Isolation and Characterization of an actinomycete strain producing an antifungal metabolite effective against Candida albicans

 

Adithya Chandrashekar, Anuraag Muralidharan, Ananthamurthy Koteshwara,

Angel Treasa Alex, V. M. Subrahmanyam*

Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal- 576104, Karnataka, India

*Corresponding Author E-mail: vm.subra@manipal.edu

 

ABSTRACT:

Novel antifungals are challenging to find as potential target may have close human orthologs. Among the pathogenic fungi, Candida albicans is the most prevalent pathogen. Screening of actinomycetes for antifungals has been an aggressive area of research since decades. For discovering newer metabolites against Candida albicans, work was oriented in isolation of actinomycetes for evaluating the antifungal activity of their cultural extracts. A total of 115 strains were isolated from different soil samples using Starch Casein Agar. Mueller Hinton Agar and Glucose Soybean Meal were used as suitable assay and cultivation medium. Only 11.30% isolates were significantly active and isolate M-23 was found promising. Metabolite production reached maximum on the 7th day of incubation at 30⁰C using modified Glucose Soybean Meal medium. The culture supernatant and its ethyl acetate extract fraction had inhibitory zone diameters of 25mm and 30mm. Effect of addition sodium acetate as precursor was insignificant. M-23 was identified as Streptomyces coelicolor by 16SrDNA sequencing. The metabolite was partially characterized using various physicochemical tests. The UV spectrum showed three absorbances in 360-405nm region. The crude extract gave an IC50 of 256.5μg/ml against vero cell lines. This strain can find use in the production of newer antifungals.

 

KEYWORDS: Actinomycetes, Antifungal, Streptomyces coelicolor.

 

 


INTRODUCTION:

The incidence of fungal infections has increased since the early 1980s, leading to high morbidity and mortality rates. Worldwide, more than 800 million people suffered or are suffering from one or other type of fungal infections[1]. Organisms belonging to the Candida species are the usual habitants of the human hosts, causing diverse array of infections. There are only limited number of potential antifungal drugs that play a crucial role in combating the surging mycotic infections which has put up an intrinsic challenge on the Pharmaceutical sector to meet the dire requirement of novel antifungal molecules. Hence, new broad-spectrum agents with novel mechanism of action are required and search for these remains a daunting task[2].

 

 

 

 

Soil is being extensively screened for isolation of Streptomyces species, a class of actinomycetes. This species constitutes 50% of terrestrial actinomycetes population and interestingly 75-80% of the therapeutically useful metabolites and antibiotics have been derived from this genus[3]. Some marine actinomycetes are reported to produce various novel and complex antitumor, cytotoxic, antibacterial, and antifungal metabolites[4]. However, the production of such metabolites especially antibiotics is greatly influenced by the cultural and nutritional parameters. Improvements in the production yield could be made by incorporating essential nutrients in the cultivation medium[5]. Discovery of new potent antifungal drugs is an urgent medical need. Development of such compounds from microbial origin especially from actinomycetes is making sufficient roads in the Pharmaceutical sector over the recent years. In the present study, a systematic screening programme with terrestrial and marine soil samples was carried out to isolate actinomycetes for the production of antifungal metabolite (s) effective against Candida albicans. The potential strain was characterized and identified. Partial characterization of the metabolite was also carried out to identify the nature of the metabolite.

 

MATERIALS AND METHODS:

Materials:

All the chemicals procured were of analytical grade, unless stated otherwise.

 

Methods:

Collection of soil samples:

A total of 3 different indigenous soil samples were from different locations viz., Mumbai Juhu Beach, Chennai Marina Beach and Kemmanu-Udupi, India. The samples were dried and stored at 4⁰C[6].

 

Pre-treatment of soil samples:

The soil samples were crushed using mortar and pestle into uniform fine particles for ease in isolation of individual actinomycete colonies and prevent contamination due to fungal cells[7].

 

Isolation of actinomycetes:

Primary screening:

Actinomycetes were isolated using Starch casein agar by soil-plate technique[8]. A pinch of the soil samples was spread in separate petri plates. SCA was poured and mixed uniformly. The plates were incubated at 30±2⁰C for 10-12 days. The isolates were tested for their inhibitory activities against Candida albicans on Mueller Hinton agar using agar plug assay[9]. The appearance of clear zone around the agar plug indicated inhibitory activity.

 

Secondary screening:

The active isolates from in the primary screening process were evaluated for their antifungal activity as described in M44-A guidelines by cup-plate assay[10]. These isolates were inoculated in separate conical flasks containing 25ml of 1% Glucose Soybean medium and incubated at 30⁰C on a rotary shaker at 150rpm for 7-10 days[11]. After incubation the culture harvest was centrifuged at 10,000rpm for 20mins. The cell free supernatant was analysed for its antifungal activity by cup-plate assay[12]. The potential and promising isolates were chosen based on Kirby-Bauer method[13].

 

Metabolite Production:

The most active strain(s) selected from the secondary screening was cultivated on modified Glucose Soybean Medium (g/100ml: Glucose 1, Soybean meal extract 1, NaCl 1, KH2PO4 0.1, K2HPO4 0.1, CaCO3 0.01) with incubation at 30⁰C on a rotary shaker at 150rpm for 7-10 days. Sodium acetate as precursor was added at 0.1% level to another production flask with similar cultural conditions to study the effect on antifungal metabolite production[12,14].

 

Extraction of the metabolite:

The culture harvest of the most potent isolate was centrifuged at 10,000rpm for 20 minutes. The active metabolites were recovered from the extracellular cell-free supernatant by solvent extraction process. In this process, ethyl acetate and petroleum ether were used for extraction. The culture harvest and the solvent were mixed in 1:1 ratio and the active metabolites were extracted using rotary evaporator. The extracts were evaluated for antifungal activity. The results were compared with appropriate controls and standard antifungal antibiotics Nystatin and Amphotericin B[15].

 

Characterization of the isolate:

The isolate having the most potent antifungal activity as identified from the secondary screening was further characterized based on morphological and physiological characteristics. The actinomycete like colonies were confirmed by light microscopy by using inclined cover slip method[16]. The physiological characters were studied using ISP 6 and 7 as mentioned in International Journal of Systemic Bacteriology by Shirling and Gottlieb, 1966[17,18]. The sugar utilization ability was studied using HiIMViC Biochemical Test Kit. The identity of the organism was confirmed by 16s rDNA molecular sequencing, performed by using Fast MicroSeq 16S 500 at Gujarat State Biotechnology Mission (GSBTM), Gandhinagar, Gujarat, India.

 

Characterization of the metabolite:

Preliminary phytochemical investigation tests of the purified metabolite were carried based on the standard protocols[19,20]. Solubility of the metabolite was checked by dissolving it in solvents like water, methanol, DMSO, 5% NaOH, 5% NaHCO3 etc. Presence of unsaturation was checked by treating with neutral KMnO4 solution. The extracts were checked for the presence of Carbohydrates by Molisch’s Test and Fehling’s Solution Test, Carboxylic Acids by Litmus Test, Proteins and Free Amino Acids by Biuret Test, Phenols by Ferric Chloride Test, Flavonoids by Shinoda Test and Sterols by Salkowski Test[21]. The crude dried extract was further partially characterized using Thin Layer Chromatography and UV Spectroscopy[22].

 

Thin Layer Chromatography:

Solvent system of ethyl acetate: hexane: acetone: methanol (2:1:1:2) was used to run the sample. The purity was analysed based on the Rf values calculated.

 

UV Profile:

The UV spectrum 200-800nm region was obtained to determine the λ max. This was carried out to determine the absorbance region which could provide some information about the chromophore and nature of the metabolite[23,24].

Evaluation of the extracts for cytotoxicity:

The cytotoxic activity of the extract was determined by MTT assay using Vero cells. The absorbance was read at 540nm using micro-plate reader[6]. IC50 values were determined by calculating % of viability.

 

RESULTS AND DISCUSSION:

Results:

Collection of soil samples:

Actinomycetes are extensively studied for the exploration of their bioactivities. Therefore, with this objective of isolation of actinomycetes, soil samples from various localities were collected aseptically. 

 

Pre-treatment of soil samples:

Pre-treatment of the soil sample is an important step in the isolation of actinomycetes. Crushing the soil samples helped in easy isolation and identification of actinomycetes as individual distinct colonies as observed by uniformity in their growth pattern on the isolation medium. It was observed that exposing the soil samples to higher temperature reduces the contamination due to unwanted vegetative bacterial and fungal cells to a minimum level.

 

Isolation of actinomycetes:

Primary Screening:

A total of 115 colonies resembling actinomycetes were isolated from primary screening. The colonies were identified on the basis of physical and morphological features viz., size, colour, shape and texture. The active isolates were evaluated for their antibiotic activity by agar plug assay Figure 1. Mueller Hinton agar (MHA) was used as an antibiotic assay medium for the evaluation of antifungal activity. The appearance of clear zone around the agar plug indicated the antibiotic activity due to diffusion of the metabolite produced. Out of these isolates, 70 isolates were found to have inhibitory activity against Candida albicans.

 

Figure 1: Agar Plug assay

 

Secondary Screening:

The antifungal activity of the 70 active isolates was further evaluated by cup-plate assay method. Based on research data available 1% GSM was found suitable for the growth of actinomycetes. The culture supernatants showing average inhibitory zone diameter of 15mm and above were considered significant and chosen for further studies. A total of 13 isolates had this inhibitory zone diameter and among these M-23 which exhibited the highest average inhibitory zone diameter of 25mm was chosen for further cultivation and extraction of the metabolites Table 1

 

Table 1: Cup-plate assay for 13 isolates

Isolates from Soil Samples

*(M-Mumbai Juhu Beach, C-Chennai Marina Beach, U-Udupi-Kemmanu)

Average zone diameter (mm) triplicate readings

M-11

16

M-12

15

M-13

18

M-23

25

M-36

16

M-38

12

M-46

15

M-47

12

M-53

13

M-54

12

M-64

14

C-11

15

U-11

14

 

Metabolite Production:

Modified Glucose Soybean meal medium with the addition of few essential trace elements and salts was employed for the growth of M-23 and production of the metabolite. It was observed that the addition of trace elements to 1% GSM increased the rate of production of the metabolite on 7th day. The addition of sodium acetate as precursor did not alter the yield significantly. The antifungal activity of the culture supernatants was evaluated by cup-plate assay and reported in Table 2.

 

Table 2: Cup-plate assay

Parameter

Inhibitory zone diameter (mm)

1% GSM

25

Modified 1% GSM 

25

Modified 1% GSM with Precursor 

22

 

Extraction of the metabolite:

The culture harvest of M-23 was centrifuged and the metabolite (s) was extracted using solvent extraction. Ethyl acetate and Petroleum ether were the choice of solvents. The crude extracts were then evaluated for their antifungal activity by cup-plate assay- Table 3. It was observed that the ethyl acetate extract had promising and stable activity with inhibitory zone diameter of 30mm compared to the activity of petroleum ether extract with 26mm zone diameter- Figure 2. Hence, the ethyl acetate fraction was chosen for further analysis. Due to the intermediate polarity of the solvent, and with relevant research data available it was assumed that majority of the metabolites produced could be extracted using ethyl acetate.

Table 3: Cup-plate assay results of extracts of M-23

Parameter

Inhibitory zone diameter (mm)

Ethyl Acetate Extract

30

Petroleum Ether Extract

26

Nystatin 25µg

18

Amphotericin B 25µg

20

 

Figure 2: Cup-plate assay

 

Characterization of the isolate:

The strain M-23 was chosen for further characterization based of International Journal of Systemic Bacteriology described by Shirling and Gottlieb, 1966. The morphological and cultural characteristics viz., aerial and substrate mycelium characteristics were studied employing ISP media 2, 4 and 5 as shown in Figures 3, 4 and 5. The organism was capable of utilizing carbon, nitrogen sources and other nutrients present in the respective medium. Based on these studies it can be inferred that the isolate belonged to the genus Streptomyces. The organism exhibited yellowish grey aerial mycelium and the substrate mycelium was dark brown in colour. The physiological characters for melanin production studied using ISP 6 and 7 showed that the melanoid pigments were absent. The sugar utilization and biochemical characteristics studied using the Biochemical Test Kit are illustrated in Table 4. Phenotypically the strain was observed to be non-motile and Gram Positive. The molecular characterization by 16s rDNA further confirmed the identity of the isolate as Streptomyces coelicolor provided by Gujarat State Biotechnology Mission, Gandhinagar. 

 

Figure 3: ISP 2

 

Figure 4: ISP 4   

 

Figure 5: ISP 5  

 

Table 4: Physiological Sugar Utilization and Biochemical Tests

Tests

Results

Glucose 

++

Adonitol 

+

Arabinose 

+

Lactose 

-

Sorbitol 

-

Mannitol 

-

Sucrose 

+

Rhamnose 

+

Catalase Test

-

Urease Test

-

Gelatin Liquefaction 

-

H2S Test

+

IMViC Test

-

 

Characterization of the metabolite:

The physical properties were studied based on the basic investigation tests as given above. The metabolite could possibly be aromatic in nature. The yellow colour of the compound indicated the possibility of nitro groups. The compound was semi-solid with a faint characteristic odour at room temperature. The presence of unsaturation was confirmed on the basis of reaction with potassium permanganate solution. The extract was partially soluble in methanol, water, 5% NaOH, 5% and NaHCO3, completely soluble in ethyl acetate and acetonitrile which indicated that the metabolite could be slightly non-polar. The extract was further analysed for detection of various functional groups as discussed in Table 5. The presence of conjugation and steroidal moiety explains the complexity of the structure. The phenolic group may be attributed to the antimicrobial activity of the compound.

 

Table 5: Chemical Tests

Chemical Tests

Results

Phenolic Compounds

+

Free Amino Acids and Proteins

-

Sterols

+

Flavonoids

-

 

Thin Layer Chromatography:

Separation of the active compound (s) from the extract was carried out as mentioned in the methodology. The TLC plate was observed under UV light. Three distinct spots with Rf values 0.725, 0.55 and 0.675 were visible in the ethyl acetate fraction. The Rf values were compared with standard drugs to get an idea about the type of compound that could be present.

UV Profile:

Three peaks were observed in 360-405nm range, with high intense peak at 405nm. The three different components seem to be closely related. Further characterization is needed to predict the structure and identity of the compound. The metabolite could be polyenic in nature as majority of the antifungal antibiotics are polyenic- Figure 6

 

Figure 6: UV Profile

 

Cytotoxicity Assay:

The cytotoxic studies were carried out on Vero cell lines to check the toxicity potential and the IC50 value was found to be 256.5µg/ml by using appropriate formula-

 

Table 6: Cytotoxicity Assay

Concentration (µg/ml)

IC50 Value

500

94.022

250

47.353

125

24.520

62.6

9.191

 

DISCUSSION:

Its well-known fact that fungi are opportunistic organisms that will invade only if the host’s immune response is weakened[1]. With the exponential emergence of resistant Candida albicans to the available antifungal drugs and patient sensitivity issues, the issue has acquired a great impetus for the continuous search of novel antibiotics. Research reports on antibiotics from rare forms of actinomycetes are very few and there is an imperative need to isolate such antibiotics. Generally, rare forms of this genera are found in marine sediments [5,12]. As such, marine soils were explored for possible isolation, Okazaki and Okami investigated the antibiotic production potential of actinomycetes from Sagami Bay, Japan and observed the diversity of the strains and compounds produced by them. Also, they had reported few novel active substances having unique antimicrobial spectrum. Soil selection thus plays an important role in isolation of bioactive actinomycetes. In the present study it was found during the isolation process, Mumbai Juhu Beach soil offered a promising source for many actinomycetes. From a total of 115 isolates, 70 strains had shown antifungal properties among which 13 strains were found to possess significant inhibitory activities with inhibitory zone diameter > 15 mm. The strain M-23 further characterized and reported as Streptomyces coelicolor, was investigated as it had shown the maximum inhibitory zone diameter of 25 mm in the secondary screening and its ethyl acetate extract gave 30 mm diameter. The pH of the soil was 7.2 and was found to be ideal in the isolation process of actinomycetes. Streptomyces coelicolor had 77.5% similarity with Streptomyces anulatus[18]. The antifungal activity of Streptomyces coelicolor has not been investigated in detail. Literature reports predict that there are some derived mutants of this species viz., Streptomyces violaceruber exhibited antimicrobial activity due to a compound celiomycin[6,14]. Microorganisms are the biggest source for wide variety of therapeutically useful agents which may not only serve as direct drugs but also as lead compounds for structural modifications and templates for rational drug design. In this aspect the microbial sources offer better chances for satisfactory scale-up. Antibiotics, especially the microbial metabolites have several common structural features, the most common being cyclopeptide frameworks and various macrocyclic lactone ring systems, particularly in the actinomycetales products[23]. The ethyl acetate fraction showed a zone diameter of 30mm which seems promising. Ethyl acetate was used, as maximum amount of metabolite was retained in the organic phase. Similar methodology was adopted by Gebreselema and Feleke during the isolation of actinomycetes[11]. The metabolite was found to be non-toxic with an IC50 of 256.5µg/ml against Vero cell lines which indicates that the metabolite has minimal cytotoxic effects. This cytotoxic study is similar to the study carried out by Sudha during characterization of a metabolite obtained from an actinomycete[6]. The strain Streptomyces coelicolor can thus find further use in the discovery of newer antifungal metabolites.

 

SUMMARY AND CONCLUSION:

The metabolic potential of actinomycetes offers a strong area of research. The actinomycete isolated and identified as Streptomyces coelicolor offers a great platform to discover promising antifungal compounds. Further studies on characterization of the organism pertaining to the gene responsible and the structure of the metabolite need to be explored.

 

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India for providing the necessary facilities to carry out the study. The authors are thankful to DBT for sanctioning a project in the area of antifungal drugs, Project ID: BT/PR10827/AAQ/3/661/2014. Also, the authors are thankful to Gujarat State Biotechnology Mission, Gandhinagar, Gujarat, India for identification of the microorganism.

 

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Received on 31.01.2019           Modified on 10.03.2019

Accepted on 28.05.2019         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(10):4601-4606.

DOI: 10.5958/0974-360X.2019.00791.1