ACTINOMYCETES FROM BRACKISH ENVIRONMENT WITH

ANTIBACTERIAL COMPOUND WHICH ANTAGONISTIC TO

METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA)

Eva Luviriani1*, Ari Asnani2, Oedjijono3

1
Postgraduate student of Biology Faculty, Unsoed, Purwokerto 53123
2
Departement of Chemistry, Faculty of Mathematic amd Natural Science,
Jenderal Soedirman University, Purwokerto 53123
3
Departement of Biology, Faculty of Biology, Jenderal Soedirman University,
Purwokerto 53123
*
coresponding author: evaluviriani12@gmail.com (Eva Luviriani)

ABSTRACT

Actinomycetes can be isolated from brackish environments such as mangrove

ecosystems. Several species of Actinomycetes are able to inhibit the growth of
Methicillin Resistant Staphylococcus aureus (MRSA). The epidemiological
changes of MRSA have become a public concern and important for researchers to
look for new antibacterial compounds derived from natural sources.The purposes
of this research are to examine antagonism, the minimum inhibition concentration
of antibacterial compounds, and identify the Actinomycetes isolate from the
brackish environment against MRSA. Actinomycetes from Brackish Environment
shows antibacterial activity against MRSA with weak inhibition capability. MIC
of antibacterial compound from three isolates W-5A, W-5B, and P-7D are 4
mg/mL, 2 mg/mL, and 8 mg/mL. The identity of brackish environment
Actinomycetes W-5A, W-5B, and P-7D which shows antagonism against MRSA
is Streptomyces.

Keywords : Actinomycetes, antibacterial compound, MRSA, 16S rRNA Gene

INTRODUCTION
Actinomycetes can live in various environmental conditions that contain
many nutrients. Some species of Actinomycetes can be isolated from brackish
environments such as mangrove ecosystems, which haveaccumulation of organic
and nutrient that are affected by tidal influences [1]. Several species of
Actinomycetes are able to inhibit the growth of Methicillin Resistant
Staphylococcus aureus (MRSA) like Micromonospora sp. ICN36 [2].
Streptomyces sp. CFJ2, Streptomyces antibioticus strain 1022-257, Streptomyces
flaveolus strain NRRL B-1334, Streptomyces psammoticus strain NBRC 13971,
and Streptomyces sp. can inhibit 10 clinical MRSA isolate [3]. Streptomyces sp.
which isolated from coastal areas can inhibit Multi Drug Resistant
Staphylococcus aureus (MDRSA) [4].
MRSA is a Staphylococcus aureus bacterium that is resistant to β-lactam
group antibiotics such as penicillin and its derivatives, for example, methicillin,
oxacillin, dicloxacillin, nafcilin and cephalosporin. S. aureus is commonly found
in human epithelials and its an opportunistic pathogen involved in nosocomial
infections. MRSA infections can be severe disease and fatal to the sufferer [5].
The infection is not only in the hospital but also in the community [6].
The epidemiological changes of MRSA and the widespread of antibiotic
resistance have become a public concern and important for researchers to look for
new antibacterial compounds derived from natural sources. Actinomycetes
isolated from the marine areas potentially to produce antibacterial compounds
with new active compounds [7]. Therefore, the exploration of marine
actinomycetes with antibacterial compund which capable of resisting evolutionary
changes of target bacteria in suppressing MRSA problems is important.

MATERIAL AND METHODS

The materials used in this study were mangrove sediments from Kalipanas
and Kutawaru, MRSA from microbiology culture collection of Biology Unsoed
Purwokerto.

1) Sites of Sampling
Sampling was done by simple random sampling. Samples used were
sediments from mangrove ecosystems that were taken randomly in two different
areas, Kalipanas (P) and Kutawaru (W), Cilacap Tengah.

2) Isolation and selection of actinomycetes antagonistic to MRSA

The isolation was carried out with pre treatment method, there was dry-
heating, phenol chemical germicide, and OS (Oil Separation). Actinomycetes was
cultivated on SCN agar by pour plate. Incubation was carried out at room
temperature for 7-9 weeks.
The selection of actinomycetes isolates antagonistic to MRSA was
performed by inoculating the MRSA bacteria on the NA medium directly to pour
plate, then made to block the 7 day actinomycetes isolate with diameter of 6 mm
and placed on the MRSA inoculated NA medium. The culture was incubated at
28°C for 24 hours. The resulting response is the inhibitory zone around the
actinomycetes colony.
The selection of actinomycetes isolates antagonistic to MRSA was done by
inoculating the MRSA bacteria on NA medium directly to pour plate, the
actinomycetes isolate age of 7 days was made block agar with cork drill
laboratory and placed on the inhaled NA medium of MRSA. Incubation was
carried out at 28 °C for 24 hours. The resulting response is the inhibit zone around
the block of the actinomycetes colony.

3) Determination of optimum incubation time

Actinomycetes was cultivated on SCN broth in glass bottle and incubated at
37 oC for 0, 7, 14, 21, and 28 days. A total of 10% (10 mL) of actinomycetes
inoculum was inoculated on a new SCN broth. The fermented inoculum of the
incubator shaker was filtered with Whatman filter paper to obtain filtrate and
precipitate. Filtrate was tested on MRSA by Kirby Bauer method.

4) Fermentation and during optimum incubation

A culture of pure actinomycetes isolates age 7 days which produced the best
inhibitory zone against MRSA inoculated into 100 mL SCN broth. Incubation
carried out at the incubator shaker at 30 °C for 8 days. A total of 10% (10 mL) of
actinomycetes were cultivated into 100 mL of sterile SCN broth and incubated at
the shaker incubator at 30 °C for the known optimum incubation time [8]. Then
the liquid culture was centrifuged with speed of 4.000 rpm and 20 minutes to
obtained supernatant. Supernatant extraction was performed use etil acetat (1:1
v/v). Concentrated ethyl acetate extract was obtained by evaporation.
Extracts of antibacterial compounds on each isolate were dropped on a test
disk of 15 μL with different concentrations (10, 8, 6, 2 mg / mL) and incubated at
37 ° C for 24 hours. The diameter of the inhibit zone formed around the test disk
is measured.
5) Identification of phenetically actinomycetes isolates
The phenetic character of actinomycetes isolates was known through
morphological observations of colonies and cells, Gram staining, and catalase
enzyme activity test.

6) Identification of actinomycetes isolates was based on 16S rRNA gene

Identification of actinomycetes isolates was based on 16S rRNA gene, DNA
isolation used PrestoTM Mini gDNA Bacteria Kit (Geneaid Instruction Manual).
Measurement of concentration and DNA purity used UV spectrophotometer at
absorbance λ 260 nm and λ 280 nm. DNA obtained was amplified with 27F and
1492R primers using KAPPA Taq Extra Hotstart ReadyMix with dye. DNA
electrophoresis used 1% of agarose gel submerged in 1x TAE buffer. PCR
conditions are first heating at 95 oC for 3 minutes, denaturation for 15 seconds at
94 oC, annealing 15 seconds at 55 oC and 1 minute temperature 68 oC, cooling at 4
o
C. The cycle is repeated 30 times [9]. Sequencing process carried out by Genetika
Science laboratory. The obtained sequence results were analyzed by BioEdit
application to determine the sequence of base pairs of DNA. Species
determination used Basic Local Alignment Search Tool (BLAST) program.

7) Identification of phytogenetic actinomycetes isolates

Reconstruction of phylogenetic trees used MEGA software 5 with the
Neighbor-Joining method 1000 bootstrap replications was based on 16S rRNA
gene sequence.

RESULTS AND DISCUSSION

1) Isolation and selection of actinomycetes antagonistic to MRSA

Three isolates of W-5B, W-5A, and P-7D showed the best inhibitory zone
to MRSA with Inhibition zone of 2.40 mm, 1.20 mm, and 0.80 mm. Ability of
inhibiton of actinomycetes isolates caused by several factors such as test media
type, incubation conditions, antibacterial type, and bacteria resistance to
antibacterial tests [10]. Inhibition zone and percentage of inhibition activity
actinomycetes against MRSA presented in Table 1.
 Tabel 1. Inhibition zone and percentage of inhibition activity
actinomycetes against MRSA
Persentase Aktivitas
Zona Hambat
No. Kode Isolat Penghambatan
(mm)
(%)
1. W-5B 2,40 28,6
2. W-5A 1,20 16,70
3. P-7D 0,80 11,80
4. P-3A 0,70 10,40
5. W-7A 0,70 10,40
6. W-6A 0,60 9,10
7. W-1A 0,60 9,10
8. P-6B 0,50 7,70
9. P-6E 0,50 7,70
10. P-7I 0,50 7,70
11. W-1A 0,50 7,70
12. W-8A 0,50 7,70
13. W-3A 0,50 7,70
14. W-9A 0,50 7,70
15. W-6B 0,00 0,00
16. P-6D 0,00 0,00

2) Determination of optimum incubation time

The optimum incubation time of the three isolates is for 14 days [figure 1].
The incubation time was the stationary phase of actinomycetes and at the end of
the stationary phase the actinomycetes produces secondary metabolites such as
antibiotics, vitamins, and hormones [11]. The high production of metabolites is
related to the growth phase of microorganisms [12]. Seven day incubation period
is the initial phase of the exponential phase, in this phase antibacterial secondary
metabolites was a little. Decreasing of the substrate concentration results in the
growth of the cell begins to decrease. Therefore, the accumulation of metabolic
products will occur and enter in the stationary phase. In this phase the isolates did
not show significant growth even stalled. It is associated with availability of
nutrients and competition between microorganisms. Therefore, secondary
metabolite production occurs in the form of antibacterial [13].
Figure 1. Inhibit zone curve of actinomycetes isolate extract with different
incubation time
3) Minimum Inhibitory Concentrations (MIC) of antibacterial compounds
against MRSA
MIC of W-5A, W-5B, and P-7D antibacterial compounds against MRSA
were 4 mg / mL, 2 mg / mL, and 8 mg / mL with inhibition zone of 1.75 + 0.50
mm; 2.25 + 0.50 mm; and 2.25 + 0.50 mm. The antibacterial compound of W-5B
isolate has the best antibacterial activity in inhibiting MRSA. MIC values in this
study different with research conducted by Kumar and Rao (2012), that is activity
of antibacterial compounds of ethyl acetate extract from actinomycetes isolates
against MDRSA showed MIC of 1 mg / mL. This is thought to be due to
differences in concentration variations and the content of antibacterial compounds
produced by actinomycetes [14].

4) Identification of actinomycetes isolates based on phenetic characters

The morphology of W-5A isolate colonies was circular, small, white air
mycelium, dark brown mycelium substrate, pigmentation of medium, and phenol
method. W-5B isolate has circular colonies, small, colonies wrinkled, raised,
entire, cream colour aerial mycelium, dark brown substrate mycelium,
pigmentation in medium, and phenol method. The P-7D isolates had irregularly
shaped colonies, small, colonies surface like dust, umbonate, undulate, gray aerial
mycelium, dark brown substrate mycelium, medium pigmentation, and phenol
method. Characteristic of colony morphology presented in Figure 2.
 [W-5A] view from top [W-5A] view from bottom

[W-5B] view from top [W-5B] view from bottom

[P-7D] view from top [P-7D] view from bottom

Fig 2. The colony morphology of W-5A, W-5B, dan P7-D isolates

The results of gram staining on three isolates of actinomycetes (W-5A, W-

5B, and P-7D) were positive Gram and showed the presence of catalase enzyme
activity. W-5A cell morphology has branched substrate hyphae and hasn’t septate,
a gray-branched aerial hyphae formed short spore chains with monosporous spore
structures. The hyphae of W-5B isolate were branched and has septate. Aerial
hyphae grows on a medium-gray surface with long spiral shapes like spore chains.
Type of spore chain was Retinaculiaperti with open loops spore chain structure.
The P-7D isolate has branched substrate hyphae and hasn’t septate. Aerial hyphae
are grows on a surface medium with grey colour and forming spore chains.
Rectiflexibiles spore type with open loops spore chain structure. The morphology
cell of actinomycetes isolates (W-5A, W-5B, and P-7D) can be seen in Figure 3.
 [W-5A] substrate hyphae Aerial hyphae Spore

[W-5B] substrate hyphae Aerial hyphae Spore

[P-7D] substrate hyphae Aerial hyphae Spore

Fig 3. The morphology of actinomycetes isolates with magnification of 400x

5) Identification of actinomycetes based on 16S rRNA gene

The highest DNA purity was shown by P-7D of 2.00 with DNA
concentration of 30 μg / mL. The DNA purity levels of W-5B and W-5A isolates
were 1.83 and 1.55 with DNA concentration of 55 μg / mL and 70 μg / mL. The
high levels of DNA purity if the value of 1.8 - 2.0 [15]. The higher of the DNA
concentration was better quality of DNA sample. The DNA concentration of
0.001-0.1 μg/μL or 10-100 μg / mL was enough for DNA amplification [16].
The result of BLAST analysis showed that W-5B sequence has high
similarity with S. malaysiensis with highest identity percentage of 83%. Sequence
of P-7D has a high similarity with S. griseoplanus with the highest identity
percentage of 81%. The sequence of W-5A isolates has no significant similarity to
the sequence of actinomycetes isolates present in GenBank. That showed that W-
5A possibly a new species of actinomycetes. A similarity score of 89-93%
indicates that both W-5B and P-7D isolates show a different family [17].
A similarity percentage of 98% or less means that the sample isolate is a different
species or may be considered a new species [18,19]. The results of the analysis
using BLAST W-5A and W-5B are presented in Table 1 and Table 2.

Table 1. The analysis results of W-5B isolate by BLAST program

Max Total Query Max
Species E-value Accession
Score Score Cover Identity
Streptomyces
malaysiensis strain 815 815 63% 0,0 83% NR_041410.1
NBRC 16446
Streptomyces catbensis
804 804 63% 0,0 82% NR_125457.1
strain NBRC 107860
Streptomyces katrae
795 795 63% 0,0 82% NR_041136.1
strain NBRC 13447

Table 2. The analysis results of P-7D isolate by BLAST program

Max Total Query E- Max
Species Accession
Score Score Cover value Identity
Streptomyces
griseoplanus strain 1014 1014 95% 0,0 81% NR_118415.1
NRRL -ISP 5009
Streptomyces
aomiensis strain 977 977 92% 0.0 80% NR_112998.1
M24DS04
Streptomyces
seranimatus strain YIM 957 957 92% 0.0 80% NR_132286.1
45720

6) Identification of phylogenetic actinomycetes isolates

The results of phylogenetic tree reconstruction and inter-species distance
analysis showed that P-7D with W-5A isolates and W-5B of 0,013 (Figure 4).
Similiarity percentage of P-7D with W-5A and W-5B of 99,9% and 99,8%. The
W-5B and P-7D Streptomyces catbensis strain NBRC 107860 and Streptomyces
aomiensis strain M24DS04 of 82,6% dan 83,7%. Similarity percentages of 98%
or less are defined as distinct species or can be categorized as new species [20,21].
It shows that W-5B and P-7D was a new species of the genus Streptomyces.
Phylogenetically Gram positive bacteria are divided into groups that have
high GC content and low GC. GC content is the percentage of GC couples in a
DNA organism. Streptomyces is a Gram-positive bacterium that has a high GC
content (> 50%) [22]. Based on the results of phenetic and phylogenetic
identification showed that W-5A, W-5B, and P-7D isolates are species of
actinomycetes members belonging to the genus Streptomyces. The result of
phylogenetic tree reconstruction and similarity percentage of analysis result are
presented in Figure 4.

0,00
0,003 0

0,007

0,006
0,00
0

0,0
0,016
01

0,013
0,012 0,00
0,006 2
0,007

0,01 0,00
0,00 2
2
3
0,00
9
0,00
5
0,00
0
Jarak Genetik

Figure 4. Phylogenetic tree of selected actinomycetes isolates based on full

sequence of 16S rRNA gene, Neighbor-Joining method with 1000
Genetic distance
replication bootstrap

CONCLUSION

Actinomycetes from Brackish Environment shows antibacterial activity

against MRSA with weak inhibition capability. MIC of antibacterial compound
from three isolates W-5A, W-5B, and P-7D are 4 mg/mL, 2 mg/mL, and 8 mg/mL.
The identity of brackish environment Actinomycetes W-5A, W-5B, and P-7D
which shows antagonism against MRSA is Streptomyces.
AKNOWLEDGE

The authors are grateful to Indonesian Higher Education for providing

research grant under National Strategic Research of Jenderal Soedirman
University in 2016.

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