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, KH 2PO4 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 Average zone
*(M-Mumbai Juhu Beach, diameter
C-Chennai Marina Beach, (mm)
U-Udupi-Kemmanu) 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 22
with Precursor

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 30
Extract
Petroleum Ether 26
Extract
Nystatin 25µg 18
Amphotericin B 20
25µg

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