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Antifungal activity of Streptomyces VITSVK5 spp. against drug resistant Aspergillus clinical isolates from pulmonary tuberculosis patients
Journal de Mycologie Médicale (2010) 20, 101—107
ORIGINAL ARTICLE / ARTICLE ORIGINAL
Antifungal activity of Streptomyces VITSVK5 spp. against drug resistant Aspergillus clinical isolates from pulmonary tuberculosis patients ´ antifongique de Streptomyces VITSVK5 sp. sur des isolats d’Aspergillus Activite ´ sistants isole ´ s de patients atteints de tuberculose pulmonaire mutire S. Kumar, K. Kannabiran * Biomolecules and Genetics Division, School of biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India Received 17 November 2009; received in revised form 14 April 2010; accepted 16 April 2010
KEYWORDS Streptomyces spp.; Marine sediments; Antifungal activity; Aspergillus
Summary Objective. — Research of isolates producing antifungal activity. Material and methods. — An actinomycetes strain having anti-Aspergillus activity against multidrug drug resistant clinical isolates was isolated from marine sediment samples collected at Marakkanam coast of Bay of Bengal, India. The strain was characterized by polyphasic taxonomic approach and identified as a member of the genus Streptomyces. Based on the phenotypic and phylogenetic analysis, the strain was classified as a new species of the genus Streptomyces and designated as Streptomyces VITSVK5 spp. The G + C content of the isolated DNA is 71.3 mol% and the 16S rDNA sequence of the strain showed 93% similarity with Streptomyces MSU2261. The media and growth conditions were optimized for maximal growth under shake flask conditions by measuring the dry weight of mycelium. Eleven multidrug resistant (MDR) Aspergillus strains were isolated from sputum samples collected voluntarily from the pulmonary tuberculosis patients. Results. — The isolated MDR strains showed the MIC value between 4—16 mg/ml against the tested antifungal drugs, amphotericin B, itraconazole, ketoconazole and fluconazole. Ethyl acetate crude extract of Streptomyces VITSVK5 spp. exhibited significant antifungal activity against MDR strains with the zone of inhibition ranging from 22 to 28 mm and the MIC value of 0.125—4 mg/ml. Conclusion. — The isolated Streptomyces is a good candidate for the extraction and the purification of its antifungal metabolite. # 2010 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (K. Kannabiran). 1156-5233/$ — see front matter # 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2010.04.005
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MOTS CLÉS Streptomyces sp. ; Sédiments marins ; Activité antifongique ; Aspergillus
S. Kumar, K. Kannabiran Re ´sume ´ Objectif. — Recherche de souches à activité antifongique. Mate´riel et me´thodes. — Une souche d’actinomycète ayant une activité antifongique sur des isolats d’Aspergillus multirésistants a été isolée à partir d’échantillons de sédiments marins récoltés sur la côte de Marakkanam de la baie du Bengale en Inde. La souche a été identifiée comme appartenant au genre Streptomyces. Sur des critères phénotypiques et phylogénétiques, elle a été considérée comme une nouvelle espèce du genre Streptomyces et dénommée Streptomyces VITSVK5. Son G + C est de 71 % et la séquence de son ADNr 16S montre 93 % de similarité avec Streptomyces MSU2261. Le milieu de culture et les conditions de croissance ont été optimisés en culture agitée par mesure du poids sec du mycélium. Onze souches d’Aspergillus multirésistantes (MDR) ont été isolées des crachats de patients volontaires atteints de tuberculose pulmonaire. Re ´sultats. — Les antifongiques testés sur ces souches résistantes (amphotéricine B, itraconazole, ketoconazole et fluconazole) ont montés des CMI comprises entre 4 et 16 mg/ml. L’extrait à l’éthyl acétate de l’extrait brut de Streptomyces VITSVK5 présente une activité antifongique significative sur les souches MDR avec des zones d’inhibition comprises entre 22 et 28 mm et des CMI comprises entre 0,125 et 4 mg/ml. Conclusion. — La souche isolée est un bon candidat pour la purification et l’identification de l’antifongique en cause. # 2010 Elsevier Masson SAS. Tous droits réservés.
Introduction Aspergillus is a genus of fungi with worldwide distribution and at present approximately 200 officially recognized species are reported. A relatively small number of Aspergillus strains cause human diseases [17]. Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger are the most common human pathogens and inhalation of air borne spores (2— 3 mm) of these fungal pathogens along with prevailing host characteristics or predisposing factors causes pulmonary aspergillosis. Identification of pathogenic Aspergillus strains are relatively more simple and easily distinguished by microscopic examination of conidia colour. A. fumigatus conidia germinates in 5—14 hours at 37 8C and most species of Aspergillus are incapable of growing at 37 8C and this property is therefore a key characteristic feature that distinguishes pathogenic species from non pathogenic species [20]. Invasive aspergillosis causes 100% mortality if untreated [3]. Currently, two antifungal agents, amphotericin B and itraconazole, are often prescribed to treat aspergillosis. But they are often associated with serious side effects such as nephrotoxicity, electrolyte disturbance and hypersensitivity reactions. Continuous use of these antifungal drugs is associated with emergence of multidrug resistant (MDR) Aspergillus strains. The decreased sensitivity of Aspergillus species against available drugs and lack of effective drugs to combat such pathogens continue to be a major challenge for health care providers. Hence, there is an urgent need to search for natural sources with novel bioactive compounds having a broad spectrum of activity with less side effects against drug resistant Aspergillus for effective control and management of aspergillosis. Microorganisms from extreme environments have gained considerable attention in recent years because of its diversity and biological activities, mainly due to its ability to produce novel chemical compounds of high commercial value [1,8]. Actinomycetes are capable of producing several secondary metabolites are a group of prokaryotic organisms; they are gram-positive bacteria that grow extensively in soils
rich with organic matter [2,8]. Majority of the studies on extremophilic organisms however have been confined to the isolation and characterization of extremophilic bacteria but in general marine actinomycetes are relatively less explored for novel bioactive secondary metabolites till date. The marine environment remains as a virtually untapped source for novel actinomycetes [7]. The distribution and abundance of actinomycetes are generally dependent on various ecological habitats, which include beach sand [27] and seawater [28]. In Indian Peninsula, only 41 species of actinobacteria belonging to eight genera have been isolated and studied for its potential role. Only a very few reports are available about their presence, ecological distribution and the diversity of actinomycetes in the southern coastal parts of Indian peninsula. Marine actinomycetes have been shown as the novel source for secondary metabolites and antagonistic activity of isolated secondary metabolites was already reported [5,11— 14]. Despite extensive exploration of actinomycetes for their antimicrobial products in the past, the search for novel molecules having unique therapeutic properties continues to be an active area of research. In this study, we have reported the isolation, characterization and optimization of alkaliphilic and NaCl-tolerant marine actinomycetes; identified and designated as Streptomyces VITSVK5 spp. having antifungal activity against MDR Aspergillus spp. isolated from pulmonary TB patients.
Materials and methods Sampling and isolation of multidrug resistant Aspergillus strains Sputum samples (exporated morning) were collected in a sterile container on two consecutive days from pulmonary TB patients after obtaining approval from the hospital ethical committee and informal consent from patients admitted to the Government Vellore Medical College and Hospital (GVMCH), Vellore, Tamil Nadu, India. The samples were
Antifungal activity of Streptomyces VITSVK5 spp. cultured on sabouraud’s dextrose agar and Aspergillus strains were isolated. Further, it was differentiated and identified using lactophenol cotton blue staining technique [6]. Positive samples were screened for their susceptibility pattern to antifungal drugs namely amphotericin B, itraconazole, fluconazole and ketoconazole and the Aspergillus spp. which showed resistance to these drugs were selected for further studies.
Preparation of drug dilution Stock solution of antifungal drugs, amphotericin B (Himedia, India) was prepared by dissolving 0.032 g in 10 ml distilled water to make a concentration of 3200 mg/ml. Similarly, Itraconazole/Ketoconazole/Fluconazole were prepared by dissolving 0.032 g in 10 ml acetone and 0.2 M HCl (1:1) to make a concentration of 3200 mg/ml. It was then further diluted using RPMI-1640 (Himedia, India) in microtitre assay plate to give varying concentrations (16—0.015 mg/ml) [19].
Isolation and screening of marine actinomycetes Actinomycetes were isolated from marine sediment samples collected at the depth of 20 cm in the Marakkanam coast, (128 12’ 0’’ North, 798 57’ 0’’ East), Bay of Bengal, India. Using a large sterile spatula, the upper soil was removed and with small sterile spatula about 5—10 g of sediment sample (between 5 and 15 cm of depth) was collected in a sterile polythene bag covered with aluminium sheet and transported as quickly as possible to the laboratory. The sediment samples were dried in laminar airflow for 8—12 hours and then kept at 42 8C for 10 to 30 days in a sterile petri dish. These preheated samples were used for the isolation of actinomycetes [26]. The International Streptomyces Project (ISP) No. I, Starch Casein agar and Kuster’s agar medium with 25% sea water and 25% soil extract was used for the isolation of actinomycetes and the growth media was supplemented with the antibiotics cycloheximide (25 mg/ml) and nalidixic acid (25 mg/ml) (Himedia, India). The soil extract was prepared by mixing 400 g of sediments with 1000 ml of distilled water and centrifuged. The supernatant was filtered through 0.45 mm membrane filters (Millipore, India). The clear supernatant obtained was adjusted to a pH of 7—9 and used as a soil extract. Plates prepared with different medium and varying NaCl concentration (3, 5, 7, 9 and 11% [w/v]) were incubated at 28 8C and observed after 2—4 weeks of incubation. The colonies were recognized according to their cultural characteristics and then transferred to slant culture at 4 8C as well as at 20% (v/v) glycerol stock at 80 8C. The isolated actinomycetes were screened for anti-Aspergillus activity against the standard ATCC cultures A. fumigatus (ATCC 6645) and A. niger (ATCC 6404) as well as MDR clinical isolates by well diffusion method.
Culturing conditions The potential isolate was inoculated on different media, SCA, ISP 1, ISP 2, ISP 3, ISP 4, ISP 5, ISP 6, ISP 7, modified Bennett’s agar, Kuster’s agar (Himedia, India) and observed for maximal growth at 7, 14 and 21 days [25]. Different parameters (temperature, pH, sodium chloride, carbon
103 source and organic nitrogen sources) were considered to investigate the influence of various constituents on the growth of the isolate. Media composition was optimized in batch culture and for each parameter the growth was measured as dry weight of the mycelium. Actinobacteria spores (107/mL) were used to inoculate the Bennett medium (peptone 2 g/L, yeast extract 1 g/L and beef extract 1 g/L) in 1000 mL Erlenmeyer flask with five intends; carbon sources at 1% (w/v) (D-mannitol, D-galactose, dextrose, maltose, lactose, raffinose, D-melibiose and sucrose), nitrogen sources, temperature, pH, NaCl concentration for 72 hours in an orbital shaker incubator with shaking at 200 rpm. The influence of magnesium, potassium and trace minerals on the growth of the isolate was also investigated.
Taxonomy The morphological, cultural, physiological and biochemical characterization of the isolate was carried out as described in ISP [25]. The morphology of the spore bearing hyphae with entire spore chain along with substrate and aerial mycelium was examined under light microscope as well as scanning electron microscope (Hitachi, S-3400N). The DNA was isolated from the strain using HiPurA bacterial DNA isolation and purification kit (Himedia, India) and the 16 s rDNA was amplified by PCR using a master mix kit, Medoxmix (Medox, India) as per user manual. The primers and the PCR conditions were adapted from previous reports [21]. The design of the sequencing primers and the methodology for the sequencing were adapted from previous reports [24,18,12]. The 16 s rDNA sequence of the strain was determined on both strands using dideoxy chain termination method. The similarity and homology of the sequence was compared with the existing sequences available in the data bank using BLAST search. The DNA sequences were aligned and phylogenetic tree was constructed by neighbor joining method [10]. The phylogenetic tree based on Maximum-parsimony method was also carried out using Mega 4.0.2—biologist-centric software for evolutionary analysis of DNA and protein sequences [24]. For G + C content determination the DNA was isolated by the method of Marmur [15] and the G + C content was determined using the thermal denaturation method of Marmur and Doty [16].
Extract preparation Well grown slant culture of the potential isolate was used for seed culture preparation by inoculating it in 50 ml medium containing the optimized production medium prepared with sea water 50%, distilled water 50%, pH 8.2 and incubated for 2 days in rotary shaker (200 rpm) at 30 8C. The inoculums (10%) were transferred into 200 ml production medium in 1 liter Erlenmeyer flasks and kept for fermentation for a week. After fermentation the broth was centrifuged at 4000 rpm for 10 minutes at 10 8C and the supernatant was extracted twice with ethyl acetate (400 ml) and washed with 500 ml water. The extract was then concentrated in rotary vacuum and lyophilized. The lyophilized ethyl acetate extract was used to prepare stock using DMSO (16 mg/L). The appropriate concentration of the extract for the antifungal assay was made by serial dilution.
104
Assay of anti-Aspergillus activity of antifungal drugs Conidia of Aspergillus species were taken from well-grown slant and it was adjusted to 1 106 conidia/ml using RPMI medium. It was diluted further with PBS/Tween 20 to make a suspension of 2 103 conidia/ml. It was plated and spread over blood agar plates and incubated at 37 8C for 48 hours and 100 ml of inoculums was added into all the wells in the appropriate row, which makes the final inoculums concentration to 5 105 conidia/ml. The microtitre plates containing varying concentration of antifungal drugs with inoculums were incubated for 48 hours at 37 8C in a moist chamber and the MIC was read visually. As no breakpoints have been established for antifungal drugs, we have followed the following breakpoints: MIC values 0.125—2 mg/ml was considered as sensitive, 4—8 mg/ml as intermediate and 8—16 mg/ ml as resistant and the value > 16 mg/ml was considered as minimal fungicidal concentration (MFC).
Anti-Aspergillus activity of the isolate against MDR clinical isolates The anti-Aspergillus activity (against MDR clinical isolates) of culture broth supernatant and ethyl acetate extract of the isolate was tested by well diffusion technique, and also further evaluated by broth micro dilution method using the standard protocol (CLSI M38—A). The lyophilized ethyl acetate extract was dissolved in DMSO (16 mg/ml) [26] and it was serially diluted with distilled water to get the varying concentration (0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/ ml) and 100 ml of each concentration was placed in the well. The drug-free positive control was also included for comparison. For each MDR isolates, 100 ml of 5 x 105 conidia/ml
Figure 1 The phylogram showing the position of strain VITSVK5 with other Streptomyces based on 16S rDNA sequence. Phylogenetic tree based on neighbor joining analysis of 1000 replicated data. Number at nodes indicates the percent level of bootstrap support. Score bar represents one nucleotide substitution per 100 nucleotides. Bootstrap values of 50 and above are shown. ´ sur les se ´ quences d’ADNr 16S montrant la Dendogramme base position de la souche VITSVK5 par rapport aux autres souches de ´ sente une substitution pour Streptomyces. La barre repre ´ otides. 100 nucle
S. Kumar, K. Kannabiran culture was added into the respective wells. The antifungal activity of ethyl acetate extract of isolate against the standard ATCC cultures A. fumigatus (ATCC 6645) and A. niger (ATCC 6404) was compared with standard antifungal drugs. Microtitre plates were incubated for 48 hours at 37 8C in a moist chamber and the MIC was read visually. Same breakpoints were used to calculate the MIC values as described for antifungal drugs.
Results MDR Aspergillus species from pulmonary TB patients Out of 188 sputum samples collected from TB patients, 69 samples (37%) were positive for Aspergillus spp. and the distribution of individual species in sputum culture was almost same in all the samples. Aspergillus fungal strains were differentiated microscopically and found that A. niger was more predominant followed by A. fumigatus and A. flavus. Aspergillus spp. having MIC value 4—16 mg/ml with at least three tested antifungal drugs were considered as MDR strains. Out of 11 MDR Aspergillus clinical isolates, MDR 1—8 belonged to A. niger and MDR 9—11 were A. fumigatus.
Isolation and identification of the potential strain The potential strain showed maximal growth when cultivated on optimized medium consisting of glucose 0.5%, soluble starch 2%, meat extract 0.4%, yeast extract 0.5%, peptone 0.5%, calcium carbonate 0.4%, sodium sulfate 0.1%, potassium chloride 0.05%, magnesium chloride 0.2%, dipotassium phosphate 0.05%, sodium chloride 22%, sea water 50% and distilled water 50%. Maximal growth was also observed when D-galactose and D-mannitol was used as carbon source. Among the six different amino acids used as nitrogen source, maximal growth was seen with L-cysteine. The potential strain grew well in NaCl at a concentration ranging from 2—10% but maximal growth was observed at 9%. Being a mesophilic organism, it showed a narrow range of incubation temperature for relatively good growth and secondary meta-
Figure 2 Maximum-parsimony tree based on 16S rDNA sequences showing Streptomyces VITSVK5 spp. relationship with other Streptomyces species. Bootstrap values of 50 and above alone are indicated. ´ sur les se ´ quences d’ADNr Arbre de parcimonie maximum base 16S montrant les relations entre les souches VITSVK5 et les ` ces de Streptomyces. autres espe
Antifungal activity of Streptomyces VITSVK5 spp.
105
Table 1 The MIC values of Streptomyces VITSVK5 spp. and standard antifungal drugs against ATCC Aspergillus strains. ´ moins d’Aspergillus. CMI de l’extrait de Streptomyces VITSVK5 spp. et de divers antifongiques sur deux souches te ATCC strains
VITSVK5 (mg/ml)
AMB (mg/ml)
ITR (mg/ml)
FLU (mg/ml)
KET (mg/ml)
A. fumigatus (ATCC 6645) A. niger (ATCC 6404)
0.5 0.25
1.0 2
0.5 1
2 4
4 2
bolite production. The maximal mycelia growth and bioactivity was seen at 30 8C. The strain was also able to grow in the pH range of 7—9. However, the maximal activity was observed at pH 8.2. In the optimized conditions and production medium, the strain produces white spore mass initially and turns to grey after 72 hours. Spores are spherical in shape; arranged in long chains and each chain contains 10—25 spores. The mature spores are 0.5—1.0 mm in diameter and the length is between 0.8 and 1.0 mm.
Taxonomy The isolated strain is a gram positive, non-acid fast, nonmotile, aerobic actinomycete. The aerial mycelium is branched, white to grey in color and the substrate mycelium is branched. Comparison of biochemical and cultural characteristics of the isolate with those of known actinomycetes species described in Bergey’s manual of Systematic Bacteriology strongly suggests that the strain belongs to the genus Streptomyces. The partial sequencing of 16 s rRNA gene of the strain on both directions yielded 16 s rDNA nucleotide sequence with 1424 base pairs. The 16 s rDNA sequence of the strain was deposited in the GenBank (NCBI, USA) under the accession number GQ848482. The BLAST search of 16 s rDNA sequence of the strain showed highest similarity (93%) with Streptomyces MSU2261 (AY23229) and phylogenetic tree was constructed with bootstrap values (Fig. 1). A neighbor-joining tree based on 16 s rDNA sequences showed that the isolate occupies a distinct phylogenetic position within the radiation including representatives of the Streptomycetes family. The phylogenetic tree based on Maximumparsimony method also showed that the isolate forms a separate clade (Fig. 2). Based on the molecular taxonomy
and phylogeny, the strain was identified as novel Streptomyces spp. and designated as Streptomyces VITSVK5 spp.
Anti-Aspergillus activity The MIC value of widely used antifungal drugs against ATCC Aspergillus strains is given in Table 1. The ethyl acetate extract of Streptomyces VITSVK5 spp. showed significant antifungal activity against ATCC Aspergillus strains, A. fumigatus (MIC: 0.5 mg/ml) and A. niger (MIC: 0.25 mg/ ml) when compared to standard antifungal drugs. The MIC value of ethyl acetate extract against MDR Aspergillus clinical isolates is given in Table 2. The antifungal activity was compared with different antifungal drugs, amphotericin B, itraconazole, ketaconazole and fluconazole with MIC values ranging from 4—16 mg/ml. However, MDR strains showed more susceptibility to the ethyl acetate extract with MIC values ranging from 0.125—4 mg/ml. The ethyl acetate extract of Streptomyces VITSVK5 spp. showed least MIC values (0.125 mg/ml) against MDR3, MDR4 and MDR5 (A. niger) clinical isolates.
Discussion The marine sediment sampling site, Marakkanam, is situated in southeast coast of India. It is a narrow sandy coastal belt exhibits tidal flats and marsh zones. It is perched at a height of 14 m (45 feet) above mean sea level and has large areas of saltpans near wide backwater with salinity of 32—38 parts per thousand. A total of 69 actinomycetes were isolated and screened for antifungal activity against Aspergillus species. In this paper, we report the isolation and characterization of Streptomyces VITSVK5 spp. capable of producing secondary
Table 2 The MIC values of ethyl acetate extract of culture broth supernatant of Streptomyces VITSVK5 spp. and of standard antifungal drugs against drug resistant clinical isolates. ´ sistants. CMI de l’extrait ethyl acetate de Streptomyces VITSVK5 sp. et de divers antifongiques sur les isolats d’Aspergillus multire MDR clinical isolates
VITSVK5 (mg/ml)
AMB (mg/ml)
ITR (mg/ml)
FLU (mg/ml)
KET (mg/ml)
MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR
2 0.5 0.125 0.125 0.125 4 0.25 2 4 0.25 2
8 0.25 8 16 8 2 16 1 0.125 0.25 0.25
2 8 4 2 8 4 8 16 8 4 2
4 8 2 8 1 1 8 4 0.5 4 8
16 8 16 4 2 8 8 16 4 4 4
1 2 3 4 5 6 7 8 9 10 11
AMB: amphotericin B; ITR: itraconazole; FLU: fluconazole; KET: ketoconazole.
106 metabolite having antifungal activity against MDR clinical isolates of Aspergillus species. Cultural characteristics and media composition were optimized in batch culture based on the growth of the strain. As the samples were collected from the ocean near-salt pans, the salt concentration in the media was increased and the growth was evaluated at varying concentration of NaCl and pH. The cultural and morphological characterization strictly indicates that the strain belongs to Streptomyces spp. It is evident from the phylogenetic tree that the strain is a novel species, which forms a separate subclade from other members of Streptomyces. A tree constructed by the Maximum-parsimony method also showed that the strain forms a distinct phyletic line from other closest related strains available in the database. The strain is 93% similar with Streptomyces MSU2261 available with the culture data bank. Due to the unavailability of phenotypic data of the strain (MSU2261) in the culture data bank, we are unable to do the comparative phenotypic studies and DNA relatedness studies. The ethyl acetate extract of Streptomyces VITSVK5 spp. showed significant activity against MDR Aspergillus strains (A. fumigatus and A. niger) when compared to standard antifungal drugs. The MIC values exhibited by the ethyl acetate extract were better than the standard antifungal drugs tested. The most frequently diagnosed fungal infections are caused by pathogens from the genera Aspergillus and Candida [22]. These fungi are ubiquitous and can be acquired from host surroundings (A. fumigatus). Exposure of A. fumigatus conidia frequently causes nosocomial acquired infections and community acquired infections in immunocompromised as well as in immunocompetent people and often results in pulmonary aspergillosis [9]. In the long list of currently available anti-infective drugs in the market, antifungal drugs are very few, but they form a significant group of drugs having an important role in the control of life-threatening fungal infections [30]. Most of the studies conducted in Indian Peninsula have been restricted to isolation, identification and maintenance of actinobacteria in different culture media and studied for their antagonistic properties against different microbial pathogens. An actinobacteria, Streptomyces spp. PM-32 isolated from offshore sediments collected at the Bay of Bengal coast has been shown to possess antimicrobial activity against a group of bacterial and fungal pathogens with moderate antagonistic activity against Aspergillus species [29]. Antagonistic activity of different Streptomyces species isolated from soil samples has already been reported earlier [23]. Anti-Aspergillus (A. niger) activity of Streptomyces species isolated from coastal water of Dhanushkodi, Tamil Nadu India has been reported earlier [4]. It is suggested that the frequent and systematic screening of marine actinomycetes in the Indian peninsula could provide novel species as well as novel bioactive compounds which could be developed as lead compound for future therapeutic applications. Extraction and identification of the lead compound from Streptomyces VITSVK5 spp. and its mechanism of action on MDR Aspergillus strains are in progress.
Conflicts of interest The authors have not declared any conflicts of interest.
S. Kumar, K. Kannabiran
Acknowledgements Authors thanks the management of VIT University for supporting research and also thanks the Dean Government Vellore Medical College and Hospital (GVMCH), Vellore, Tamil Nadu, India for providing clinical samples.
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ORIGINAL ARTICLE / ARTICLE ORIGINAL
Antifungal activity of Streptomyces VITSVK5 spp. against drug resistant Aspergillus clinical isolates from pulmonary tuberculosis patients ´ antifongique de Streptomyces VITSVK5 sp. sur des isolats d’Aspergillus Activite ´ sistants isole ´ s de patients atteints de tuberculose pulmonaire mutire S. Kumar, K. Kannabiran * Biomolecules and Genetics Division, School of biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India Received 17 November 2009; received in revised form 14 April 2010; accepted 16 April 2010
KEYWORDS Streptomyces spp.; Marine sediments; Antifungal activity; Aspergillus
Summary Objective. — Research of isolates producing antifungal activity. Material and methods. — An actinomycetes strain having anti-Aspergillus activity against multidrug drug resistant clinical isolates was isolated from marine sediment samples collected at Marakkanam coast of Bay of Bengal, India. The strain was characterized by polyphasic taxonomic approach and identified as a member of the genus Streptomyces. Based on the phenotypic and phylogenetic analysis, the strain was classified as a new species of the genus Streptomyces and designated as Streptomyces VITSVK5 spp. The G + C content of the isolated DNA is 71.3 mol% and the 16S rDNA sequence of the strain showed 93% similarity with Streptomyces MSU2261. The media and growth conditions were optimized for maximal growth under shake flask conditions by measuring the dry weight of mycelium. Eleven multidrug resistant (MDR) Aspergillus strains were isolated from sputum samples collected voluntarily from the pulmonary tuberculosis patients. Results. — The isolated MDR strains showed the MIC value between 4—16 mg/ml against the tested antifungal drugs, amphotericin B, itraconazole, ketoconazole and fluconazole. Ethyl acetate crude extract of Streptomyces VITSVK5 spp. exhibited significant antifungal activity against MDR strains with the zone of inhibition ranging from 22 to 28 mm and the MIC value of 0.125—4 mg/ml. Conclusion. — The isolated Streptomyces is a good candidate for the extraction and the purification of its antifungal metabolite. # 2010 Elsevier Masson SAS. All rights reserved.
* Corresponding author. E-mail address: [email protected] (K. Kannabiran). 1156-5233/$ — see front matter # 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.mycmed.2010.04.005
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MOTS CLÉS Streptomyces sp. ; Sédiments marins ; Activité antifongique ; Aspergillus
S. Kumar, K. Kannabiran Re ´sume ´ Objectif. — Recherche de souches à activité antifongique. Mate´riel et me´thodes. — Une souche d’actinomycète ayant une activité antifongique sur des isolats d’Aspergillus multirésistants a été isolée à partir d’échantillons de sédiments marins récoltés sur la côte de Marakkanam de la baie du Bengale en Inde. La souche a été identifiée comme appartenant au genre Streptomyces. Sur des critères phénotypiques et phylogénétiques, elle a été considérée comme une nouvelle espèce du genre Streptomyces et dénommée Streptomyces VITSVK5. Son G + C est de 71 % et la séquence de son ADNr 16S montre 93 % de similarité avec Streptomyces MSU2261. Le milieu de culture et les conditions de croissance ont été optimisés en culture agitée par mesure du poids sec du mycélium. Onze souches d’Aspergillus multirésistantes (MDR) ont été isolées des crachats de patients volontaires atteints de tuberculose pulmonaire. Re ´sultats. — Les antifongiques testés sur ces souches résistantes (amphotéricine B, itraconazole, ketoconazole et fluconazole) ont montés des CMI comprises entre 4 et 16 mg/ml. L’extrait à l’éthyl acétate de l’extrait brut de Streptomyces VITSVK5 présente une activité antifongique significative sur les souches MDR avec des zones d’inhibition comprises entre 22 et 28 mm et des CMI comprises entre 0,125 et 4 mg/ml. Conclusion. — La souche isolée est un bon candidat pour la purification et l’identification de l’antifongique en cause. # 2010 Elsevier Masson SAS. Tous droits réservés.
Introduction Aspergillus is a genus of fungi with worldwide distribution and at present approximately 200 officially recognized species are reported. A relatively small number of Aspergillus strains cause human diseases [17]. Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger are the most common human pathogens and inhalation of air borne spores (2— 3 mm) of these fungal pathogens along with prevailing host characteristics or predisposing factors causes pulmonary aspergillosis. Identification of pathogenic Aspergillus strains are relatively more simple and easily distinguished by microscopic examination of conidia colour. A. fumigatus conidia germinates in 5—14 hours at 37 8C and most species of Aspergillus are incapable of growing at 37 8C and this property is therefore a key characteristic feature that distinguishes pathogenic species from non pathogenic species [20]. Invasive aspergillosis causes 100% mortality if untreated [3]. Currently, two antifungal agents, amphotericin B and itraconazole, are often prescribed to treat aspergillosis. But they are often associated with serious side effects such as nephrotoxicity, electrolyte disturbance and hypersensitivity reactions. Continuous use of these antifungal drugs is associated with emergence of multidrug resistant (MDR) Aspergillus strains. The decreased sensitivity of Aspergillus species against available drugs and lack of effective drugs to combat such pathogens continue to be a major challenge for health care providers. Hence, there is an urgent need to search for natural sources with novel bioactive compounds having a broad spectrum of activity with less side effects against drug resistant Aspergillus for effective control and management of aspergillosis. Microorganisms from extreme environments have gained considerable attention in recent years because of its diversity and biological activities, mainly due to its ability to produce novel chemical compounds of high commercial value [1,8]. Actinomycetes are capable of producing several secondary metabolites are a group of prokaryotic organisms; they are gram-positive bacteria that grow extensively in soils
rich with organic matter [2,8]. Majority of the studies on extremophilic organisms however have been confined to the isolation and characterization of extremophilic bacteria but in general marine actinomycetes are relatively less explored for novel bioactive secondary metabolites till date. The marine environment remains as a virtually untapped source for novel actinomycetes [7]. The distribution and abundance of actinomycetes are generally dependent on various ecological habitats, which include beach sand [27] and seawater [28]. In Indian Peninsula, only 41 species of actinobacteria belonging to eight genera have been isolated and studied for its potential role. Only a very few reports are available about their presence, ecological distribution and the diversity of actinomycetes in the southern coastal parts of Indian peninsula. Marine actinomycetes have been shown as the novel source for secondary metabolites and antagonistic activity of isolated secondary metabolites was already reported [5,11— 14]. Despite extensive exploration of actinomycetes for their antimicrobial products in the past, the search for novel molecules having unique therapeutic properties continues to be an active area of research. In this study, we have reported the isolation, characterization and optimization of alkaliphilic and NaCl-tolerant marine actinomycetes; identified and designated as Streptomyces VITSVK5 spp. having antifungal activity against MDR Aspergillus spp. isolated from pulmonary TB patients.
Materials and methods Sampling and isolation of multidrug resistant Aspergillus strains Sputum samples (exporated morning) were collected in a sterile container on two consecutive days from pulmonary TB patients after obtaining approval from the hospital ethical committee and informal consent from patients admitted to the Government Vellore Medical College and Hospital (GVMCH), Vellore, Tamil Nadu, India. The samples were
Antifungal activity of Streptomyces VITSVK5 spp. cultured on sabouraud’s dextrose agar and Aspergillus strains were isolated. Further, it was differentiated and identified using lactophenol cotton blue staining technique [6]. Positive samples were screened for their susceptibility pattern to antifungal drugs namely amphotericin B, itraconazole, fluconazole and ketoconazole and the Aspergillus spp. which showed resistance to these drugs were selected for further studies.
Preparation of drug dilution Stock solution of antifungal drugs, amphotericin B (Himedia, India) was prepared by dissolving 0.032 g in 10 ml distilled water to make a concentration of 3200 mg/ml. Similarly, Itraconazole/Ketoconazole/Fluconazole were prepared by dissolving 0.032 g in 10 ml acetone and 0.2 M HCl (1:1) to make a concentration of 3200 mg/ml. It was then further diluted using RPMI-1640 (Himedia, India) in microtitre assay plate to give varying concentrations (16—0.015 mg/ml) [19].
Isolation and screening of marine actinomycetes Actinomycetes were isolated from marine sediment samples collected at the depth of 20 cm in the Marakkanam coast, (128 12’ 0’’ North, 798 57’ 0’’ East), Bay of Bengal, India. Using a large sterile spatula, the upper soil was removed and with small sterile spatula about 5—10 g of sediment sample (between 5 and 15 cm of depth) was collected in a sterile polythene bag covered with aluminium sheet and transported as quickly as possible to the laboratory. The sediment samples were dried in laminar airflow for 8—12 hours and then kept at 42 8C for 10 to 30 days in a sterile petri dish. These preheated samples were used for the isolation of actinomycetes [26]. The International Streptomyces Project (ISP) No. I, Starch Casein agar and Kuster’s agar medium with 25% sea water and 25% soil extract was used for the isolation of actinomycetes and the growth media was supplemented with the antibiotics cycloheximide (25 mg/ml) and nalidixic acid (25 mg/ml) (Himedia, India). The soil extract was prepared by mixing 400 g of sediments with 1000 ml of distilled water and centrifuged. The supernatant was filtered through 0.45 mm membrane filters (Millipore, India). The clear supernatant obtained was adjusted to a pH of 7—9 and used as a soil extract. Plates prepared with different medium and varying NaCl concentration (3, 5, 7, 9 and 11% [w/v]) were incubated at 28 8C and observed after 2—4 weeks of incubation. The colonies were recognized according to their cultural characteristics and then transferred to slant culture at 4 8C as well as at 20% (v/v) glycerol stock at 80 8C. The isolated actinomycetes were screened for anti-Aspergillus activity against the standard ATCC cultures A. fumigatus (ATCC 6645) and A. niger (ATCC 6404) as well as MDR clinical isolates by well diffusion method.
Culturing conditions The potential isolate was inoculated on different media, SCA, ISP 1, ISP 2, ISP 3, ISP 4, ISP 5, ISP 6, ISP 7, modified Bennett’s agar, Kuster’s agar (Himedia, India) and observed for maximal growth at 7, 14 and 21 days [25]. Different parameters (temperature, pH, sodium chloride, carbon
103 source and organic nitrogen sources) were considered to investigate the influence of various constituents on the growth of the isolate. Media composition was optimized in batch culture and for each parameter the growth was measured as dry weight of the mycelium. Actinobacteria spores (107/mL) were used to inoculate the Bennett medium (peptone 2 g/L, yeast extract 1 g/L and beef extract 1 g/L) in 1000 mL Erlenmeyer flask with five intends; carbon sources at 1% (w/v) (D-mannitol, D-galactose, dextrose, maltose, lactose, raffinose, D-melibiose and sucrose), nitrogen sources, temperature, pH, NaCl concentration for 72 hours in an orbital shaker incubator with shaking at 200 rpm. The influence of magnesium, potassium and trace minerals on the growth of the isolate was also investigated.
Taxonomy The morphological, cultural, physiological and biochemical characterization of the isolate was carried out as described in ISP [25]. The morphology of the spore bearing hyphae with entire spore chain along with substrate and aerial mycelium was examined under light microscope as well as scanning electron microscope (Hitachi, S-3400N). The DNA was isolated from the strain using HiPurA bacterial DNA isolation and purification kit (Himedia, India) and the 16 s rDNA was amplified by PCR using a master mix kit, Medoxmix (Medox, India) as per user manual. The primers and the PCR conditions were adapted from previous reports [21]. The design of the sequencing primers and the methodology for the sequencing were adapted from previous reports [24,18,12]. The 16 s rDNA sequence of the strain was determined on both strands using dideoxy chain termination method. The similarity and homology of the sequence was compared with the existing sequences available in the data bank using BLAST search. The DNA sequences were aligned and phylogenetic tree was constructed by neighbor joining method [10]. The phylogenetic tree based on Maximum-parsimony method was also carried out using Mega 4.0.2—biologist-centric software for evolutionary analysis of DNA and protein sequences [24]. For G + C content determination the DNA was isolated by the method of Marmur [15] and the G + C content was determined using the thermal denaturation method of Marmur and Doty [16].
Extract preparation Well grown slant culture of the potential isolate was used for seed culture preparation by inoculating it in 50 ml medium containing the optimized production medium prepared with sea water 50%, distilled water 50%, pH 8.2 and incubated for 2 days in rotary shaker (200 rpm) at 30 8C. The inoculums (10%) were transferred into 200 ml production medium in 1 liter Erlenmeyer flasks and kept for fermentation for a week. After fermentation the broth was centrifuged at 4000 rpm for 10 minutes at 10 8C and the supernatant was extracted twice with ethyl acetate (400 ml) and washed with 500 ml water. The extract was then concentrated in rotary vacuum and lyophilized. The lyophilized ethyl acetate extract was used to prepare stock using DMSO (16 mg/L). The appropriate concentration of the extract for the antifungal assay was made by serial dilution.
104
Assay of anti-Aspergillus activity of antifungal drugs Conidia of Aspergillus species were taken from well-grown slant and it was adjusted to 1 106 conidia/ml using RPMI medium. It was diluted further with PBS/Tween 20 to make a suspension of 2 103 conidia/ml. It was plated and spread over blood agar plates and incubated at 37 8C for 48 hours and 100 ml of inoculums was added into all the wells in the appropriate row, which makes the final inoculums concentration to 5 105 conidia/ml. The microtitre plates containing varying concentration of antifungal drugs with inoculums were incubated for 48 hours at 37 8C in a moist chamber and the MIC was read visually. As no breakpoints have been established for antifungal drugs, we have followed the following breakpoints: MIC values 0.125—2 mg/ml was considered as sensitive, 4—8 mg/ml as intermediate and 8—16 mg/ ml as resistant and the value > 16 mg/ml was considered as minimal fungicidal concentration (MFC).
Anti-Aspergillus activity of the isolate against MDR clinical isolates The anti-Aspergillus activity (against MDR clinical isolates) of culture broth supernatant and ethyl acetate extract of the isolate was tested by well diffusion technique, and also further evaluated by broth micro dilution method using the standard protocol (CLSI M38—A). The lyophilized ethyl acetate extract was dissolved in DMSO (16 mg/ml) [26] and it was serially diluted with distilled water to get the varying concentration (0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg/ ml) and 100 ml of each concentration was placed in the well. The drug-free positive control was also included for comparison. For each MDR isolates, 100 ml of 5 x 105 conidia/ml
Figure 1 The phylogram showing the position of strain VITSVK5 with other Streptomyces based on 16S rDNA sequence. Phylogenetic tree based on neighbor joining analysis of 1000 replicated data. Number at nodes indicates the percent level of bootstrap support. Score bar represents one nucleotide substitution per 100 nucleotides. Bootstrap values of 50 and above are shown. ´ sur les se ´ quences d’ADNr 16S montrant la Dendogramme base position de la souche VITSVK5 par rapport aux autres souches de ´ sente une substitution pour Streptomyces. La barre repre ´ otides. 100 nucle
S. Kumar, K. Kannabiran culture was added into the respective wells. The antifungal activity of ethyl acetate extract of isolate against the standard ATCC cultures A. fumigatus (ATCC 6645) and A. niger (ATCC 6404) was compared with standard antifungal drugs. Microtitre plates were incubated for 48 hours at 37 8C in a moist chamber and the MIC was read visually. Same breakpoints were used to calculate the MIC values as described for antifungal drugs.
Results MDR Aspergillus species from pulmonary TB patients Out of 188 sputum samples collected from TB patients, 69 samples (37%) were positive for Aspergillus spp. and the distribution of individual species in sputum culture was almost same in all the samples. Aspergillus fungal strains were differentiated microscopically and found that A. niger was more predominant followed by A. fumigatus and A. flavus. Aspergillus spp. having MIC value 4—16 mg/ml with at least three tested antifungal drugs were considered as MDR strains. Out of 11 MDR Aspergillus clinical isolates, MDR 1—8 belonged to A. niger and MDR 9—11 were A. fumigatus.
Isolation and identification of the potential strain The potential strain showed maximal growth when cultivated on optimized medium consisting of glucose 0.5%, soluble starch 2%, meat extract 0.4%, yeast extract 0.5%, peptone 0.5%, calcium carbonate 0.4%, sodium sulfate 0.1%, potassium chloride 0.05%, magnesium chloride 0.2%, dipotassium phosphate 0.05%, sodium chloride 22%, sea water 50% and distilled water 50%. Maximal growth was also observed when D-galactose and D-mannitol was used as carbon source. Among the six different amino acids used as nitrogen source, maximal growth was seen with L-cysteine. The potential strain grew well in NaCl at a concentration ranging from 2—10% but maximal growth was observed at 9%. Being a mesophilic organism, it showed a narrow range of incubation temperature for relatively good growth and secondary meta-
Figure 2 Maximum-parsimony tree based on 16S rDNA sequences showing Streptomyces VITSVK5 spp. relationship with other Streptomyces species. Bootstrap values of 50 and above alone are indicated. ´ sur les se ´ quences d’ADNr Arbre de parcimonie maximum base 16S montrant les relations entre les souches VITSVK5 et les ` ces de Streptomyces. autres espe
Antifungal activity of Streptomyces VITSVK5 spp.
105
Table 1 The MIC values of Streptomyces VITSVK5 spp. and standard antifungal drugs against ATCC Aspergillus strains. ´ moins d’Aspergillus. CMI de l’extrait de Streptomyces VITSVK5 spp. et de divers antifongiques sur deux souches te ATCC strains
VITSVK5 (mg/ml)
AMB (mg/ml)
ITR (mg/ml)
FLU (mg/ml)
KET (mg/ml)
A. fumigatus (ATCC 6645) A. niger (ATCC 6404)
0.5 0.25
1.0 2
0.5 1
2 4
4 2
bolite production. The maximal mycelia growth and bioactivity was seen at 30 8C. The strain was also able to grow in the pH range of 7—9. However, the maximal activity was observed at pH 8.2. In the optimized conditions and production medium, the strain produces white spore mass initially and turns to grey after 72 hours. Spores are spherical in shape; arranged in long chains and each chain contains 10—25 spores. The mature spores are 0.5—1.0 mm in diameter and the length is between 0.8 and 1.0 mm.
Taxonomy The isolated strain is a gram positive, non-acid fast, nonmotile, aerobic actinomycete. The aerial mycelium is branched, white to grey in color and the substrate mycelium is branched. Comparison of biochemical and cultural characteristics of the isolate with those of known actinomycetes species described in Bergey’s manual of Systematic Bacteriology strongly suggests that the strain belongs to the genus Streptomyces. The partial sequencing of 16 s rRNA gene of the strain on both directions yielded 16 s rDNA nucleotide sequence with 1424 base pairs. The 16 s rDNA sequence of the strain was deposited in the GenBank (NCBI, USA) under the accession number GQ848482. The BLAST search of 16 s rDNA sequence of the strain showed highest similarity (93%) with Streptomyces MSU2261 (AY23229) and phylogenetic tree was constructed with bootstrap values (Fig. 1). A neighbor-joining tree based on 16 s rDNA sequences showed that the isolate occupies a distinct phylogenetic position within the radiation including representatives of the Streptomycetes family. The phylogenetic tree based on Maximumparsimony method also showed that the isolate forms a separate clade (Fig. 2). Based on the molecular taxonomy
and phylogeny, the strain was identified as novel Streptomyces spp. and designated as Streptomyces VITSVK5 spp.
Anti-Aspergillus activity The MIC value of widely used antifungal drugs against ATCC Aspergillus strains is given in Table 1. The ethyl acetate extract of Streptomyces VITSVK5 spp. showed significant antifungal activity against ATCC Aspergillus strains, A. fumigatus (MIC: 0.5 mg/ml) and A. niger (MIC: 0.25 mg/ ml) when compared to standard antifungal drugs. The MIC value of ethyl acetate extract against MDR Aspergillus clinical isolates is given in Table 2. The antifungal activity was compared with different antifungal drugs, amphotericin B, itraconazole, ketaconazole and fluconazole with MIC values ranging from 4—16 mg/ml. However, MDR strains showed more susceptibility to the ethyl acetate extract with MIC values ranging from 0.125—4 mg/ml. The ethyl acetate extract of Streptomyces VITSVK5 spp. showed least MIC values (0.125 mg/ml) against MDR3, MDR4 and MDR5 (A. niger) clinical isolates.
Discussion The marine sediment sampling site, Marakkanam, is situated in southeast coast of India. It is a narrow sandy coastal belt exhibits tidal flats and marsh zones. It is perched at a height of 14 m (45 feet) above mean sea level and has large areas of saltpans near wide backwater with salinity of 32—38 parts per thousand. A total of 69 actinomycetes were isolated and screened for antifungal activity against Aspergillus species. In this paper, we report the isolation and characterization of Streptomyces VITSVK5 spp. capable of producing secondary
Table 2 The MIC values of ethyl acetate extract of culture broth supernatant of Streptomyces VITSVK5 spp. and of standard antifungal drugs against drug resistant clinical isolates. ´ sistants. CMI de l’extrait ethyl acetate de Streptomyces VITSVK5 sp. et de divers antifongiques sur les isolats d’Aspergillus multire MDR clinical isolates
VITSVK5 (mg/ml)
AMB (mg/ml)
ITR (mg/ml)
FLU (mg/ml)
KET (mg/ml)
MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR MDR
2 0.5 0.125 0.125 0.125 4 0.25 2 4 0.25 2
8 0.25 8 16 8 2 16 1 0.125 0.25 0.25
2 8 4 2 8 4 8 16 8 4 2
4 8 2 8 1 1 8 4 0.5 4 8
16 8 16 4 2 8 8 16 4 4 4
1 2 3 4 5 6 7 8 9 10 11
AMB: amphotericin B; ITR: itraconazole; FLU: fluconazole; KET: ketoconazole.
106 metabolite having antifungal activity against MDR clinical isolates of Aspergillus species. Cultural characteristics and media composition were optimized in batch culture based on the growth of the strain. As the samples were collected from the ocean near-salt pans, the salt concentration in the media was increased and the growth was evaluated at varying concentration of NaCl and pH. The cultural and morphological characterization strictly indicates that the strain belongs to Streptomyces spp. It is evident from the phylogenetic tree that the strain is a novel species, which forms a separate subclade from other members of Streptomyces. A tree constructed by the Maximum-parsimony method also showed that the strain forms a distinct phyletic line from other closest related strains available in the database. The strain is 93% similar with Streptomyces MSU2261 available with the culture data bank. Due to the unavailability of phenotypic data of the strain (MSU2261) in the culture data bank, we are unable to do the comparative phenotypic studies and DNA relatedness studies. The ethyl acetate extract of Streptomyces VITSVK5 spp. showed significant activity against MDR Aspergillus strains (A. fumigatus and A. niger) when compared to standard antifungal drugs. The MIC values exhibited by the ethyl acetate extract were better than the standard antifungal drugs tested. The most frequently diagnosed fungal infections are caused by pathogens from the genera Aspergillus and Candida [22]. These fungi are ubiquitous and can be acquired from host surroundings (A. fumigatus). Exposure of A. fumigatus conidia frequently causes nosocomial acquired infections and community acquired infections in immunocompromised as well as in immunocompetent people and often results in pulmonary aspergillosis [9]. In the long list of currently available anti-infective drugs in the market, antifungal drugs are very few, but they form a significant group of drugs having an important role in the control of life-threatening fungal infections [30]. Most of the studies conducted in Indian Peninsula have been restricted to isolation, identification and maintenance of actinobacteria in different culture media and studied for their antagonistic properties against different microbial pathogens. An actinobacteria, Streptomyces spp. PM-32 isolated from offshore sediments collected at the Bay of Bengal coast has been shown to possess antimicrobial activity against a group of bacterial and fungal pathogens with moderate antagonistic activity against Aspergillus species [29]. Antagonistic activity of different Streptomyces species isolated from soil samples has already been reported earlier [23]. Anti-Aspergillus (A. niger) activity of Streptomyces species isolated from coastal water of Dhanushkodi, Tamil Nadu India has been reported earlier [4]. It is suggested that the frequent and systematic screening of marine actinomycetes in the Indian peninsula could provide novel species as well as novel bioactive compounds which could be developed as lead compound for future therapeutic applications. Extraction and identification of the lead compound from Streptomyces VITSVK5 spp. and its mechanism of action on MDR Aspergillus strains are in progress.
Conflicts of interest The authors have not declared any conflicts of interest.
S. Kumar, K. Kannabiran
Acknowledgements Authors thanks the management of VIT University for supporting research and also thanks the Dean Government Vellore Medical College and Hospital (GVMCH), Vellore, Tamil Nadu, India for providing clinical samples.
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