Anti-MRSA activity and Optimal Culture Condition of Streptomyces sp.

isolate, SUK 25
S. J. Ahmadc, S. Sudib, H. M. Sidekb, D. F. Basria, and N.M. Zina 1
a

School of Diagnostic and Applied Health Sciences , Faculty of Health

Sciences, Universiti Kebangsaan Malaysia, UKM, Jalan Raja Muda A. Aziz,

50300 Kuala Lumpur, Malaysia
b

School of Bioscience & Biotechnology Study, Faculty of Science &

Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor,

Malaysia
c

School of Bomedicine and Diagnostic Sciences, Faculty of Medicines and

Health Sciences, Universiti Sultan Zainal Abidin, Jalan Sultan Mahmud,
20400, Kuala Terengganu, Terengganu, Malaysia

I.

Abstract
a) Background of study:
The potential of secondary metabolites extracted from Streptomyces
sp. for treatment of bacterial infections including infections with
Staphylococcus aureus has previously been documented. In our own
laboratory, we showed significant antimicrobial activities associated
with endophytic Streptomyces sp. isolated from medicinal plants in
Peninsular Malaysia.
b) Objectives:
This

work

aimed

to

determine

anti-Methicillin

Staphylococcus aureus (MRSA) activities

resistant-

from Streptomyces sp.

isolates.
c) Materials & Methods:
Disc diffusion and Minimum Inhibitory Concentration (MIC) assay
were used to determine the antibacterial activity. Optimization of
1

Corresponding author; email: nora@medic.ukm.my

2
fermentation parameters for the most potent anti-MRSA extract in
terms of medium type, pH, aeration rate and culture period was also
carried out. Lastly, toxicity of the extract against Chang liver cells was
determined employing an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) assay.
d) The results: The screening results showed most potent extract as antiMRSA was SUK 25, while SUK 27 was found as least potent extract.
Disc diffusion assay revealed that spread plate technique was more
efficient in screening anti-MRSA activity compared to pour plate (pvalue < 0.05) and thus used in further studies. For SUK 25, Thronton
media was demonstrated to be the best medium for enhancing anti
MRSA activity in SUK 25 with MIC value, 2.44 0.01 g/ml. The
lowest MIC of 1.95g/mL was obtained when SUK 25 was cultured
for 7 days and aerated at at pH 7 using this medium with an aeration
rate of 140 rpm. The crude extract was not toxic towards Chang liver
cells (IC50 value = 43.31 1.24 g/ml).
e) Conclusion: The SUK 25s culture condition was optimized using
Throntons media, at initial pH 7 and aerated at 140rpm. Further
isolation and identification of bioactive compounds will allow
development of the isolate for anti-MRSA therapeutics.
f) Significance and impact of this study: Nowdays, MRSA infection
treatment

from

commercial

drug

could

dosage.Therefore,alternative medicine from

be

toxic

at

high

natural source highly

recommended.
Keywords: Streptomyces, MRSA, Thronton media, MIC
II.

Introduction:
Endophyte bacteria is an organism that lives in plant tissues, act as
symbiont with host and secreted benificial products (27 ). For
examples, metabolites with medicinal value were taxol, cryptocin and
cryptocandin (28).These secondary metabolites were extensively

3
studies and antibiotic compounds obtained were reported by
researchers and pharmaceutical agencies (26).
Treatments for MRSA infection for alternative ways need to be
initiated. Metabolites from Streptomyces sp. were reported to act as
antibacterial agents against pathogenic bacteria. Our previous work
had found, endophytic Streptomyces sp isolated from medicinal plants
in Peninsular Malaysia had shown significant antimicrobial activities
(13). Therefore, the present study was carried out to determine the
most potent Streptomyces sp. isolates as anti-MRSA agent and evaluate
its optimal culture conditions as well as the cytotoxic effect.
III.

Materials and Methods:

a) Test microorganism:
The sample of endophytic actinobacteria, SUK (Strain of UKM) and
MRSA were obtained from the Novel Antibiotic laboratory at UKM, Kuala
Lumpur. The SUK collection namely SUK 25, SUK 27, SUK 28, and SUK
30 and MRSA culture were MRSA ATCC 33591, MRSA ATCC 43300 and
MRSA 49476. These endophytic Streptomyces sp. was previously isolated
from medicinal plants in Peninsular Malaysia( 13). The SUK 25 and SUK
27 was isolated from Zingiber spectabile,while SUK 28 from

The

actinobacteria spore from Sarcandra glabra and SUK 30 from Oroxylum

indicum. The 14 days culture on ISP International Streptomycetes Project
2 agar for 14 days at 28 C and maintained at -80 C in 20% glycerol
solution (25), while MRSA strain was maintained on Mueller Hinton Agar
(MHA) supplement with 2% Sodium Chloride, NaCl at 8C. actinobacteria
were cultured on ISP 2 agar for 14 days prior antimicrobial testing.
b) Anti-bacterial screening :
Bioassay screening of 4 isolates of Actinobacteria from various sources of
medicinal plants (SUK 25, SUK 26, SUK 27,SUK 28, and SUK 30) were
screened against MRSA ATCC strain 33591, 43300 and 49476 (13). A
cubic (1 cm3) of matured actinobacteria was placed on nutrient agar that

4
was lawn with MRSA. The inhibition zone was measured after overnight
incubation in which Vancomycin was used as positive control. Out of these
nine actinomyces, 4 isolate (SUK 25, SUK 27. SUK 28, SUK 30) were
proceeded for fermentation in nutrient broth followed by extraction and
tested against MRSA through disc assay method. The inhibition zone was
measured for each plate and Vancomycin was used as a positive control.
c) Cultural condition of SUK isolates:
Culture conditions for the production of anti- MRSA was determined by
inoculation of 5-6 cubic (~1 cm 3) matured SUK 25 from ISP2 media into
one-third of 1 L Erlenmeyer flasks which each flask contained a sterilized
400ml broth. The flask was incubated for 7 days at 28C with aeration rate
160rpm. Eight fermentation medium with formula modification were used
namely A3M Media (20), Bn-2 Media (20), ISP 9 Media (22), CzapekDox Media (24), Bennette Media (14), Throntons Media (30), Arney
Heydorn Media (15) and Nutrient Broth (Merck). After that, selected
media was optimized based on anti-MRSA activities, the parameters
involved were incubation period, pH level of the media and aeration rate.
d) Ethyl acetate extraction
Ethyl acetate extraction (34) was employed to harvest secondary
metabolite from fermented broth after 7 days incubation. Culture filtrates
were extracted with three half-volume of ethyl acetate. After that, solvent
phase was concentrated with rotary evaporator (Buchi, Switzerland) at 40
C and was left to dry. Crude extracts obtained were suspended with
methanol and were used for MIC testing against MRSA ( 9). The extracts
of SUK 25 exploited from different media (namely, A3M Media, Bn-2
Media, ISP 9 Media Czapek-Dox Media, Bennette Media Throntons
Media, Arney Heydorn Media and Nutrient Broth) were proceeded with
MIC test. The concentration used at 0.488 g/mL-1000g/mL.
e) Cytotoxicity Test

5
Cytotoxicity effect of SUK 25 extracts were tested against mammalian
Chang liver cells, following a method described by Babu et al. 2010 ( 2).
Chang liver cells were grown in complete Dulbeccos Modified Eagle
Medium (DMEM) (GIBCO, NY) supplemented with 10% fetal bovine
serum and 1% pencillin/streptomycin, seeded to 96-well plates at 5 x 10 4
cells/well and incubated at 37C, 5% CO2 for 48 h. At the end of the
incubation, the culture medium was replaced with 100 L of SUK 25
extracts at concentrations ranging from 0.001 1000 g/ml diluted serially
using DMEM. Incubation was carried out for another 48 h. Cells incubated
in 100 L DMEM without extracts were used as controls. Following
incubation with extracts, 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich Co., USA) solution
in phosphate buffered saline (PBS) was added to each well and incubated
further for 3-4 h. The medium was then carefully replaced with 100 L
dimethyl sulfoxide (DMSO) and mixed thoroughly to dissolve the
formazan crystal product. Absorbance was then measured at 540 nm using
a

microtiter

plate

reader

(SLT-Labinstruments,

Germany).

Cell

survivability was calculated using the following formula:

% Cell surivivability :
Mean absorbance in test wells

x 100

Mean absorbance in control wells

The IC50 values were obtained from a dose-response curve for each extract
tested.
f) Statistical Analysis
The data obtained were assessed using IBM SPSS version 21.
IV.

Results:

As documented in Fig. 1(ii), the crude extracts of 4 isolates namely SUK

25, SUK 27, SUK 28 and SUK 30 were used to screen anti-MRSA
metabolites. These four isolates were screened to determine their

6
capability in producing secondary metabolites. At this stage, the SUK 25
isolate was found to as anti-MRSA agent. The inhibition zone of MRSA
ATCC 49476, MRSA ATCC 43300 and MRSA ATCC 33591 were 30mm,
20mm and 21mm respectively when react against SUK 25 extracts
compared to Vancomycin action, their inhibition zone were 16mm, 15mm
dan 15mm respectively.
In addition, analysis of one-way ANOVA of data for the inhibition
zone of MRSA ATCC 43300 produced by SUK 25 extracts were
significantly different between

pour plate technique and spread plate

technique (p-value <0.05). The spread plate method (SPM) exhibited

significantly higher inhibition zone value against MRSA ATCC 43300
compared to pour plate method (PPM) at 19 0.26mm and 15 0.38mm,
respectively by Post Turkey HSD test. SPM was selected for screening
secondary metabolite against MRSA as this technique produced better
inhibition zone than PPM with significant difference (<0.05) in Fig. 1(i).
Parameters used to determine the optimal culture condition for best
anti-MRSA activities of SUK 25 include type of media, fermentation
period, pH media and aeration rate for optimal oxygen sources. From the
data in Fig. 2, eight media namely A3M, ISP 9, Bennette, Nutrient Broth,
Arney Hedrons, Bn-2,Throntons and Czapek-Dox were screened for the
enrichment of secondary metabolite production of SUK 25. It can be
assessed by measuring its capability to produce lowest MIC value to
inhibit MRSA ATCC 43300 as well as high weightage of the dry weight of
crude extracts produced by SUK 25 with constant volume 400ml, initial
pH 7 and an incubating period of 7 days.
The Fig. 4 showed the lowest MIC value (2.44 0.01 g/ml),
is through incubating the SUK 25 in Throntons media followed by
Czapek-Dox with MIC value 5.86 0.01 g/ml, Arney Hedrons with
MIC value 6.25 0.1 g/ml, Nutrient Broth and ISP 9 with same MIC
value of1000 g/ml, Bennette and Bn-2 with MIC value of 8000 g/ml
and A3M media with MIC value of 32000g/ml. While the trends in
producing weightage of crude extracts exploited from 400ml media was

7
lead by A3M, then Bn-2, ISP 9, Arney Hedrons, Bennette, Nutrient
Broth, Czapek-Dox and Throntons with crude extract weightage 1040.1
mg , 7 0.2mg 5.80.1 mg, 2.80.2 mg, 2.40.2 mg, 2.30.1 mg, 0.90.1
mg and 0.50.1 mg, respectively. However, it was interesting to note that
despite the high weightage value of crude extract A3M, (1040.1 mg) but
the antibacterial effect was the lowest, (32000g/ml). Independent t-test
revealed that the MIC value of Throntons media (2.44 0.01 g/ml) was
significantly different compare with Czapek-Dox (5.86 0.01 g/ml), pvalue <0.05. Therefore, Thronton medium was selected as the best media
for producing anti-MRSA metabolites (Fig.4). This media contains only
one carbon source mannitol and two nitrogen sources namely potassium
nitrate and asparagines. These results showed that the production of crude
extracts was inversely proportional to the production of active extracts that
had potential secondary metabolite.
The Fig. 5 represent the sources of each media used, their MIC
value and weight of crude extracts produced by the media fermentation
were compared.
As shown in Fig. 4, incubation period, the initial pH medium
and aeration rate was optimized in Throntons media. The anti-MRSA
activity and weightage of crude extract of SUK 25 was assessed at 3 rd ,
7th , 14th and 21st days of fermentation. As seen in Fig. 4 (i), 7 th days
fermentation period

produced significantly (p-value < 0.05) highest

activity (lowest MIC value, 2.44 0.01 g/ml g/ml) compared to other
period of fermentation days(p-value < 0.05). While the crude extract
produced significantly highest weightage at 21st days with 1.2 0.01 mg,
(p-value < 0.05) compared to fermentation days of interval except at 14th
day fermentation (p-value >0.05). As antibacterial effect was chosen as
primary criteria, optimum fermentation period at 7th days of fermentation
period was chosen (Fig. 4).
The initial pH of Thronton media was determined through 5
interval values, starting with pH 5 until pH 9 by assessing the MIC value
and its weightage of crude extract of SUK 25. The data in Fig. 4 (ii)

8
showed that the significant highest activity, 2.44 0.01 g/ml against
MRSA ATCC 43300 was Thronton media formulated at initial pH 7 (pvalue <0.05). Independent t-test revealed that the MIC value of pH 7
media resulting significantly different by comparing with a MIC value of
pH 6 media, 7.83 0.01 g/ml (p-value < 0.05). However, weightage of
crude extract of SUK 25 produced by pH 7 media was the second highest
of the rest of the media tested with 1.97 0.01mg.

Therefore,

optimum pH for incubation condition SUK25 for secondary metabolites

was pH 7 Throntons media for 7 days at 140 rpm (Fig. 4).
Besides, the inhibitory activity of MRSA ATCC 43300 showed
significantly different between data observed when the aeration rate was
adjusted between 120 to 180 rpm, (p-value <0.05). But, the MIC value
produced when the aeration rate at 160 rpm and 180 rpm showed
insignificant different between each (p-value < 0.05) which the MIC value
3.91 0.0010g/ml and 3.91 0.0014g/ml, respectively. The significant
lowest MIC value, 0.975 0.001 g/ml produced after the aeration rate
was adjusted to 140rpm compared to other resorts (p-value < 0.05). The
data collected for weightage crude extract of SUK 25 significantly
different to each other after the aeration rate was adjusted between 120 to
180 rpm, (p-value < 0.05). The highest weightage value demonstrated
when the aeration rate was fixed to 160 rpm (p-value < 0.05) compared to
other aeration rates. However, there was an insignificant value of
weightage produced between 120rpm and 180 rpm, also between 120 rpm
and 140 rpm (p-value > 0.013).
V.

Discussion:
Antibacterial testing of the four SUK isolates to

determine the effectiveness of their extracts against MRSA inhibition. The

reaction was compared with Vancomycin as control drug. The inhibition of
MRSA growth was assumed due to active metabolite action from
actinobacteria. Ghadin et al. 2008 (13) shown inhibition zone produced
from the actinobacteria isolate in antibacterial screening assay had
potential medicinal value to be as an antibacterial agent.

9
In SPM technique, MRSA cells were spread in surface of
MHA. While PPM technique the MRSA cells was culture within
MHA.The depth of the agar in both techniques were constant, 5mm.
Therefore potency of SUK 25 extracts (in PPM technique) unable to
penetrate the target cells in the agar. Besides, the SUK 25s secondary
metabolites were extracted using ethyl acetate as a solvent in which the
solvent was categorized as similar solvent. Hence, the effectiveness of
SPM technique much better than PPM because of the action of nonpolar
compound in the extracts to be as anti-MRSA agent compared to polar
compound. In addition, Valli et al. 2012 (31) proved that antibacterial
screening of crude extracts of Streptomyces sp. had been practically
applied. Besides, the SPM was standard method in antimicrobial assay
which applying Kirby- Bauer method (9) and designed for single
antimicrobial testing.
High catabolism rate of Streptomycetes cells had been found to
inhibit the production of bioactive compound as antimicrobial agent (12).
Carbon sources in the media for incubation the bacteria was used as source
of energy in microorganism of catabolic process. Comparison of carbon
source for each media showed (Fig.5) that only Nutrient media had no
carbon source, whereas A3M media composed highest carbon source,
eight different sources as listed in Fig.5. The second highest was Bn-2
media had 2 sources and the rest media had one sources. Other studies
(7,23) reported that Streptomycetes used glucose as carbon sources in their
growth. Production of actinordin was more effectively produced by
Streptomyces coelicolor when glucose was added into fermentation media
(23). The best media that produced effective anti-MRSA activities from
SUK 25 was Thronton media (MIC=2.44g/ml in Fig. 4) with crude
extracts weight of 0.5mg. The Fig. 5 showed that the only carbon source
in Thronton media was mannitol. However, In carbon utilization test
results (data not shown) on ISP 9 standard media showed that SUK 25 was
not used mannitol as carbon source. Hence, the energy source obtained
from nitrogen source as asparagine (C4H7NO4), an amino acid. Borodina
and friends (2005) reported that asparagines was used as carbon and

10
nitrogen source in Streptomyces coelicolor A3 (2) growth (6). Whereas,
Aharonowitz & Demain,1978 (1) found that absence of glycerol in
fermentation media of Streptomyces clavuligerus causes asparagines acts
as carbon and nitrogen sources for Cephalosporin production. While
Czapek-Dox media which had sucrose as carbon source, but was not used
by SUK 25 as carbon source. Instead, sodium nitrate was used as energy
source. The MIC value obtained from extracts produced in this media was
slightly higher than extracts exploited by Thronton media, (5.86g/ml).
Voelker & Altabe, 2001 (32) reported that nitrate compound metabolism
were converted to ammonium, then to glutamine , an amino acid, amino
sugar, nucleotide and other secondary metabolites.
In some circumstances, an enzyme secreted in catabolic process
may repress the secretion of secondary metabolite. Different type of
carbon sources in A3M and Bn-2 media inhibit

SUK 25 growth.

Therefore,long incubation period is needed to reach the stationary phase

and produce active secondary metabolites. Martin and friends, 1999 (19)
stated that Carbon Catabolite Repression (CCR) occurs when variety of
carbon sources presence in medium fermentation causes inhibition of
penicillin

produced by Penicillium chrysogenum. In CCR condition,

synthesis of enzymes utilizing other substrates was repressed until the

primary substrate was exhausted. For example, production of Gentamycin
by Micromonospora purpurea was interfered by the presence of glucose
and xylose but not to fructose and maltose, ( 11). Assimilation of nitrogen
source was used for the synthesis of cellular components such as protein,
nucleic acid, cell wall, primer and secondary metabolite. Comparison of
nitrogen sources for fermentation media as stated in Fig. 7 found that
Bennette, Bn-2 and A3M media had three different sources of nitrogen.
While Bennette and Bn-2 had similar nitrogen sources, namely yeast
extract, meat extracts and N-Z case, A3M media had protein with 18 types
of amino acid and soluble nitrogen amino. Thronton, Nutrient and Arney
Hedron media had 2 nitrogen sources (Fig. 5).

11
Fermentation media which had limited nitrogen sources
were inhibited in the growth process, thus secondary metabolite production
was repressed such condition in Czapek-dox and ISP 9 media. However,
MIC value obtained from Czapek-dox media (5.86g/ml) lower than ISP 9
media (1000 g/ml). Voelker and Altabe, 2001 ( 32) stated that antibiotic
production occurs when nitrogen sources limited. While BarriosGonz~ilez et al., 2003 (3) stated, there were 4 categories of secondary
metabolites. The categories include, metabolite from aromatic amino acid
synthesis such as cancidin, a metabolite produced from amino acids such
as Cephalosporins, metabolite from the metabolism of acetyl-coA (in Kreb
Cycle) such as Erythromycin and metabolite produced by sugar such as
streptomycin. Besides, stationary phase of SUK 25 more faster occurs in
Czapek-dox media as contributing factor in secondary metabolite
production because SUK 25 did not use complex carbon source compared
glucose in ISP 9 media.
The MIC value of Arney Hedrons media (6.25g/ml) also
much lower as compared to Nutrient media (1000g/ml). Both sources of
nitrogen in Nutrient media were from organic sources, while Arney
Hedrons media nitrogen sources were formulated synthetically. A nonorganic sources of nitrogen such as ammonium sulfate causes a change of
pH media too acidic due to free acid release as fast ammonia metabolism
occurred (17). The best media, Thronton had asparagine and potassium
nitrate. According to EI-Tayeb et al., 2004 (10) potassium nitrate was used
to produce Rifamycin B by Amycolatopsis mediterranei. This fact was
supported by Voelker and Altabe, 2001 (32), nitrate compound reduced to
ammonium and nitrogen metabolism for amino acid synthesis and
secondary metabolite. In addition, the asparagine was used to supply
energy in catabolic process.
This secondary metabolite production of SUK 25 occurs during
stationary phase (Fig. 3) in response to defense mechanism for survival
and the protection of the produced spores (5). Kampen, 1997 (17) stated
that the appropriate pH level acts as buffer to any change of hydrogen ion

12
concentration (pH) when acid and alkaline formed in fermentation media.
Streptomyces sp. suitable grew in range of pH, between pH 7.0 and pH7.4
(25). As the activity of anti-MRSA effects reflected optimum secondary
metabolite production, the aeration rate of 140 rpm was chosen. Function
of aeration is to supply the cultured organism with the oxygen (33) and the
relationship between aeration rate and oxygen supplied was linear.
However, an appropriate oxygen supply was resulting positive effect on
growth of cells. As reported by Martin et al. 2004 (20), production of
secondary metabolite from

Streptomyces olindensis that producing

Retamycin could not affected by using a high rate of aeration but a

sufficient oxygen supply at 140 rpm increased the production. In this
present work, SUK 25 showed significant inhibitory activity, with MIC
value of 1.95g/ml at aeration of 140 rpm but not at the higher speed.
Evaluation of cytotoxic activities of SUK 25 extracts was
conducted against Chang liver cells. The cytotoxic effects of crude extract
exploited by Thronton media was categorized as nontoxic as recommended
by NCI (National Cancer Institute) which IC50 of crude extract over 30
g/ml was defined as noncytotoxic ( 16,29). The SUK 25 extracts tested
exhibited non toxic towards mammalian Chang liver cells with IC 50 values
was 43.31 1.24 g/ml. Therefore, it is important to further investigate this
crude extract for cytotoxicity test against cancer cells, thus to develop
bioactive compound from the nature.
From this present study, SUK 25 was a potential source
from Stretomyces sp. as anti-MRSA agent and optimum culture conditions
for anti-MRSA activities from SUK 25 extracts obtained in Thronton
media with initial pH was pH 7 and aerated at 140 rpm. Hence, the SUK
25 crude extracts has a huge potential to be explored as anti-MRSA agent
for drug development.
VI.

Conflict of interest statement:

All authors declare that they have no conflict of interest.

VII.

Acknowledgements:

13
The authors appreciate the financial support from the Ministry of Higher
Education of Malaysia under the grant no. UKM-NN-03-FRGS0042-2009
and Universiti Sultan Zainal Abidin (UniSZA).
VIII.

Financial Disclosure:
This research study had received financial support from Ministry of Higher
Education of Malaysia under the grant no. UKM-NN-03-FRGS0042-2009.

IX.

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