INTRODUCTION:
Soil
harbour the life of variety types of microorganisms, including bacteria which
serve as the sources of secondary metabolites producer that act against many
simultaneously existing human pathogenic bacteria and phytopathogenic fungi1.
Among the members of bacteria group, Actinomycetes which are gram-positive
bacteria that play an important part in the production of bioactive materials
of high profitable value2.
Besides,
the actinomycete population was determined as one of the main groups of the
soil population also they are manufacturers of important antibiotic profiles
and possess the capacity to produce a large number of secondary metabolites to
support the pharmaceutical industry3. Interestingly, Malaysia is
geographically diverse, which support the prosperity of different groups of
microorganisms as different species of actinomycetes. Antibiotic-resistant
microorganisms remain one of the central reasons for death due to infectious
diseases4. This requires the need of extra attention to find
solutions towards this problem and one of the alternative is by harnessing rare
actinomycetes as novel antibiotic producers. This is due to the fact that
actinomycetes carry the potential to produce a broad variety of compounds such
as phenols, benzopyranones, alkaloids, flavonoids, benzoquinones, steroids,
terpenoids, xanthones, and other natural products5,6. In this
project, actinomycetes were isolated from Beach Ridges Interspersed with Swales
(BRIS) soil collected from Setiu District, Terengganu, Malaysia subject to
testing for antimicrobial activities, secondary metabolite production and
compound detection using Gas Chromatography Mass Spectrophotometry (GCMS)
approach. Methanol extracts showed unusual properties of antimicrobial
activities, secondary metabolites results that indicating to may be rich in
novel bioactive compounds.
MATERIAL
AND METHODS:
Samples:
BRIS
soil samples were obtained from Setiu District, Terengganu, Malaysia, kept in
ice and transported to University Pendidikan Sultan Idris immediately.
Isolation
of actinomycetes from BRIS soil samples:
1
gram of soil samples was used and serially diluted up to 10−6.
Later, 100μl were taken from each dilution and spread on the surface of 2
types of medium (ISP Medium No.2 and Actinomyces agar)7,8 supplemented
with nalidixic acid (50μg/mL) and cycloheximide (50 μg/mL)9.
Then, the plates were incubated for a month at 30°C. Required colonies were
then subcultured onto ISP2 agar and purified using the streak plate method.
Glycerol stocks were prepared and plates containing isolated colonies were
stored at 4°C to further investigation10.
Colony
and cell morphology:
Observation
of colony morphology was based on the cultures grown on ISP3 agar11 for
14 days in 30oC. Gram staining and microscopic observation were done
to determine cell morphology and Gram type of each isolates.
Phylogenetic
analysis:
DNA
extraction was done using HiYield Genomic DNA Mini Kit (Real Biotech Corp.,
Taiwan) followed by 16S rRNA gene amplification using universal primers 27F and
1525 R11. Purified PCR products were sent for sequencing prior to
editing using BioEdit version 7.0. Used the Basic Local Alignment Search Tool
(BLAST) for the nearest match of each of the edited sequences was established
and pairwise sequence similarities well are done by using the 16S-based ID
using the EzTaxon-e web server (http://eztaxon-e.ezbiocloud.net)9.
Verified and adjusted alignment was used for building a phylogenetic tree based
on the neighbor-joining by using the MEGA 7.0 Analysis12.
Metabolite
extraction:
Isolates
Streptomyces sp. AA13, Amycolatopsis sp. AA12, and MicromonosporaspAA141
were selected for secondary metabolite analysis. The isolates we grown in ISP2
broth7 with shaking for 14 days at 30°C13 followed by
centrifugation for 30 minutes at 12,000rpm. Discard the supernatants and
transferred the precipitate into new tubes with added methanol and incubated at
37°C with shaking for 3 days to achieve complete extraction. Crude extract of
secondary metabolites were prepared by drying the preparations using Rotary
Evaporator (rotovap) at 40°C13.
Antimicrobial activity:
Antimicrobial
activity of the methanol crude extracts was determined against Bacillus
subtilis (DSM 10), Micrococcus luteus (DSM 20030), Klebsiella pneumonia
(DSM 30104), Escherichia coli (DSM 30083), Pseudomonas fluorescens
(DSM 50090) and Saccharomyces cerevisiae. The microorganisms were grown
in Muller-Hinton broth overnight at 37oC prior to testing14.
Agar
diffusion method was used to antimicrobial effects of the methanol products15.
The microbial lawn was prepared by spreading each microbial suspensions on
Muller-Hinton agar. Three dilutions of each crude extracts by methanol were
prepared to achieve the concentrations of 125, 250 and 500μg/mL which were
then applied on sterile paper discs (6mm diameter). The paper discs were placed
on the agar plate. Incubated at 37oC for 24 hours. Antimicrobial
activity of each metabolite extracts were determined by measuring the diameter
of inhibition zone16.
GC/MS
analysis of metabolite extracts:
GC/MS
analysis of the crude extracts were conducted using GC-MS (Thermo Electron
Corporation, Waltham) provided with HP-5 MS capillary column (30m × 0.25 mm ×
0.25μm). The carrier gas was helium with a flow rate of 1mL/min. The
column oven temperature was 40 °C at first for 2 minutes then changed it at
10–250 °C/min, which was kept for one minute, and 5–280 °C/min which was kept
at 8 minutes. Between m/z 40 and 450 got the electron impact spectra in
positive ionization method were acquired17.
RESULTS:
Identification
of strains:
All
of the isolates were Gram-positive with different colony morphologies as listed
in Table 1. Phylogenetic analysis based on 16S rRNA sequences revealed that
isolate AA13 belongs to genus Streptomyces and closely related to Streptomyces
phaeoluteichromatogenesT. Previous studies reported that genus Streptomyces
consist of major potential metabolite producers and carry the potential in
producing novel compounds9 Isolate AA12 belongs to genus Amycolatopsis
and closely related to Amycolatopsis samaneae RM287T with
98.16% sequence similarity while isolate AA141 belongs to genus Micromonospora
and closely related to Micromonospora aurantiaca ATCC 27029T with
99.51% sequence similarity (Figure 1). These 2 genera were reported to produce
potential novel compounds with wide range of activities3.
CONCLUSION:
Pandemic
COVID-19 has portrayed the importance of effort to be taken in order to find
new leads for the development of antibiotics and other drugs. Numbers of
research has shown that Actinomycetes carry the potential in producing
bioactive compounds3. This Study Significantly in line with previous
findings where Actinomycetes were successfully isolated from BRIS soil and
found to produce antimicrobial activities. Moreover, their metabolite extracts
showed high capacity of antimicrobial activity when tested against a penal of 5
different types of organisms from that consist of Gram positive, Gram negative
and yeast samples. GC/MS analysis of the methanol extract detected the presence
of variety antimicrobial compounds including Hexadecanoic acid, Indole,
Tridecanoic acid, Hexadecanoic acid, methyl ester, Actinomycin C2, Decanamides,
9-Octadecenoic acid, Octacosanol and Squalene. As the conclusion, the diversity
and bioactivity of the compounds produced by actinomycetes isolated from BRIS
soil offer a great possibility for future applications, mostly in the medical
field. Further analysis need to be done in order to find potential producer
that can serve as new drug leads.
ACKNOWLEDGMENTS:
This
article based on the research project designated Isolation of Soil
Actinomycetes and Its Secondary Metabolite for Potential Drugs Against
Antibiotic-Resistant Microorganisms. The authors would like to expand their
gratitude to Universiti Pendidikan Sultan Idris for the University Research
Grants (code: 2018-0142-103-01) that helped the authors for fund the research.
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