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VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY
INTERNATIONAL UNIVERSITY
PRELIMINARY DETECTION OF
ANTIMICROBIAL ACTIVITIES OF
EXIGUOBACTERIUM INDICUM
A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Pharmaceutical Biotechnology
Student name: Nguyen Huynh Tam – ID No.BTBTIU10087
Supervisor: Dr. Nguyen Hoang Khue Tu
May, 2014
Acknowledgment
Firstly, I take this opportunity to thank the school of Biotechnology,
International University – Viet Nam National University HCMC for giving me the
chance to take part in this thesis course. This course helps me to gain lots of
knowledge in both theory and practice.
Secondly, I want to give my deepest gratitude to Dr. Nguyen Hoang Khue
Tu for her fully support, valuable and professional information and guidance,
which helped me in completing this task through various stages.
Thirdly, I want to express my appreciation to the technicians of Lab 701,
702 and 101 for supporting and helping me with the resources to accomplish the
thesis.
Fourthly, I am obliged to my colleague friends: Mr. Nguyen Ngoc Huu, Mr.
Uong Nguyen Thanh Tung, Mr. Le Van Canh, Mr. Vo Duy Le Giang, Mr. Tran
Quang Manh, Mr. Nghe Van Dat, Mrs. Le Bao Thu, Mrs. Tran Ngoc Phuong
Nguyen for the valuable information provided by them in their respective fields. I
am grateful for their help during the thesis period.
Lastly, I thank my family for their constant encouragement which makes
this thesis become possible.
i
List of tables
Table 1 Result of optical density of Exiguobacterium indicum ........................10
Table 2 Diameter of inhibition zone (mm) and potency determination according
to ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant on Salmonella
typhi .......................................................................................................14
Table 3 Diameter of inhibition zone (mm) and potency determination according
to ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant on Pseudomonas
aeruginosa ...............................................................................................14
Table 4 Diameter of inhibition zone (mm) and potency determination according
to ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant on Serratia
marcescens ..............................................................................................14
Table 5 Diameter of inhibition zone (mm) and potency determination according
to ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant (pH 6, 7, 8) on
Salmonella typhi .......................................................................................16
Table 6 Diameter of inhibition zone (mm) and potency determination according
to ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant (pH 6, 7, 8) on
Pseudomonas aeruginosa...........................................................................16
Table 7 Temperature & Time effects on Salmonella typhi ..............................24
Table 8 Temperature & Time effects on Pseudomonas aeruginosa ..................24
Table 9 Temperature & Time effects on Serratia marcescens .........................25
Table 10 pH effects on Salmonella typhi .....................................................25
Table 11 pH effects on Pseudomonas aeruginosa .........................................25
ii
Table of figures
Figure 1 Diagram of experimental design .................................................... 5
Figure 2 Gram stain of Exiguobacterium indicum .........................................10
Figure 3 Exiguobacterium after 3 days .......................................................11
Figure 4 Inhibition zones of Exiguobacterium indicum supernatant after
culturing at 25 oC and 37 oC for 3 days on Salmonella typhi ............................12
Figure 5 Inhibition zones of Exiguobacterium indicum supernatant after
culturing at 25 oC and 37 oC for 3 days on Pseudomonas aeruginosa ..............12
Figure 6 Inhibition zones of Exiguobacterium indicum supernatant after
culturing at 25 oC and 37 oC for 3 days on Serratia marcescens .......................12
Figure 7 Standard curve of antimicrobial activities of ceftriaxone against
Salmonella typhi .......................................................................................13
Figure 8 Standard curve of antimicrobial activities of ceftriaxone against
Pseudomonas aeruginosa...........................................................................13
Figure 9 Standard curve of antimicrobial activities of ceftriaxone against
Serratia marcescens..................................................................................13
Figure 10 Inhibition zones of Exiguobacterium indicum supernatant after
adjusted pH 6, 7, 8 against Salmonella typhi and Pseudomonas aeruginosa. ....15
Figure 11 MS of compound A ....................................................................18
Figure 12 IR of compound A .....................................................................18
Figure 13 MS of compound B ....................................................................19
Figure 14 IR of cpompound B ...................................................................20
iii
Table of Contents
1. Introduction .......................................................................................... 3
2. Materials and Methods ............................................................................ 5
2.1 Research object and location .............................................................. 5
2.2 Experimental design .......................................................................... 5
2.2.1 Cultivation & Optimization & Extraction of Exiguobacterium indicum ... 6
2.2.2 Antimicrobial activities tests of Exiguobacterium indicum .................. 7
2.2.3 Preliminary identification of compounds extracted from
Exiguobacterium indicum ...................................................................... 9
2.3 Data analysis .................................................................................... 9
3. Results ................................................................................................10
3.1 Cultivation & Optimization & Extraction ...............................................10
3.2 Antimicrobial activities ......................................................................11
3.3 Preliminary identification ...................................................................17
3.3.1 Compound A ..............................................................................18
3.3.2 Compound B ..............................................................................19
4. Discussion ............................................................................................20
5. Conclusion ...........................................................................................22
References ...............................................................................................23
Appendix .................................................................................................24
iv
DETECTION OF PRELIMINARY ANTIMICROBIAL
ACTIVITIES OF PIGMENT PRODUCING
BACTERIA
Tam Huynh Nguyen, Tu Khue Hoang Nguyen
School of Biotechnology, International University – Vietnam National University
in HCMC

Corresponding author’s email address: [email protected]
1
Abstract
Nowadays, drug resistance is increasing. Hence, finding out the new
compound to overcome is essential in pharmaceutical fields. This study focused
on
the
antimicrobial
activities
of
the
pigment
producing
bacteria,
Exiguobacterium indicum cultured in Luria broth in different incubation time
(24h, 48h, 72h) at different incubation temperature (25 oC, 37oC, 45oC). The
antimicrobial activity test was carried out, basing on the agar diffusion method.
The 25
o
C cultured supernatant of the bacteria showed the maximal ability
against the pathogens such as Salmonella typhi (1.4 ± 0.1 mm), Pseudomonas
aeruginosa (1.27 ± 0.06 mm), and Serratia marcescens (1.13 ± 0.15 mm). By
calculating
the
equivalent
concentration
of
ceftriaxone
for
the
potency
determination of the supernatants, the highest potency on Salmonella typhi,
Pseudomonas aeruginosa, and Serratia marcescens
was 15.14 μg/ml, 3.98
μg/ml, 3.98 μg/ml, respectively. The antimicrobial activity was stable at pH6, 7,
8. The extraction step was conducted for the preliminary identification of the
pigment compounds. The study also suggested the extraction process to isolate
two different kinds of yellow pigments with different solubiity in distilled water
and chloroform, which showed a weak antimicrobial activity on Micrococcus
luteus. This was a first reports on antimicrobial activity of Exiguobacterium
indicum isolated in Vietnam.
Keywords:
Exiguobacterium indicum
Antimicrobial activities
Agar diffusion method
Preliminary identification
2
1. Introduction
Pigmentation is a characteristic that is common to many kinds of bacteria.
Pigments are compounds can absorb the light. Therefore, they are responsible
for the colors display of the organisms. Many pigments producing organisms of
bacteria domain have the role in the survival of the organisms which produce
them. Lastly, bacterial pigments can be chemically treated and used in a variety
of industrial processes such as food colorants, textile and other colorants,
fluorescence – based indicators, human health, etc.
Colorants are products that humans use every day from food to clothing
and textiles. However, there is a problem with many of these colorants is that
they are harmful to the environment because they are difficult to biodegrade.
Furthermore, they may also contain toxins that are harmful to humans. Hence,
the task of discovering pigments suitable for being used as food colorants is that
not only produce an ideal color, but also safe for human health.
Pigments used for textile colorants are not as restricted as food colorants.
That is the reason why there are more pigments used in textiles and other nonedible products than in food. For instance, the prodigiosin and violacein, known
as red and purple pigments respectively, were used as colorants on lots of
fabrics such as acrylic fiber, silk, cotton, polyester, and polyester microfiber.
Their colorfastness on the fabrics was recorded. Both pigments show high
colorfastness on to acrylic fiber, silk, and polyester microfiber. On the other
hand, they display average colorfastness to cotton and polyester. Furthermore,
there is discovered that we can reduce the amount of pigment required for
staining due to their high staining capability on all the fabrics. These tests show
the ability of specific pigments to be used successfully as colorants for textiles.
Due to the discovery of new safe and effective natural colorants, they can
replace yarn and denim which are potentially harmful synthetic dyes.
Another application of the bacterial pigment is fluorescence – based
indicators. In laboratories, the scientists used these pigments to label antibodies,
as well as indicate the progress of specific reactions. For example, phycoerythrin,
an accessory pigment to chlorophyll in photosynthetic bacteria is essential
because it captures light energy and then transfers it to the chlorophyll reaction
center.
Amazingly, the pigments’ visual properties are not related to one major
industrial application of bacterial pigments which is human health. Some
bacterial pigments are useful for human health because they can provide key
nutrients and compounds the body required. For example, carotene is
a group
of pigments that have many beneficial effects towards human health. Many
3
bacteria can produce β-carotene and astaxanthin (a xanthophyll) which are
essential in maintaining the yellow color of the retinal macula, giving it the
ability to act as sunblock on certain parts of the retina. Therefore, we can state
that the pigments play an important role in maintaining the health of the human
eyes. Moreover, usual bacteria pigments such as prodigiosin, carotene, and
xanthophylls have many other effects on human health. Progiosin can be used
for antitumor, antimicrobial activities and dyes. Actinorhodin can be used for
antimicrobial activities. Consequently, there are many microbes producing
pigment such as blue - green, yellow – orange, red, etc. Focus on the biological
pigment from microorganisms is interesting.
Exiguobacterium is a genus of bacilli and a member of the low GC phyla
of Firmicutes. Collins et al. first described the genus Exiguobacterium with the
characterization of E. aurantiacum strain DSM6208T from an alkaline potato
processing plant. It has been found in areas covering a wide range of
temperatures (-12oC—55oC) including glaciers in Greenland and hot springs in
Yellowstone, and has been isolated from ancient permafrost in Siberia. This
ability to survive in varying temperature extremes makes them an important
area of study. Currently, seven genomes from the genus have been completed
as either complete (one circular chromosome, with plasmids) or in a draft format
(containing multiple unassembled contigs).
Exiguobacterium indicum’s cells are aerobic, Gram-positive, motile and
rod-shaped. The stationary-phase cells are coccobacilli. Colonies on nutrient agar
are round, shiny, irregular, elevated and orange-coloured after 24 h at 22°C. No
spores are formed in Exiguobacterium indicum. These bacteria can grow from 5–
30°C and at pH 6–10. The optimum temperature and pH for growth are 25°C
and pH 7 (Chaturvedi, P., & Shivaji, S., 2006).
Nowadays, drug resistance is increasing. Hence, finding out the new
compound to overcome is essential in pharmaceutical fields. This project will
provide the source of antimicrobial agent, as well as the new process to isolate
and purify the intracellular pigment as orange yellow from Exiguobacterium
indicum because the information of Exiguobacterium indicum was very few.
Because of the limitation of time, I only do the preliminary detection of
antimicrobial activities. This will be the preparation for further detection in higher
level such as application of Exiguobacterium indicum.
In this thesis, I expect to obtain the result shows that Exiguobacterium
indicum has the potency and effectiveness in antimicrobial activities.
4
2. Materials and Methods
2.1 Research object and location
2.1.1 Research object
Exiguobacterium indicum (P11) and pathogens including Candida albicans
ATCC 10231 (P1), Salmonella typhi ATCC 19430 (P2), Pseudomonas aeruginosa
ATCC 27853 (P3), Staphylococcus sciuri ATCC 29061 (P5), Serratia marcescens
ATCC13880 (P6), Micrococcus luteus ATCC 10240 (P8) were supplied by Dr.
Nguyen Hoang Khue Tu, school of Biotechnology, International University – Viet
Nam National University HCMC.
2.1.2 Location
My thesis was conducted at Laboratory 701 & 702 & 101, school of
Biotechnology, International University – Viet Nam National University HCMC.
2.2 Experimental design
Cultivation
Optimization (temperature, time and pH)
Fraction and extraction by using different solvents
Antimicrobial activities in each fraction
Analysis & Writing
Figure 1 Diagram of experimental design
5
2.2.1 Cultivation & Optimization & Extraction of Exiguobacterium
indicum
2.2.1.1 Cultivation of Exiguobacterium indicum and pathogens
Luria – Bertani (LB) was prepared following Miller’s formula: 1% peptone,
0.5% yeast extract, and 1% sodium chloride (Miller, 1972). The medium was
adjusted to reach pH 7 before being autoclaved.
Exiguobacterium indicum and pathogens including Candida albicans,
Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus sciuri, Serratia
marcescens and Micrococcus luteus were cultured in 5 ml test tubes containing
LB for 24h at room temperature (25oC) separately and adjusted to a final cell
count of approximately 106 CFU/ml.
Gram positive pathogens include Candida albicans, Staphylococcus sciuri,
Serratia marcescens, Micrococcus luteus and Exiguobacterium indicum. On the
other
hand,
gram
negative
pathogens
include
Salmonella
typhi
and
Pseudomonas aeruginosa. After culturing, they were purified by streak plate
method onto LB agar plates and kept into glycerol at -80oC as stock culture. All
bacterial strains were checked by Gram stain method to check their morphology
and make sure that they are not contaminated with any substances.
2.2.1.2 Optimization of the conditions and cell free supernatant
preparation for antimicrobial activity test
Exiguobacterium indicum was transferred with the same amount, 0.1 ml
into 12 Erlenmeyer flasks containing 50 ml of LB broth. Then they were cultured
at different temperatures: 25oC, 37oC, 45oC and 50oC and different durations: 1
day, 2 days and 3 days with shaking.
Crude cell free extract (CFE) of Exiguobacterium indicum was prepared
following the method that is contructed by Ogunbanwo et al. (Ogunbanwo et al.,
2003). CFS was taken by centrifuging the culture medium at 10,000 rpm for 20
minutes at 4°C. After centrifuging, supernatants and pellets of each Erlenmeyer
flasks were separated respectively into 50 ml falcons. The supernatant of each
sample was transferred into 50 ml falcons and then adjusted at different pH: 6,
7 and 8.
2.2.1.3 Extraction of Exiguobacterium indicum
In order to study on the pigment extraction, 0.1 ml Exiguobacterium
indicum cultures were added into two Erlenmeyer flasks containing 50 ml LB.
6
Then, two Erlenmeyer flasks were cultured for 2 days in 25 oC with shaking for
extraction step. The pellets were collected after centrifugating at 12000 rpm/10
min/room temperature. The study about the pigment of Exiguobacterium
indicum has not been studied widely yet. Therefore, this study had tried to
screen the solvent for pigment isolation from the pellets.
According
to
properties
of
cell
wall
membrane
which
includes
phospholipid, the pigment on the cell wall membrane should be soluble in lipid or
nonpolar solvents. Therefore, the screening solvents were based on the high
non-polarity (petroleum ether, chloroform) to polarity solvents (methanol,
distilled water).
The pellets from 2 samples were then extracted by using petroleum ether
and chloroform. However, the pigment was not extracted. Therefore, methanol
was used to extract. 2 ml methanol was added to each falcon to extract the
pigment from the pellets. Next, falcons were shaken for 30 minutes. After that,
they were centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatants
which contain the pigment in methanol fraction were transferred into new
falcons. The methanol extraction procedure was repeated 3 times with the
pellets. All the pigment fractions were combined together and the total was 6 ml.
After these steps, the pellets which have no more color were discarded. The 6ml
supernatant from each pellet was collected and put into 45°C incubator in order
to evaporate the methanol from the solutions. The yellow orange solid powders
were collected.
Next step, an enough amount of distilled water was added into the
powder to dissolve the water soluble compound. Then the falcons were again
centrifuged 10,000 rpm for 10 minutes at 4°C. The supernatant and pellet were
collected. After that, the supernatant was lyophilized into powder which was
used for preliminary identification steps as compound A. The pellet was dissolved
into chloroform, then filtered, and evaporated by putting into 45°C incubator for
a night. The powders were also kept for preliminary identification steps as
compound B.
2.2.2 Antimicrobial activities tests of Exiguobacterium indicum
2.2.2.1 Testing with supernatant
The antimicrobial activities of Exiguobacterium indicum were assayed at 3
different durations: 1 day, 2 days and 3 days and 4 different temperatures:
25oC, 37oC, 45oC and 50oC. The antibiotic Ceftriaxone (Shinpoong Daewoo
7
Pharmaceutical Company, Vietnam) was used to compare to their CFEs for the
antimicrobial activity against Candida albicans, Salmonella typhi, Pseudomonas
aeruginosa, Staphylococcus sciuri, Serratia marcescens and Micrococcus luteus.
The antibacterial activities of Exiguobacterium indicum were tested by
applying agar well diffusion method described by Cleidson et al. (2007) with
some modifications. 20 μl of 24-hour-old culture of test microorganisms
prepared at cultivation step was inoculated onto LB agar by spread plate
method. Then, wells were made on these pathogenic containing LB plates by
using a sterile cork borer (5 mm in diameter). Each well was fulfilled by 100 μl of
crude CFEs of Exiguobacterium indicum. Next, the dishes were placed at 4oC for
1 hour to make time for the diffusion of CFEs to the agar, and then incubated at
room temperature (RT). The results of this test were observed after 16 to 24
hours of incubation. When there were antimicrobial activities, halo zones or
inhibition zones would appear around the wells since there would be no growth
of pathogen. The diameters of the inhibition zones observed were measured in
millimeter, excluded the diameter of the well.
The antibiotic ceftriaxone at
different concentration (10 mg/ml, 1 mg/ml, 0.1 mg/ml, 0.01 mg/ml, 0.001
mg/ml, 0.0001 mg/ml) were assayed their activities against Candida albicans,
Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus sciuri, Serratia
marcescens and Micrococcus luteus by the same protocol to make standard for
the potency of the crude extracts of Exiguobacterium indicum. The tests were
triplicated.
After testing with time and temperature, the best condition was chosen.
The antimicrobial activities of Exiguobacterium indicum then were assayed for
stability at 3 different types of pH (6, 7 and 8). The same procedure as 2.2.2.1
was applied. The tests were triplicated.
2.2.2.2 Testing with pellet
The pellets of Exiguobacterium indicum before extractions were also
assayed to check whether it has the antimicrobial activities or not by using disk
diffusion method. The pellets were put onto the pathogen containing plates. The
plates were incubated at room temperature (RT). The results of this test were
observed after 16 to 24 hours of incubation. When there were antimicrobial
activities, halo zones or inhibition zones would appear around the papers since
there would be no growth of pathogen. The diameters of the inhibition zones
observed were measured in millimeter.
8
2.2.3
Preliminary
identification
of
compounds
extracted
from
Exiguobacterium indicum
2.2.3.1 Mass spectrometry (MS)
The compound A was dissolved in water and compound B was dissolved
in chloroform with enough amounts. After that, the samples were sent to
University of Science. The MS instrument used was MicrOTOF-q with ESI
interface, Bruker Daltonics, Coventry, UK. The conditions of MS were ion polarity
(positive), capillary voltage (4500 V), dry gas (8.0 L min -1), nebulizer (1.2 Bar),
mass range (100 – 1000m/z), collision energy (4.0 eV) and collision cell RF (250
Vpp). After a week, the results were given back under a graph. The formulas of
the compounds were predicted base on the graph.
2.2.3.2 Infrared (IR)
The compound A and B were prepared as in the 2.2.1.3 but collected in
powder forms. Then they were sent to University of Science. In order to obtain
an infrared spectrum of a solid, it is necessary to get light, mainly infrared
through the sample. A thin layer of a solid deposited as a solution on an infrared
cell and allowed to evaporate has proven successful with many solids. Solvents
such as CHCl3, CH2Cl2 and CCl4 have been frequently been used. The solid
sample should have an appreciable solubility in one of these solvents. A drop of
a solution left to evaporate will deposit a thin film of crystal that will often
transmit sufficient light to provide an acceptable infrared spectrum. After a
week, the results were given back under a graph. The main functional groups of
the compounds were predicted base on the graph.
2.3 Data analysis
The computer program Statistical Package for the Social Sciences version
16.0 (SPSS ver 16.0) (IBM Corporation, USA) was applied to analyze data.
Results were expressed as mean ± standard deviation (SD). Statistical
significance of the results was calculated to at P < 0.05.
9
3. Results
3.1 Cultivation & Optimization & Extraction
To prevent the contamination of cultures, Exiguobacterium indicum in
each culture was gram stained. Exiguobacterium indicum which was Grampositive and rod shape, was showed under light microscope (figure 2).
Figure 2 Gram stain of Exiguobacterium indicum
After culturing Exiguobacterium indicum for 3 days, the OD were measured in table 1.
Table 1 Result of optical density of Exiguobacterium indicum
Temp
Time
OD (600 nm)
25
24
0.865
25
48
1.217
25
72
1.438
37
24
0.905
37
48
0.943
37
72
1.223
At 45 oC and 50 oC, the optical density of Exiguobacterium indicum was
almost zero. It was meant that there was no Exiguobacterium indicum growth at
45 oC and 50 oC. The figure 3 illustrated for the growth at different temperatures
after 3 days. The third and fourth flasks were still transparent after culturing at
10
45 oC and 50 oC, respectively (Figure 3). Therefore, these conditions were not
selected for antimicrobial activities tests.
Figure 3 Exiguobacterium after 3 days
3.2 Antimicrobial activities
3.2.1 Testing with supernatant
The supernatant of Exiguobacterium indicum did not show the effects
against Candida albicans, Staphylococcus sciuri, and Micrococcus luteus because
there was no inhibition zones observed onto their plates. However, the
supernatant of Exiguobacterium indicum showed the antimicrobial ability against
Salmonella typhi, Pseudomonas aeruginosa and Serratia marcescens (figure 4,
5, 6). The inhibition zones were measured and presented with the tables 2.
The antibiotic ceftriaxone at different concentration (10 mg/ml, 1 mg/ml,
0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml and 0.0001 mg/ml) were assayed their
activities against Salmonella typhi, Pseudomonas aeruginosa and Serratia
marcescens to make standard for the potency of the crude extracts of
Exiguobacterium indicum (figure 7, 8 and 9).
11
25
25
37
25
37
37
Figure 4 Inhibition zones of Exiguobacterium indicum
supernatant after culturing at 25 oC and 37 oC for 3 days
on Salmonella typhi
37
37
25
25
37
25
Figure 5 Inhibition zones of Exiguobacterium indicum
supernatant after culturing at 25 oC and 37 oC for 3 days
on Pseudomonas aeruginosa
37
25
37
25
37
25
Figure 6 Inhibition zones of Exiguobacterium indicum
supernatant after culturing at 25 oC and 37 oC for 3 days
on Serratia marcescens
12
Log of concentration (ug/ml)
Ceftriaxone against Salmonella
typhi
y = 0.128x - 0.6118
R² = 0.9666
5
4
3
2
1
0
0
5
10
15
20
25
30
35
40
Diameter of inhibition zone (mm)
Figure 7 Standard curve of antimicrobial activities of ceftriaxone against Salmonella typhi
Log of concentration (ug/ml)
Ceftriaxone against Pseudomonas
aeruginosa
5
y = 0.1214x - 0.9376
R² = 0.959
4
3
2
1
0
0
10
20
30
40
50
Diameter of inhibtion (mm)
Figure 8 Standard curve of antimicrobial activities of ceftriaxone against Pseudomonas
aeruginosa
Log of concentration (ug/ml)
Ceftriaxone against Serratia
marcescens
5
y = 0.1423x - 1.4837
R² = 0.9959
4
3
2
1
0
0
5
10
15
20
25
30
35
40
Diameter of inhibition zone (mm)
Figure 9 Standard curve of antimicrobial activities of ceftriaxone against Serratia
marcescens
13
Table 2 Diameter of inhibition zone (mm) and potency determination according to
ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant on Salmonella typhi
Salmonella typhi
25
37
Hrs
Zone
1
(cm)
Zone
2
(cm)
Zone
3
(cm)
Mean of
inhibition
zone diameter
(cm)
SD
24
0.7
0.8
0.9
0.8
0.1
Equivalent
concentration (in
comparison to
ceftriaxone)
(μg/ml)
2.58
48
0.9
1
1.1
1
0.1
4.66
72
1.3
1.4
1.5
1.4
0.1
15.14
24
0.4
0.6
0.7
0.57
0.15
1.30
48
0.7
0.8
0.8
0.77
0.06
2.34
72
0.8
0.9
0.9
0.87
0.06
3.14
Table 3 Diameter of inhibition zone (mm) and potency determination according to
ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant on Pseudomonas aeruginosa
Pseudomonas aeruginosa
25
37
Hrs
Zone
1
(cm)
Zone
2
(cm)
Zone
3
(cm)
Mean of
inhibition
zone diameter
(cm)
SD
24
0.9
0.9
1
0.93
0.06
Equivalent
concentration (in
comparison to
ceftriaxone)
(μg/ml)
1.57
48
0.8
0.9
1
0.9
0.1
1.43
72
1.2
1.3
1.3
1.27
0.06
3.98
24
0.8
0.8
0.9
0.83
0.06
1.19
48
0.6
0.7
0.7
0.67
0.06
0.74
72
0.7
0.8
0.8
0.77
0.06
0.98
Table 4 Diameter of inhibition zone (mm) and potency determination according to
ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant on Serratia marcescens
Serratia marcescens
25
37
Hrs
Zone
1
(cm)
Zone
2
(cm)
Zone
3
(cm)
Mean of
inhibition
zone diameter
(cm)
SD
24
0.8
0.8
0.9
0.83
0.06
Equivalent
concentration (in
comparison to
ceftriaxone)
(μg/ml)
0.50
48
0.9
0.9
1
0.93
0.06
0.70
72
1
1.1
1.3
1.13
0.15
1.35
24
0.7
0.8
0.8
0.77
0.06
0.40
48
0.7
0.8
0.8
0.77
0.06
0.40
72
0.7
0.8
0.8
0.77
0.06
0.40
14
SPSS was used to indicate whether temperature and time affect the result
of antimicrobial activities. The tables 7, 8, 9 in appendixes which are based on
tests of subjects between effects would prove that.
The significant differences which are from table 7, table 8 and table 9 in
appendixes showed that the temperature and the time have affected the
diameters of inhibition zones.
Base on the table 2, table 3 and table 4, the best condition which gave
the
most
effectiveness
antimicrobial
activities
against
Salmonella
typhi,
o
Pseudomonas aeruginosa and Serratia marcescens were at 25 C for 3 days.
After the best condition was chosen, the antimicrobial activities of
Exiguobacterium indicum were tested for the stability at 3 different types of pH
(6, 7 and 8). The supernatants of Exiguobacterium indicum showed the
antimicrobial ability against Salmonella typhi, Pseudomonas aeruginosa and no
effect against Serratia marcescens (figure 7). The inhibition zones were
measured and presented with the tables 8.
7
6
7
6
8
6
8
8
8
6
7
7
Figure 10 Inhibition zones of Exiguobacterium indicum supernatant after adjusted pH 6,
7, 8 against Salmonella typhi and Pseudomonas aeruginosa. Left: Salmonella typhi;
Right: Pseudomonas aeruginosa
15
Table 5 Diameter of inhibition zone (mm) and potency determination according to
ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant (pH 6, 7, 8) on Salmonella
typhi
Salmonella typhi
pH
Zone
1
(cm)
Zone
2
(cm)
Zone
3
(cm)
Mean of
inhibition
zone diameter
(cm)
6
1
1.1
1.2
1.10
0.10
Concentration
(in
comparison
to
ceftriaxone)
(μg/ml)
6.25
7
1.3
1.5
1.8
1.53
0.25
22.43
8
0.8
1.1
1.2
1.03
0.21
5.14
SD
Table 6 Diameter of inhibition zone (mm) and potency determination according to
ceftriaxone (μg/ml) of Exiguobacterium indicum supernatant (pH 6, 7, 8) on
Pseudomonas aeruginosa
Pseudomonas aeruginosa
pH
Zone
1
(cm)
Zone
2
(cm)
Zone
3
(cm)
Mean of
inhibition
zone diameter
(cm)
6
0.9
0.9
0.9
0.90
0
Concentration
(in
comparison
to
ceftriaxone)
(μg/ml)
1.43
7
1.3
1.4
1.4
1.33
0.06
4.80
8
0.9
1
1
0.97
0.06
1.72
SD
SPSS was used to indicate whether temperature and time affect the result
of antimicrobial activities. The tables below which are based on tests of subjects
between effects would prove that.
The significant differences which are from table 10 and table 11 in
appendixes showed that the pH have affected the diameters of inhibition zones.
Base on the table 5 and table 6, the best condition which gave the most
effectiveness antimicrobial activities against Salmonella typhi, Pseudomonas
aeruginosa and Serratia marcescens were at pH 7.
In conclusion, the optimum condition for Exiguobacterium indicum to have
the greatest antimicrobial activities was at 25 oC, pH 7 for 3 days.
16
Potency of Exiguobacterium indicum compare to Ceftriaxone
Base on the ceftriaxone curve on Salmonella typhi, Pseudomonas
aeruginosa and Serratia mercescens, the potency of Exiguobacterium indicum
supernatant was measured according to ceftriaxone (figure 12, 13 and 14 in
appendixes)
For antimicrobial ability against Salmonella typhi, the minimum potency
of Exiguobacterium indicum compare to Ceftriaxone is 1.30 μg/ml at 37 oC for 1
day and the maximum is 22.43 μg/ml at 25 oC, pH 7 for 3 days.
For antimicrobial ability against Pseudomonas aeruginosa, the minimum
potency of Exiguobacterium indicum compare to Ceftriaxone is 0.74 μg/ml at 37
o
C for 2 days and the maximum is 4.8 μg/ml at 25 oC, pH 7 for 3 days.
For antimicrobial ability against Serratia marcescens, the minimum
potency of Exiguobacterium indicum compare to Ceftriaxone is 0.4 μg/ml at 37
o
C for 1 day and the maximum is 1.35 μg/ml at 25 oC, pH 7 for 3 days.
3.2.2 Testing with pellet after pigment extraction
There was no effect of pellet against Candida albicans, Salmonella typhi,
Pseudomonas aeruginosa, Staphylococcus sciuri, Serratia marcescens and
Micrococcus luteus.
3.2.3 Testing the pigment extract
The pigment after extracted from the pellet showed a weak antimicrobial
activity against Micrococcus luteus (Data was not shown).
3.3 Preliminary identification
In order to check the purity of the pigment extraction process, the
compound A and B were identified by using MS and IR.
17
3.3.1 Compound A
The result of mass spectroscopy of compound A was displayed in figure 8.
Figure 11 MS of compound A
Base on the figure 8, the molar weight of the compound A were about 257
Da.
Using
mass
calculator
(http://www.massbank.jp/MassCalc.html),
predicted formula were C10H21N6S and C13H23NO4.
N-H
C-H
C=C
Figure 12 IR of compound A
18
the
Base on figure, there were band around 3000-2850 cm-1, 1680-1640 cm-1 and 3500-3300
cm-1 which indicated that compound would have C-H, C=C and N-H stretch respectively.
Moreover, there were 2 bands around 3400 - 3200 cm-1 which indicated that there was a
primary amine. Hence, the predicted main functional group was primary amine with
double bond.
In conclusion, the compound A had the formula C10H21N6S with amine
group and double bond.
3.3.2 Compound B
The result of mass spectroscopy of compound B was displayed in figure 10.
Figure 13 MS of compound B
Base on the figure 8, the molar weight of the compound B were about
540 Da. Using mass calculator (http://www.massbank.jp/MassCalc.html), the
predicted formula were C15H20N5O13P2 and C24H28O14.
19
C=C
C=O
C-H
Figure 14 IR of cpompound B
Base on figure, there were band around 3000-2850 cm-1, 1680-1640 cm-1
and 1760-1665 cm-1 which indicated that compound would have C-H, C=C and
C=O stretch respectively. Moreover, with the present of C-H, it meant that C=O
was presented aldehyde group. Therefore, the predicted main functional group
was aldehyde group with double bond.
In conclusion, the compound B had the formula C24H28O14 with aldehyde
group and double bond.
4. Discussion
Base on the research of Chaturvedi & Shivaji (2006), the maximum growth
temperature of Exiguobacterium indicum was less than 41 oC. That was the
reason why the bacteria could not grow at 45 oC and 50 oC. The optical density of
Exiguobacterium indicum seems similar when culturing at 25 oC and 37 oC (Table
1). However, the antimicrobial activities will not be similar. Therefore, the study
had tried to test on pathogens with the cultures incubated at different
temperature and time incubation (1 day, 2 days and 3 days). Because of the
time limitation of the study, the range of incubation time was chosen for the
study was short and only the LB medium was used in this study. From the
results and analysis, the effects of cultivation temperatures on the antimicrobial
activities were not correlated to their effects on the growth of the bacteria.
20
According to the research of Chaturvedi & Shivaji (2006), the pH for growth
are from 6 -10 was proved. Moreover, after cultivation, their pH were checked and they
o
o
varied from 6 - 7 at 25 C and 37 C in this study. From this reason, the stability of
the antimicrobial activity focused on pH 6, 7 and 8. By statistical analysis, pH7
makes the activity stable. Therefore, more detail research should be done to
clarify the production of antimicrobial activity.
Exiguobacterium
indicum
presented
activity
against
Gram-negative
(Salmonella typhi, Pseudomonas aeruginosa) and Gram-positive pathogens
(Serratia mescescens). This is a positive sign for the application of this
Exiguobacterium
isolated
in
Vietnam
conditions
to
pharmaceutical
field.
However, the mechanism of these bacteria was not researched. There were
some
previous
experiments
conducted
as
procedure
but
there
was
no
information on antimicrobial activities of this strain. For better experiment, the
mechanism must be studied.
The potency of Exiguobacterium indicum supernatant on Salmonella
typhi, Pseudomonas aeruginosa and Serratia mescescens could be determined
based on ceftriaxone activity. Therefore, we could conclude that Exiguobacterium
indicum has the antimicrobial activities on Salmonella typhi, Pseudomonas
aeruginosa and Serratia mescescens. This study is a first on antimicrobial
activity of Exiguobacterium indicum isolated in Vietnam.
The yellow pigment was supposed to have the ability to against the
pathogens. However, the result showed a weak activity against Micrococcus
luteus. Probably, the amount of the pigment used for testing was not enough or
the stability of this pigment was lost during extraction. Therefore, the test should
be optimized condition for pigment activity.
Although the antimicrobial activity of the pigment was weak on
Micrococcus luteus, this study gave a signal that the pigment will give the
antimicrobial activities on the other strains in a suitable culture conditions.
Therefore, the compound A and B were extracted and preliminarily identified,
using MS and IR. With the two clear peaks at 147 Da and 257 Da in the mass
spectrum when measuring compound A, the molar weight of the compound A
was expected about 257 Da base on the functional groups determined in IR
spectrum. The peak at 147 Da might be present for the chain or branch of
compound A. More structural identification should be performed. Similarly,
compound B structure gave 5 peaks on the mass spectrum, but the molar weight
of the compound B was expected about 540 Da base on the functional groups
21
determined in IR spectrum. The other peaks might be present for the chain or
branch of compound B. More structural research should be studied to understand
why two compounds A and B are yellow but the solubility in solvent are different.
5. Conclusion
The research indicated that the supernatant of Exiguobacterium indicum
had the antimicrobial activities against pathogens such as Salmonella typhi,
Pseudomonas aeruginosa and Serratia marcescens. The pigment showed a weak
activity on Micrococcus luteus only. The most effective condition for antimicrobial
test of these bacteria was at 25 oC for 3 days. The activity was stable at pH7.
Moreover, the pigment was successfully extracted out of the cell wall membrane
of bacteria.
22
References
Ahmad, W.A., Ahmad, W.Y.W., Zakaria, Z.A., and Yusof, N.Z. Application of
Bacterial Pigments as Colorant: The Malaysian Perspective. Springer,
2012, New York, pp. 29-38, 59-70
Andrighetti-Frohner, C. R., et al. (2003). "Cytotoxicity and potential antiviral
evaluation of violacein produced by Chromobacterium violaceum" Mem
Inst Oswaldo Cruz 98(6): 843-848.
Barja JL, Lemos ML, Toranzo EA (1989) Purification and characterization of an
antibacterial substance produced by a parine Alteromonas Species.
Antimicrob Agents Chemother 33(10):1674–1679
Chaturvedi, P., & Shivaji, S. (2006). Exiguobacterium indicum sp. nov., a
psychrophilic bacterium from the Hamta glacier of the Himalayan
mountain
ranges of
India. International
journal
of systematic and
evolutionary microbiology, 56(12), 2765-2770.
Journal of Immunological Methods: author and subject indexes (1991). Volumes
136-145." J Immunol Methods: 1-71.
Şerban, E. S., et al. (2011). "Screening of the antibacterial and antifungal
activity of eight volatile essential oils" Farmacia 59: 440-446.
Valgas, C., Souza, S. M. D., Smânia, E. F., & Smânia Jr, A. (2007). ―Screening
methods to determine antibacterial activity of natural products‖ Brazilian
Journal of Microbiology, 38(2), 369-380.
Venil, C.K., and Lakshmanaperumalsamy, P. ―An Insightful Overview on Microbial
Pigment, Prodigiosin.‖ Electronic Journal of Biology, 2009, Vol. 5(3): 4961.
Wang H, Jiang P, Lu Y, Ruan Z, Jiang R, Xing XH, Lou K, Wei D (2009)
Optimization of culture conditions for violacein production by a new strain
of Duganellasp. B2. Biochem Eng J44:119–124
W. A. Ahmad et al.,Application of Bacterial Pigments as Colorant, SpringerBriefs
in Molecular Science, DOI: 10.1007/978-3-642-24520-6_2, 2012
23
Appendix
Table 7 Temperature & Time effects on Salmonella typhi
Tests of Between-Subjects Effects
Dependent Variable:Zone
Type III Sum of
Source
Squares
df
Mean Square
F
Sig.
Corrected Model
1.200
a
5
.240
24.000
.000
Intercept
14.580
1
14.580
1.458E3
.000
Temp
.500
1
.500
50.000
.000
Time
.610
2
.305
30.500
.000
Temp * Time
.090
2
.045
4.500
.035
Error
.120
12
.010
Total
15.900
18
1.320
17
Corrected Total
a. R Squared = .909 (Adjusted R Squared = .871)
Table 8 Temperature & Time effects on Pseudomonas aeruginosa
Tests of Between-Subjects Effects
Dependent Variable:Zone
Type III Sum of
Source
Squares
df
Mean Square
F
Sig.
a
5
.127
28.625
.000
14.401
1
14.401
3.240E3
.000
Time
.164
2
.082
18.500
.000
Temp
.347
1
.347
78.125
.000
Time * Temp
.124
2
.062
14.000
.001
Error
.053
12
.004
Total
15.090
18
.689
17
Corrected Model
Intercept
Corrected Total
.636
a. R Squared = .923 (Adjusted R Squared = .890)
Table 9 Temperature & Time effects on Serratia marcescens
Tests of Between-Subjects Effects
Dependent Variable:Zone
Type III Sum of
Source
Squares
df
Mean Square
F
Sig.
a
5
.064
9.600
.001
13.520
1
13.520
2.028E3
.000
Temp
.180
1
.180
27.000
.000
Time
.070
2
.035
5.250
.023
Temp * Time
.070
2
.035
5.250
.023
Error
.080
12
.007
Total
13.920
18
.400
17
Corrected Model
Intercept
Corrected Total
.320
a. R Squared = .800 (Adjusted R Squared = .717)
Table 10 pH effects on Salmonella typhi
ANOVA
Zone
Sum of Squares
df
Mean Square
Between Groups
.442
2
.221
Within Groups
.233
6
.039
Total
.676
8
F
5.686
Sig.
.041
Table 11 pH effects on Pseudomonas aeruginosa
ANOVA
Zone
Sum of Squares
df
Mean Square
Between Groups
.327
2
.163
Within Groups
.013
6
.002
Total
.340
8
F
73.500
Sig.
.000

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