1 sur une ´ lose d’isolement pour Streptomyces. ge
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P. Sanjenbam et al. Table 1 Cultural and biochemical characteristics of Streptomyces sp.VITPK1. ´ ristiques culturelles et biochimiques de StreptomyCaracte ces sp.VITPK1.
Table 2 Cultural characteristics of Streptomyces sp.VITPK1 with reference to the closest neighbour. ´ ristiques culturelles de Streptomyces sp.VITPK1 avec Caracte ´ fe ´ rence `a son voisin le plus proche. re
Media
VITPK1
Biochemical test
VITPK1
Characteristics
VITPK1
AIA ISP 1 ISP 2 ISP 3 ISP 4 ISP 5 ISP6 ISP7
Good growth Good growth Good growth Poor growth Good growth Good growth No melanin Good growth
Catalase Oxidase Indole Methyl red Voges proskauer Citrate utilization Starch hydrolysis Gelatin liquefaction
+ + + + +
Streptomyces variabilis
Morphological characterization Gram staining Motility Spore mass white Diffusible pigment Spore surface
+ + Smooth
+ Smooth
Physiological characterization Growth at 45 8C Growth at NaCl 7% (w/v)
+ +
Biochemical characterization Hypoxanthine Starch Urea Oxidase H2S production Casein Gelatin liquefaction Sucrose 1% (w/v) Mannitol 1% (w/v) Rhamnose 1% (w/v)
+ + + + + + + +
+ + + + + + + + + +
+: positive; : negative.
AgNPs showed a prominent activity against Candida strains. The anticandidal activity of cell-free supernatant and synthesized AgNPs are given in Table 3. The culture supernatant (50 mg/ml) of the isolate showed high inhibition zone against C. tropicalis (15 0.06) followed by C. krusei (13.5 1.0) and C. albicans (13 0.21). The synthesized AgNPs (50 mg/ ml) showed maximum zone of inhibition against C. albicans (20 0.067) followed by C. tropicalis (18 0.23) and C. krusei (16 1.07). The observed result indicated that synthesized AgNPs showed higher zone of inhibition when compared to the cell-free supernatant of the isolate (Fig. 3). Silver nanoparticles produced a strong and broad UVvisible spectra observed at 425 nm, indicating the presence of AgNPs. The SPR bands of AgNPs solution remain close to 420—450 nm throughout the reaction period (Fig. 4). XRD patterns showed the crystalline peaks at 2u values of 38.158, 44.358, 64.528 and 77.498 which can be indexed as 111, 200, 220, 311 planes of FCC silver (Fig. 5). The size of the nanoparticles was found to be in the range of 21—45 nm.
+: positive; : negative.
The surface morphology and topographical structure of the synthesized AgNPs were measured by AFM (Fig. 6). The surface morphology of silver nanoparticles was also observed under SEM imaging (Fig. 7A). The EDX spectrum obtained is shown in Fig. 7B. The purity of the biosynthesized AgNPs was
Figure 2 Phylogenetic tree analysis of the Streptomyces sp.VITPK1. ´ ne ´ tique de Streptomyces sp.VITPK1. Arbre phyloge Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1 Table 3 Anticandidal activity of synthesized silver nanoparticles from Streptomyces sp.VITPK1. ´ anticandidosique de nanoparticules d’argent synActivite ´ tise ´ es de Streptomyces sp.VITPK1. the Pathogen
Cell-free supernatant
AgNPs
Candida albicans Candida tropicalis Candida krusei
13 0.21 15 0.06 13.5 1.0
20 0.067 18 0.23 16 1.07
Values are mean S.D. (n = 3).
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examined and the EDX spectrum obtained revealed a strong signal for silver. The FTIR analysis revealed the presence of functional groups like amines or proteins leading to the formation of silver nanoparticles from Streptomyces sp.VITPK1 (Fig. 8). The peak at 3662.82 cm1and 3410.15 cm1 due to stretching of C = O, 2956.87 cm1 and 2926.1 cm1 due to stretching of anti-symmetric CH3, 1674.21 cm1 stretching of C = C, 1616.35 cm1 scissoring of —NH2, 1454.33 cm1 aromatic ring, 1402.25 cm 1 and 1274.95 cm1 due to —OH and —COOH groups present in the cell-free supernatant of Streptomyces sp.VITPK1 (Fig. 8A). The functional groups which leads to the reduction of Ag+ ions where observed at peaks 3695.61 cm1, 1585.49 cm1,
Figure 3 A. Antifungal activity of cell-free supernatant of Streptomyces sp.VITPK1 against Candida albicans (MTCC227). B. Activity shown by AgNPs. ´ antifongique du surnageant acellulaire de Streptomyces sp.VITPK1 contre Candida albicans (MTCC227). B. Activite ´ illustre ´ A. Activite par AgNPs.
Figure 4 UV-visible spectra of synthesized silver nanoparticles at regular intervals. ´ guliers. Spectre dans l’UV-visible des nanoparticules d’argent `a intervalles re Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Figure 5 XRD pattern of synthesized silver nanoparticles confirming the formation of silver nanoparticles. ´ tise ´ es confirAspect XRD des nanoparticules d’argent synthe mant la formation de nanoparticules d’argent.
1398.39 cm1, 1151.50 cm1 and 1068.56 cm1 due to the stretching of a C-N groups (Fig. 8B).
Discussion Currently, nanosciences are developing drastically because of its application in various fields of microelectronics and
biomedical stream [18,38]. Biologically synthesized nanoparticles are simpler and eco-friendly as compared to those produced by physical and chemical methods [19]. The nanoparticulate system proved to be of great potential to convert the labile bioactive compounds to promising leads [1]. Both bacteria and fungi have the ability to produce nanoparticles as they can easily reduce metal ions to metallic nanoparticles [2]. In this context, Streptomyces is known for its ability to produce numerous bioactive metabolites [10] and silver is well known for its antimicrobial properties [35] which could be used to produce new leads. It is advantageous to combine both the antimicrobial property and nanoparticle synthesizing ability of the compound to be considered as one of the powerful antimicrobial agent [16]. There are few reports on the extracellular biosynthesis of silver nanoparticles using cell filtrate of Streptomyces sp. [7,40]. In our study, AgNPs were synthesized successfully from the cell-free supernatant of Streptomyces sp.VITPK1. The secondary metabolites produced by the Streptomyces sp.VITPK1 reduce silver nitrate leading to the formation of nanoparticles (NPs). The peak at 450 nm may be due to the excitation of longitudinal plasmon vibrations and formation of quasi-linear superstructures of nanoparticles [32]. The intensity gradually increased in the range of 350—600 nm as the incubation time increased [19]. The functional groups involved in interaction with silver was analysed by FTIR. The FTIR spectra showed the presence of proteins in the secondary metabolites which interact with AgNPs by acting as capping agent as reported earlier [2]. Our results support the observation that the
Figure 6 Atomic force micrographs of synthesized silver nanoparticles from aqueous filtrate of Streptomyces sp.VITPK1. ` se `a partir de la phase aqueuse de filtrat de Streptomyces Micrographes de la force atomique des nanoparticules d’argent de synthe sp.VITPK1. Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Figure 7 A. SEM imaging of silver nanoparticles. B. EDAX spectrum of silver nanoparticles from Streptomyces sp.VITPK1. A. Images SEM des nanoparticules d’argent. B. Spectre EDX des nanoparticules d’argent de Streptomyces sp.VITPK1.
AgNPs can interact with the amine residues [9]. Interaction of amines with the AgNPs was already reported [37]. It was also reported that Streptomyces produces proteins as extracellular metabolites which easily interact with the AgNPs as a capping agent [26].
Thus exploration of actinomycetes for the formation of NPs could be beneficial for screening their ability against various pathogens and antibiotic resistant microorganism [29]. Candida being a polymorphic organism, it is very difficult to control its infection in immunocompromised patients.
Figure 8 FTIR spectra of (A) cell-free supernatant of Streptomyces sp.VITPK1 and (B) synthesized silver nanoparticles. ´ tise ´ es. Spectre FTIR de (A) surnageant acellulaire de Streptomyces sp.VITPK1 et (B) nanoparticules d’argent synthe Please cite this article in press as: Sanjenbam P, et al. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp.VITPK1. Journal De Mycologie Médicale (2014), http://dx.doi.org/10.1016/j.mycmed.2014.03.004
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Several reports are available on existence of drug-resistant Candida species. It is more significant to have AgNPs with profound inhibition over drug-resistant C. albicans. In our study, the synthesized AgNPs showed high anticandidal activity when compared with the activity shown by the cell-free supernatant. The fungi static effect exhibited by the synthesized AgNPs against Candida species highlights the significance of this work. The mode of action of AgNPs against the fungal pathogens such as Candida species may be by destructing the integrity of cell membrane and stopping the process of budding [20]. The results suggest that green synthesis of nanoparticles finds extensive pharmaceutical and biomedical applications, but the mechanism of action needs to be explored.
Conclusion Based on the results, it can be concluded that the actinomycetes isolate Streptomyces sp.VITPK1 is an excellent microbial resource for the extracellular synthesis of AgNPs. Inhibition of pathogenic Candida strains by biologically synthesized AgNPs from VITPK1 isolate needs to be studied further.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgement The authors thank the management of VIT University for providing facilities to carry out this study.
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