Beni-Suef University Journal of Basic and Applied Sciences 7 (2018) 250–256

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Beni-Suef University
Journal of Basic and Applied Sciences
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Full Length Article

Isolation and molecular characterization of antifungal metabolite producing T

actinomycete from mangrove soil
⁎
Mary Sulakshana Pallaa, , Girija Shankar Guntukua, Murali Krishna Kumar Muthyalaa,
Sirisha Pingalia, Prafulla Kumar Sahub
a
Department of Biotechnology, Andhra University College of Pharmaceutical Sciences, Visakhapatnam 530013, Andhra Pradesh, India
b
Department of Pharmaceutical Analysis, Raghu College of Pharmacy, Dakamarri, Bheemunipatnam, Visakhapatnam 531162, Andhra Pradesh, India

A R T I C L E I N F O A B S T R A C T

Keywords: The main object of the present study is to isolate and characterize the actinomycetes from Koringa mangrove soil
Streptomyces samples near Kakinada, Andhra Pradesh, India. Isolated actinomycetes were assessed for antagonistic activity
Dermatophytes against various bacteria and fungi. The potent strain KMFA-1 having activity against dermatophytes Candida
Antagonistic activity albicans and Pectinotrichum llanense was characterized. Based on physiological, morphological characteristics and
16S rRNA gene sequencing
16s rRNA gene sequencing the isolated strain was identified as Streptomyces hydrogenans. Extraction was done
Bioactive metabolite
with different solvents and the activity was evaluated for each solvent extract. The antibiotic that is responsible
for activity is found to be highly water soluble. The bioactive metabolite present in crude supernatant was found
to be stable at 80 °C for 1 h, 90% activity was retained after boiling at 100 °C for 1 h, but 53.4% activity was
reduced after autoclaving (at 121 °C for 15 min). The activity was not lost when the crude supernatant was
subjected to wide range of pH (2–10). Furthermore, analytical techniques must be employed to isolate the
molecule of interest. The result of present study revealed that the Streptomyces hydrogenans produced an inter-
esting antifungal metabolite.

1. Introduction material. Actinomycetes particularly Streptomyces are well known as

Mangroves are saline resistant forest ecosystems found in tropical
and sub – tropical intertidal regions around the world (Ottoni et al.,
2015). Mangrove soils provide a unique ecological niche for the growth
of diversified microorganisms which find use in recycling environ-
mental nutrients and production of exclusive secondary metabolites of
pharmaceutical importance. Several factors constantly vary in man-
grove ecosystem which cause adaptations in metabolic pathways that
led to the biosynthesis of unique metabolites (Thatoi et al., 2013). Di-
verse groups of microorganisms such as actinomycetes, fungi, bacteria,
macro and micro algae and fungus-like protozoa harbour in mangrove
forests. The total microbial community of tropical mangrove forest
comprise 91% of bacteria and fungi, 7% of algae and 2% of protozoa.
Though there is an extensive microbial diversity in mangrove eco-
system, only less than 1% of the microbial community has been re-
vealed, remaining 99% of the microorganisms were not discovered.
Only 5% of the isolated microbes have been examined chemically and
these include bacteria, fungi and actinomycetes (Xu, 2015).
Actinomycetes are group of aerobic, branched, unicellular Gram-
positive bacteria with high percentage of GC (70%) in their genetic
major sources of secondary metabolites particularly antibiotics (Berdy,
2005). Commercially important bio-molecules are majorly produced by
Streptomyces, Micromonospora, Saccharopolyspora, Amycolatopsis and
Actinoplanes. Approximately, 80% of known antibiotics have been
produced from streptomyces. Thus the great importance was given to
genus Streptomycetes as they are numerous, has high metabolic pro-
duction rate, their role in recycling organic matter and their capability
to degrade chitin, lignocelluloses etc. (Khanna et al., 2011). Isolation of
actinomycetes from unexplored soil samples may provide novel bioac-
tive metabolites. Mangrove soils are potential source of rare actino-
mycete possessing antimicrobial activity against pathogenic micro-
organisms. There is a plenty of scope to isolate such rare strains that
produce novel compounds beneficial to mankind. Although existence of
actinomycetes in mangrove soils is reportedly less when compared to
terrestrial regions, attempts were made to isolate exclusive bioactive
products of pharmaceutical importance.
Many researchers discovered novel actinobacteria from poorly ex-
plored mangrove habitats such as isolation of Asanoa iriomotensis
(Tamura and Sakane, 2005), Nonomuraea maheshkhaliensis (Ara et al.,
2008) and Streptomyces xiamenensis (Xu et al., 2009). Fungi are found to

⁎
Corresponding author.
E-mail address: mary.pharma114@gmail.com (M.S. Palla).

https://doi.org/10.1016/j.bjbas.2018.02.006
Received 15 September 2017; Received in revised form 27 January 2018; Accepted 28 February 2018
Available online 06 March 2018
2314-8535/ © 2018 Beni-Suef University. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M.S. Palla et al.
be important pathogens and among which C. albicans is the most
common pathogen in several nosocomial infections and control efforts
should target such fungal infections (Sagure et al., 1993). The present
work involves the isolation of a streptomyces strains from Koringa
mangrove soil, with potential to produce a potent antifungal compound
exclusively against Candida albicans was described. Further the isolated
actinomycete was characterized and identified based on morphological,
physiological, biochemical, and genomic features.
2. Materials and methods
2.1. Collection of mangrove soil sample
In the present study, mangrove soil samples were collected from
Koringa Mada Forest in Kakinada. The sampling area was located be-
tween 16°-30′ to 17°-00′ N latitudes and 82°-14′ to 82°-23′E longitudes.
Several sizes of samples from various places (east, west, north and south
directions) within the sampling area were collected in sterilized poly-
thene bags.
2.2. Isolation and maintenance of culture
Following collection, the samples were screened for isolation of
microorganisms. One gram of soil sample was accurately weighed and
transferred to 50 mL of sterile water, mixed well and then placed on
rotary shaker at 120 rpm for 30 min. The resultant solution was serially
diluted up to 10−10 (10−1, 10−2 ……… 10−10) with sterile water.
One millilitre of each intermediate dilution (10−1, 10−2 ……… and
10−10) was added to 50 mL of sterile molten Starch Casein Agar
medium with 50% sea water individually in separate flasks.
Antimicrobial agents such as Rifampicin (25 µg/mL) and
Cycloheximide (50 µg/mL) were used as supplements to the nutrient
media in order to prevent contamination from surrounding environ-
ment. Rifampicin inhibits the growth of bacteria and Cycloheximide
facilitates inhibition of fungi. 1 mL each of these antimicrobial agents
was added to 50 mL of above medium and plated in sterile petriplates of
30 cm size. The plates were incubated for the growth of actinomycetes
colonies at 28 °C and observed intermittently during incubation (Bizuye
et al., 2013). After 7 days of incubation, the colonies showing the
characteristics of actinomycetes (rough, chalky, powdery appearance
radiating growth and leathery texture) were observed. The pure co-
lonies were selected, isolated and maintained on ISP-2 (yeast extract
malt extract medium, HIMEDIA) at 28 °C for 7–14 days. (Malt Extract
1.0%, Yeast Extract 0.4%, Dextrose 0.4%, Agar 2%, Distilled water
50%, Seawater 50%, pH adjusted to 7.2).
2.3. Test organisms
All the actinomycetes were tested for antibacterial activity and an-
tifungal activity. Antibacterial activity against Bacillus subtilis MTCC
441, Escherichia coli MTCC 443, Pseudomonas aeruginosa MTCC 424,
Klebsiella pneumonia MTCC 109, Micrococcus luteus MTCC 106,
Staphylococcus aureus MTCC 3160, Proteus vulgaris NCIM 2813 and an-
tifungal activity against Aspergillus niger MTCC 282, Aspergillus oryzae
NCIM 643, Penicillium chrysogenum MTCC 161, Candida albicans MTCC
183. The bacterial cultures were subcultured on nutrient agar medium
and incubated at 37 °C for 24 h, fungal cultures were subcultured on
sabouraud dextrose agar medium at 25 °C for 48 h.
2.4. Preliminary screening of actinomycetes for antimicrobial activity
The preliminary screening of the isolates against various test or-
ganisms mentioned above (section 2.3.) was performed by cross streak
and agar overlay methods. In cross streak method, yeast extract malt
extract (YEME) agar (HIMEDIA) plates were used, inoculated with the
isolates by single streak at the centre of petriplate and incubated at
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Beni-Suef University Journal of Basic and Applied Sciences 7 (2018) 250–256
28 °C for 7 days. The test organisms were streaked perpendicularly to
the actinomycetes and incubated (bacteria were incubated overnight at
37°C and fungi at 25 °C for 2–5 days). The antagonism against each test
organism was recorded.
In agar overlay method, actinomycete isolates were spot inoculated
on YEME agar plates and incubated 28 ± 2 °C for 7 days. Subsequently,
the plates were overlaid with nutrient agar and sabouraud dextrose
agar medium with low concentration of agar (0.75 g /100 mL) seeded
with bacterial and fungal cultures respectively. The plates were again
incubated at 37 °C for 18–24 h in case of bacterial cultures and at 25 °C
for 48–120 h in case of fungal cultures. The inhibition zone diameter (in
mm) around the colonies was recorded.
2.5. Production of antifungal antibiotic
The production of secondary metabolite was carried out by sub-
merged fermentation using a suitable production medium. YEME broth
was used as inoculum medium to increase the cell mass of the isolate.
The inoculum was developed by cultivating 5 mL of suspension of mi-
croorganism in 45 mL of the sterilized YEME broth medium kept on
rotary shaker (150 rpm) at 28 ± 2 °C for 2 days. After 2 days, 5–10% of
metabolically active inoculum was transferred to sterile production
medium and kept on rotary shaker (150 rpm) at 28 ± 2 °C for 7 days.
Both the inoculum and production media were supplemented with 50%
Sea water in its composition. After the fermentation, supernatant was
collected by centrifugation in cooling centrifuge at 5000 rpm for
15 min. The cell mass was discarded and the activity of crude super-
natant was tested against test fungi.
2.6. Secondary screening of the isolate KMFA-1
Among the actinomycetes isolated, the strain KMFA-1 (Koringa
Mangrove Forest -Actinomycete) that exhibited potent antagonistic
activity against C. albicans and P. llenense was tested for its extracellular
bioactive metabolite production. The selected isolate was grown in
seven different media for the production of bioactive metabolite
(150 rpm) at 28 °C for 7 days. After growth, the culture broth was se-
parated by centrifuge (5000 rpm/20 min) and the cell free clear su-
pernatant was used for secondary screening.
The antimicrobial activity of crude supernatant was tested by agar
well diffusion method against Candida albicans and Pectinotrichum lla-
nense. The wells were excavated by using sterile cork borer (6 mm
diameter). The activity was evaluated by adding 50µL of the super-
natant to solidified agar medium seeded with 50µL of test organism
suspensions. The plates were placed in refrigerator for 20 min to allow
diffusion of antibiotic and then kept for incubation at 25 °C for 48 h.
The clear inhibition zone diameter around the well was recorded and all
the assays were done in triplicate.
2.7. Characterization of the potent actinomycete (KMFA-1)
The potent actinomycete (KMFA-1) was identified based on cultural,
morphological, physiological and biochemical characteristics according
to standard protocols of International Streptomyces Project (Shirling and
Gottlieb, 1966). The cultural characteristics such as colour of substrate
and aerial mycelia, growth pattern, colony texture, and colony reverse
colour were examined by culturing the potent actinomycete on ISP-2
medium (Pridham, 1965).
2.7.1. Morphological characterization
The isolate characteristics such as arrangement of spores, spore
bearing hyphae, substrate and aerial mycelium were observed visually
using a trinocular microscope (LABOMED USA CXR3 9122100, Labo
America Inc.) under 400× magnification. The Scanning Electron
Microscopy (SEM) (Model-JEOL_JSM 6610 LV) Studies were performed
to reveal the micro-morphology of the strain.
M.S. Palla et al.
Fig. 1. Preliminary screening of antimicrobial activity: (A) antibacterial (Cross streak method); Agar over lay method of the KMFA-1 isolate against (B) A. niger, (C) P. chrysogenum (D) C.
albicans, and (E) P. llanense.
Table 1
Antifungal activity of KMFA-1 by agar diffusion method.
S.No. Sample Inhibition zone Diameter (mm)
C. albicans
1 Crude extract of the isolate KMFA-1 30 ± 0.28
2 Ketoconazole (50 µg/mL) 41 ± 0.57
The preparation of the sample was done by inclined coverslip
method (Williams and Davies, 1967). For SEM, slides were cut into 1 cm
pieces and sterilized. The pieces were inserted into solidified starch
casein agar medium at an angle of 45°. The inoculum was spread along
the glass-agar medium interface. During incubation, the organisms
grew over the surface of the glass pieces. The growth from the glass
pieces were removed carefully using a brush and affixed onto the
copper stud, washed with serial grades of 30, 50, 70 and 90% alcohol.
The studs were then kept in a desiccator for final drying. The surface
containing organisms was coated with a film of gold about 150-200A
thickness and observed under SEM for spore surface ornamentation.
2.7.2. Chemotaxonomy
Cell wall composition was analyzed according to the method of
Boone and Pine (Boone and Pine, 1968). Culture was grown in YEME
broth for 7 days. After the incubation period the mycelia was separated
by centrifugation at 10,000 rpm for 15 min and washed thrice with
sterile water. The cells were then examined for the composition of
amino acids and sugars.
2.7.2.1. Identification of sugars:. The cell wall sample (0.7 mL) was
taken and HCl was added to it (to give a final concentration of 2 N HCl)
in screw capped tubes, sealed tightly and placed in a boiling water bath
for 2 h. The hydrolyzed materials were transferred to small beakers and
dried over a boiling water bath. To this water was again added, the
process was repeated four times and the materials were finally
suspended in 500 µL of water. Then 50 µL of sample was spotted on
paper (Whatman No. 1) and ascending chromatography was run using
the solvent system n-butanol, acetic acid and water (3:1:1). The
chromatogram was sprayed with a solution containing 0.5 g silver
Beni-Suef University Journal of Basic and Applied Sciences 7 (2018) 250–256
P. llanense
17 ± 0.5
32 ± 0.57
Fig. 2. Spore chain morphology of isolate KMFA-1 under 400× magnification.
nitrate, 1 mL water and 25 mL of 95% ethanol. The papers were then
dried for 2–3 min until the appearance of dark spots.
2.7.2.2. Identification of amino acids:. The cell wall samples (0.3 mL)
were taken in a sealed tube and hydrolyzed with HCl (to give a final
concentration 6 N HCl) at 110 °C for 18 h. The hydrolyzed material was
dried in the same way as mentioned for sugar identification procedure.
One-tenth mL of the same sample was spotted on Whatman No. 1 paper
and ascending chromatography was run using the solvent n-butanol,
acetic acid and water (4:1:1). The presence of amino acids is identified
by spraying with 0.25% Nihydrin and drying at 100 °C for 5 min.
2.7.3. Physiological and biochemical characterization of the selected isolate
The growth of the isolate was tested at different temperatures
within 12–40 °C, pH ranges (pH 5.0 to 10.0) and at different con-
centrations of NaCl (2–10%w/v). The carbohydrate utilization was
tested on various carbon sources such as galactose, glucose, fructose
arabinose, sucrose, mannitol, xylose, raffinose, salicin, rhamnose, and
meso-inositol. Biochemical tests such as fermentation of carbohydrates,
starch hydrolysis, nitrate reduction, casein hydrolysis, citrate utilization
tests, methyl red, indole, oxidase, catalase, voges-proskauer, urea, were
also carried out for the isolate KMFA-1.
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Fig. 3. Scanning electron micrographs of the KMFA-1 isolate grown on Yeast extract-malt extract agar medium at 28 °C for 7 days (A) Bar, 1 µm (B) Bar, 5 µm.

Fig. 4. 16S rRNA sequencing of the isolate KMFA-1.

Fig. 5. Phylogenetic analysis based on 16S rRNA gene sequencing showing the position of KMFA-1 in Neighbour-joining tree.

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Table 2 2.8. Extraction of bioactive compound from the isolate KMFA-1

Physiological and Biochemical characteristics of isolate KMFA-1.
The selected isolate KMFA-1 culture broth was collected and cen-
Test Result
trifuged at 5000 rpm for 15 min. The supernatant containing anti-
Gram staining + microbial compound was extracted with various solvent such as
Casein hydrolysis + hexane, ethyl acetate, methanol and each time antifungal activity for
Starch hydrolysis +
organic and aqueous fractions were determined using cup plate
Citrate utilization −
Voges proskauer − method.
Methyl red −
Indole production − 2.9. Stability of bioactive metabolite in culture supernatant
Nitrate reduction −
Catalase +
Urea − The stability of bioactive metabolite in crude supernatant was tested
Oxidase utilization − by subjecting it to different temperatures −20 °C, 37 °C, 80 °C, 100 °C
Cell wall composition (presence of LL-DAP) + for 1 h and autoclaving at 121 °C/15 lbs pressure for 20 min. The sta-
bility of the compound was also tested by adjusting pH ranges from 2 to
+: positive; −: negative.
10. All the samples were then tested for antagonism effect against C.
albicans.
Table 3
Utilization of carbon, Tolerance to temperature, pH and NaCl of
The stability of the bioactive metabolite during storage was eval-
the isolate KMFA-1. uated by maintaining the crude supernatant under refrigerator condi-
tions and assessing the antifungal activity for a period of 24 months.
Test Result

Carbon utilization 3. Results

Arabinose +
Glucose − 3.1. Isolation and screening of actinomycete from mangrove soils
Galactose −
Fructose +
Mannose − The present study involves the screening of novel antibiotic pro-
Meso-inositol − ducing actinomycetes from Mangrove soils. Since population density of
Rhamnose − actinomycete in mangrove soils is comparatively less than terrestrial
Salicin − regions, few colonies of actinomycete were isolated on starch casein
Raffinose −
Sucrose +
agar medium supplemented with sea water (50%v/v). All the isolates
Xylose − were subjected to preliminary and secondary screening against various
bacteria and fungi. Interestingly, the isolate KMFA-1 exhibited selective
Temperature
Growth at 12 °C − action against pathogenic dermatophytes (Fig. 1 and Table 1). The
Growth at 25–40 °C + isolate KMFA-1 was subcultured on YEME agar medium supplemented
pH tolerance with sea water (50%v/v) and preserved under refrigerated conditions
Growth at pH 5–10 + for further studies.
NaCl tolerance
NaCl tolerance at 2–7% + 3.2. Characterization of the isolate
NaCl tolerance at 10% −

The isolated strain was analyzed for physiological, biochemical and

molecular characteristics. The isolated strain was aerobic and non-
2.7.4. Genotypic characterization of actinomycete
motile Gram positive, cell wall chemotype I, glabrous or chalky, rela-
The 16S rRNA gene sequencing and phylogenetic tree construction
tively rapid growing, earthy odour with aerial and substrate mycelium
of the selected isolate KMFA-1 were done at Microbial Type Culture
formation. The diameter of the colony ranges from 1 to 2 cm and the
Collection, Institute of Microbial Technology (IMTECH), Chandigarh,
growth rate of the isolate was fairly rapid. The colour of the colony was
India. The Basic Local Alignment Search Tool (BLAST), database of
white initially becoming reddish brown on the reverse side. Thin, short,
NCBI in silico was conducted to assess the similarity between the se-
wavy mycelia with 3 to 4 turns were observed under 400× (Fig. 2).
quences.
Presence of LL-diaminopimelic acid (LL-DAP) in the peptidoglycan cell
wall and absence of whole-cell sugars were observed from the chemo-
taxonomic studies. The SEM analysis (Fig. 3) of the isolate showed thin,

Fig. 6. Antimicrobial activity of different solvent extracts and aqueous fractions of the KMFA-1 isolate by cup plate method (A) Ethylacetate extract, (B) Methanolic extract, (C) Hexane
extract.

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M.S. Palla et al.
Fig. 7. Effect of temperature variation on stability of bioactive metabolite present in culture supernatant of KMFA-1 strain. Culture supernatant was subjected to different temperatures.
entangled spirals, aerial mycelia of various lengths with matured hy-
phae showing 10 to 50 spores per chain. Thin, spiny spores were ob-
served in 14 day culture grown on YEME agar medium. These are found
to be the unique characteristics of the isolate. The isolated strain was
identified as Streptomyces hydrogenans on the basis of 16S rRNA se-
quencing (Fig. 4) and phylogenetic tree (Fig. 5). The phylogenetic tree
of the selected isolate clearly revealed its evolutionary relationship with
a group of streptomyces species generated by a neighbour-joining
method with the aid of MEGA 6.0 program (Tamura et al., 2013).
The isolate was mesophilic and exhibited amylase, catalase, pro-
tease activities. Oxidase, indole and hydrogen sulphide were not pro-
duced, also exhibits negative for citrate utilization, urea reaction,
voges-proskauer test, methyl red test, nitrate reduction (Table 2). The
isolate KMFA-1 utilized the carbon sources such as arabinose, fructose
and sucrose. However, glucose, galactose, xylose, raffinose, rhamnose,
mannitol, salicin, and meso-inositol were not utilized. The isolate can
grow up to 40 °C with pH 10 and 7% NaCl, the optimum growth tem-
perature was found to be 28 ± 2 °C (Table 3).
3.3. Extraction of active metabolite
Among the various solvent extracts, the methanolic and aqueous
crude extracts of active strain KMFA-1 showed good antifungal activity
against C. albicans and P. llanense, whereas very low activity was ex-
hibited by ethylacetate and no activity with hexane crude extracts
(Fig. 6).
3.4. Stability of bioactive metabolite in crude supernatant
The bioactive metabolite in the crude supernatant was subjected to
wide range of temperatures and pH. Active metabolite responsible for
antagonistic activity was completely stable at temperatures ranging
from -20°C to 80°C for 1 h. However, 10% of activity was reduced after
boiling (100°C for 1 h) and 53% of the activity was reduced after au-
toclaving (121°C for 15 min) (Fig. 7). There is no loss in activity over a
broad range of pH indicating its pH stability.
4. Discussion
Fungal infections have significantly increased in the past decade.
Due to limited availability of antifungal drugs in clinical use and de-
velopment of drug resistance, there is a need for development of new
class of antifungal drugs with minimal side effects (Gozalbo et al.,
2004). The potential antifungal activity of Streptomyces strains from
different sources is well known. Nevertheless, limited studies on acti-
nomycetes are available and worldwide surveys have recognised that
mangrove ecosystems are rich in microbial diversity. Hence, searching
for effective antibiotics from the mangrove ecosystem is the primary
objective of this endeavour. Very little information is available
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Beni-Suef University Journal of Basic and Applied Sciences 7 (2018) 250–256
regarding isolation of actinomycetes from mangrove soils. Sengupta
and coauthors isolated nine distinct and biologically active actino-
bacteria from mangroves (Sengupta et al., 2015). In a study, it was
highlighted that mangroves are becoming an important source for
discovering unique actinomycetes that produce robust novel bioactive
metabolites due to their adaptation towards high salinity and tidal
gradients (Hong et al., 2009, Malek et al., 2014).
The preliminary screening by cross streak and agar overlay method
showed that the selected isolate KMFA-1 was a good antifungal pro-
ducer against C. albicans and P. llanense. The secondary screening ex-
hibited the antifungal activity of the crude supernatant, where the clear
zone surrounding the wells on inoculated plates is an indication of
antibiotic (extracellular metabolite) release from the actinomycete.
Morphological and biochemical characteristics revealed the uniqueness
of the isolate KMFA-1. The isolate is found to be salt tolerant, meso-
philic and having an excellent potential to produce antifungal meta-
bolite and enzymes. The crude antifungal metabolite produced by the
streptomyces spp. isolate KMFA-1 is found to thermostable and the
antifungal activity was not lost over a wide range of pH (2–10) in-
dicating that it is active at various physiological pH. The antifungal
activity of crude supernatant was not lost even after 24 months at 4 °C,
suggesting that the bioactive metabolite possess longer shelf life at re-
frigerated temperature.
C. albicans was found to be the third most common pathogen
causing superficial infections such as oral candidiasis or vaginal can-
didiasis and systemic life-threatening disease, so there is a need of novel
antifungal agent preferably with unique mode of action (Diekema et al.,
2002; Mayer et al., 2013). This study is the first report on the isolate
Streptomyces hydrogenans having potent antagonistic activity against C.
albicans and P. llanense. The specific nature of this bioactive metabolite
produced by the selected isolate might be a diversified molecule and
exerts unique mode of action to inhibit the growth of C. albicans.
Generally, the possible mechanisms of antifungal activity are due to
degradation of cell wall hydrolytic enzymes and/or production of an-
tifungal metabolites. Chitinases, proteases, or β-1,3 glucanase are im-
portant hydrolytic enzymes that cause degradation of fungal cell wall
(Vicente et al., 2003; Kaur and Manhas, 2014; Jayamurthy et al., 2014).
To assess whether Streptomyces hydrogenans produced hydrolytic en-
zymes or antifungal compounds, the crude supernatant was extracted
with different solvents. Since the activity was retained in the aqueous
portion, it indicates that the antifungal activity may be due to an en-
zyme or a highly polar antifungal metabolite. Thermo stability of the
product further indicated the presence of heat stable antifungal com-
pound. It has been reported that Streptomyces violaceusniger YCDE9
(Prapagdee et al., 2008) and Streptomyces hygroscopicus (Trejo-Estrada
et al., 1998) have the ability to produce hydrolytic enzymes during
exponential phase as well as antifungal metabolites during stationary
phases. The antifungal activity of Streptomyces hydrogenans is attributed
to the presence of highly polar secondary metabolite that was produced
M.S. Palla et al.
during stationary phase. In addition, the activity remained stable for
5 days and then gradually decreased.
Recent reports by Kaur and co-authors have largely focussed on
isolation of actinobacterium strain and metabolites from soil
(Dalhousie, 32.53°N, 75.98° Himachal Pradesh, India) and exploration
of its potency against fungal phyto-pathogens (Kaur and Manhas,
2014); (Kaur et al., 2016). There is scanty literature available regarding
the production of anti-fungal compounds from mangrove soil isolates.
Comprehensive studies were not carried out for isolation of strains
producing novel secondary metabolites and their characterization. In
contrast, our current findings demonstrate a distinct line of objectivity
in terms of source, isolation, application, mechanism of action and
stability.
5. Conclusion
The present study suggests that actinomycete that was isolated from
mangrove ecosystem can have an immense potential to produce unique
high quality novel bioactive compounds against dermatophytes.
Further investigations are necessary for isolation and chemical char-
acterization of the compound by chromatographic and other spectral
analysis.
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