ECOLOGY AND FISHERIES ISSN 0974-6323

Vol. 3(1).pp.9-18 2010

ANTIMICROBIAL ACTIVITY OF PSEUDOMONAS SPECIES

ISOLATED FROM MARINE
ENVIRONMENT OF KARWAR, WEST COAST OF INDIA

P. V.Khajure and J.L. Rathod

Department of Marine Biology,
Karnatak University P.G & Research Centre, Kodibag, Karwar -581303

ABSTRACT

A deep Sea sediment and water samples were collected from the Karwar region,
West Coast of India, microorganisms were isolated from the samples. A total 28
isolates were subjected to primary screening by perpendicular streak method against
Gram-positive (Bacillus subtilis and Staphylococcus aureus), Gram-negative
(E.coli, Klebsiella species, Proteus species and Salmonella typhi) test bacteria. It
was observed that 4 isolates were active against only Gram-negative bacteria, 3
against Gram-positive and 8 against both Gram-negative and Gram-positive
bacteria.
Altogether 15 putative isolates were subjected to secondary screening by
Agar well diffusion method to further test the capabilities of primarily screened
organisms. Selected isolates (10) from the secondary screening belong to the genera
Pseudomonas (03), Proteus (02), Aeromonas (03) Vibrio (02). Finally species were
selected for the further study on the basis of (a) broad spectrum activity and (b)
larger zone of inhibition in comparison to others. The antibacterial substances were
extracted with Ethyl acetate from isolate-inoculated production medium fermented
for 48 hours at 30oC by solvent extraction method. The isolate grew in the presence
of 20 % (w/v) NaCl, antibiotic production being maximum with 5% (w/v) Nacl in
the production medium. Natural seawater stimulated the antibiotic biosynthesis.
The absence of catabolic repression during the synthesis of the antimicrobial
substance was demonstrated by the utilization of glucose by this isolates. The
highly stable, active principle was purified by Ethyl acetate extraction and thin
layer chromatography (TLC) and each single compound from two species was
found to posses the broad-spectrum activity. Considering all of the above evidences
and based on comparison with the current literature, the bioactive compound
isolated from Pseudomonas spp. was responsible for Antimicrobial activity.

Key Words: Pseudomonas species, Antimicrobial activity, Fermentation.

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INTRODUCTION
The Ocean, which is called the ‘Mother of origin of life’, is also the source of structurally unique
natural products that are mainly accumulated in living organisms. Several of these compound
show pharmacological activities and are helpful for the invention and discovery of bioactive

compound, primarily for deadly diseases like cancer, acquire immuno-deficiency syndrome, etc.
The lives saving drugs are mainly found abundantly in microorganisms, algae and invertebrates
and vertebrates. Modern technologies have opened vast areas of research for the extraction of
biomedical compounds from ocean and seas to treat the deadly diseases.
For more than two decades, there has been an ongoing quest to discover new drugs from
the sea. Most efforts have been directed towards chemical studies of marine invertebrates.
Although these studies have indeed proven that marine invertebrates are an important source of
new biomedical leads, a fact well demonstrated by the number of compounds currently in clinical
trails, it has proven notoriously difficult to obtain adequate, reliable supplies of these compound
from nature. Because of these problems, anew avenue of study focusing on marine
microorganisms has been gaining considerable attention. At first sight thus, the expectable
enormous biodiversity of marine microorganisms might have been the reason for the interest in
their study. Although marine microorganisms are not well defined taxonomically, preliminary
studies indicate that the wealth of microbial diversity in the world’s oceans, make this a
promising frontier for the discovery of new medicines.
Screening of marine bacteria isolated from the surface of marine algae and invertebrates
has shown that a high percentage produce antimicrobial metabolites. In addition, bacteria in
biofilms formed on the surface of marine organisms have been documented to contain a high
proportion of antibiotic producing bacteria than some other marine environment. Marine
epiphytic bacteria, associated with nutrient rich algal surfaces and invertebrates, have also been
shown to produce antibacterial secondary metabolites, which inhibit the settlement of potential
competitors.

MATERIALS AND METHODS

Sample collection: The deep-sea sediment and water sample were collected from the Karwar
region, west coast of India at a depth of 20 meter. (Lat. 14o 45.46'N. Long. 074o 02.84'E). The
samples were brought to the laboratory in aseptic condition. Than the microorganisms were
cultivated on Zobell Marine Agar 2216, than it was sub cultured on Modified Nutrient agar
(MNA).
Screening of Isolates for antimicrobial activity: The screening method consists of two steps:
primary screening and secondary screening. In primary screening the antimicrobial activity of
pure isolates was determined perpendicular streak method on Nutrient agar (NA) (Egorov, 1985).
The test organisms used were; Bacillus subtilis, Staphylococcus aureus, E.coli, Klebsiella
species, Proteus species and Salmonella typhi.
Secondary screening was performed by Agar well diffusion method against the standard test
organisms E.coli, Staphylococcus aureus, Salmonella typhi, Bacillus subtilis and Proteus species.
Effect of Sugar and NaCl on growth of Isolates: The effect of slow and rapidly metabolized
sugar was tested by replacing starch and glucose by an equivalent amount of glucose, maltose and
lactose. As NaCl is the major constituent of seawater, a kinetic study of growth and antibiotic
production with varying concentration of this salt added to Production medium was done as
shown in fig (1). The effect of composition of the water used for cultivation on growth and
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antimicrobial activity of the isolates was determined by using varying amount of natural seawater
collected from the site where the samples was obtained. During the experiment on the
optimization of the growth A.niger and S.aureus were used as test organisms. The isolates were
cultivated on a shaker (200rpm) at 30oC.The determinations were done at least thrice and the
averages of the values are reported. For the determination of wet biomass, 1ml of sample was
centrifuged and the weight of the pellet was determined.

Fig(1): Effect of NaCl on Growth and Antimicrobial activity of

Pseudomonas spp.

5
4.5
Growth Zone of Inhibition

4
3.5
3 Activity against A.niger
2.5 Activity against S.aureus
2 Growth
1.5
1
0.5
0
6h

6h
8h

2h

8h
h

2h
6h
0% l 48

l4

l7

l4
l9

l9
l7
l9

aC

aC
aC
ac

ac
aC

aC
ac

N
N

N
N

%
%

%
5%
0%

10

20
10

15

20

Characterization of Isolates: The potent isolates selected from the secondary screening were
characterized by morphological and biochemical methods. The results of microscopic
examination were compared with Bergey's manual of Determinative Bacteriology, Ninth edition
(2000) and the organism was identified. Various biochemical tests were performed for the
identification of the potent isolates are as follows; Fermentation of sugars, Hydrolysis of starch,
Indole production, Methyl red, Vogues Prauskauer, Citrate utilization, Urease test, 2%Peptone
water, Nitrate reduction test, Gelatin liquefaction, Catalase test, Oxidase test.
Fermentation process: Fermentation was carried out in a 1L Erlenmeyer flask following the
procedure as described by Liu et.al (1992)
Isolation of antibacterial metabolites: Antibacterial compound was recovered from the filtrate
by solvent extraction method following the process described by Westley et.al, 1979.and Liu
et.al1986. Ethyl acetate was added to the filtrate in the ratio of 1:1(v/v) and shaken vigorously for
1 hour for complete extraction. The Ethyl acetate phase that contains antibiotic was separated
from the aqueous phase. It was evaporated to dryness in water bath at 80°-90°C and the residue
obtained were weighed. Thus obtained compound was used to determine antimicrobial activity,
minimum inhibitory concentration and to perform bioautography.
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Determination of the antimicrobial activity: The antimicrobial activity was determined by agar
well diffusion method (Sen. et al., 1995). The partially purified extract obtained by the
evaporation of the ethyl acetate extract was dissolved in 1 ml 0.2M phosphate buffer (pH 7.0).

Then 100µl of it was loaded into well bored and test organism (0.5 McFarland turbidity standard)
swabbed Muller Hinton agar plates. The plates were incubated at 37°C for 18-24 hrs and
examined. The diameter of the zones of complete inhibition was measured to the nearest whole
millimeter.
Determination of minimum inhibitory concentration: It was determined by the serial dilution
of the antimicrobial in nutrient broth, two fold dilutions at each time, against Staphylococcus
aureus.
Thin layer chromatography and Bioautography: Silica gel plates, 10X20 cm, 1mm thick,
were prepared. They were activated at 150°C for half an hour. Ten microliters of the ethyl acetate
fractions and reference antibiotics were applied on the plates and the chromatogram was
developed using chloroform: methanol (4:1) as solvent system. The plates were run in duplicate;
one set was used as the reference chromatogram and the other was used for bioautography. The
spots in the chromatogram were visualized in the iodine vapour chamber and UV chamber.
Muller Hinton agar inoculated with Staphylococcus aureus was poured over the chromatogram
and the plate was incubated overnight at 37°C in sterile condition. The next day the inhibition
zones were noted and the Rf values of the antimicrobials were determined.
Toxicity: Hemolytic activities of the bioactive compounds towards marine and human
erythrocytes were tested. Three milliliter of 1% washed erythrocytes suspension was incubated
with the active compounds at the concentration ten times MIC values at 37oC for 45min. Distilled
water and normal saline were taken as a positive and negative controls, respectively. After
incubation, the erythrocytes were separated and the amount of hemoglobin released was
determined by measuring the absorbance of the supernants at 540nm.

RESULTS
Out of 28 isolates were subjected for primary screening process, only 17 isolates showed the
activity against test organisms. Of the 17 isolates, 4 were active against only gram-negative
organism, 3 against gram-positive organisms and 8 against both gram positive and gram-negative
organisms.
Out of the 15 isolates that were subjected for the secondary screening, 9 isolates were
active against Bacillus subtilis, 5 against Staph.aureus, 3 against E. coli, 4 against Proteus
species and 5 against Salmonella typhi.
Identification: The identification of the potent antibiotic producing strains reveals that most of
the specimens belong to the genus Pseudomonas (03), Proteus (02), Aeromonas (03) Vibrio (02).
One potent isolates were selected for fermentation on the basis of their broad spectrum of activity
and largest zone of inhibition. They were found belonging to the genera spp.
Minimum inhibitory concentration: The minimum inhibitory concentration for the extract from
spp was 5mg/ml.
Thin layer chromatography and Bioautography: The spot given by the extract of spp. was a
circular with Rf value 0.88 and the fluorescence colours of the spots were greenish yellow. The
reference antibiotic, Tetracycline, didn’t move with the solvent system. In bioautography the spp
gave an inhibition zone of 20mm diameter. The reference antibiotic Tetracycline gave the
inhibition zone of 25mm diameter at the origin.
Toxicity: The active compound from Pseudomonas spp. lysed only 1.9% of marine erythrocytes
and 1.4% of human erythrocytes at concentration ten times the MIC values.
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Table 1: Morphological and Biochemical tests for taxonomical studies of the Isolates

Tests Results
Temperature 30oC
Time 24h
Morphology
Size 1mm
Shape Circular
Colour White
Margin Entire
Elevation Raised
Consistency Butyrous
Opacity Opaque
Gram-Staining Gram –ve short rods
Motility Motile
Biochemical Tests
Aerobic/Anaerobic test Aerobic
Fermentation of Sugar
Glucose +Ve
Lactose -Ve
Maltose -Ve
Mannitol +Ve
Xylose -Ve
Sorbitol -Ve
Sucrose -Ve
Hydrolysis of Starch -Ve
Indole production -Ve
Methyl Red -Ve
Vogues Prauskauer Test -Ve
Simmon Citrate Test +Ve
Urease Test -Ve
2% Peptone +Ve
Nitrate Peptone -Ve
Gelatin liquefaction +Ve
Catalase +Ve
Oxidase +Ve
Pseudomonas species.
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Fig (2): Showing Antimicrobial activity of

Pseudomonas species

25

20
Zone of Inhibition

15
Zone of Inhibition (mm)
10

0
Bacillus pumilis
Bacillus subtilis
Staphylococcus

Methicillin
Salmonella

Aspergillus
Klebsiella
Escherichia

Lactococcus

Candida
Proteus

Serratia

Test organisms

DISCUSSION
The putative isolates of primary screening when subjected to secondary screening, showed
different activity from that of primary screening; some of the active isolates didn’t show the
activity in the secondary screening while some showed little activity and some showed improved
activity. According to Bushell (1993), during the screening of the novel secondary metabolite,
Marine isolates are often encountered which show antibiotic activity on agar but not in liquid
culture.
The result of primary and secondary screening revels that most of the active isolates were active
against gram-positive bacteria (Bacillus subtilis and S.aureus) than gram-negative bacteria. The
reason for different sensitivity between gram positive and gram-negative bacteria could be
ascribed to the morphological differences between these microorganisms, gram-negative bacteria
having an outer polysaccharide membrane carrying the structural lipopolysaccharide components.
This makes the cell wall impermeable to lipophilic solutes; the gram positive should more
susceptible having only an outer peptidoglycan layer which is not an effective permeability
barrier (Scherrer and Gerhardt, 1971).
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Table 2: Antimicrobial activity of isolates

Test Organisms Zone of MIC

Inhibition (�g ml-1)
(mm)
Escherichia coli 15 0.50
Bacillus subtilis 16 0.45
Staphylococcus aureus 17 0.45
Bacillus pumilis 18 0.45
Lactococcus lactis 18 0.45
Klebsiella pneumoniae 20 0.45
Proteus mirabilis 18 0.45
Salmonella typhi 17 0.45
Serratia marcescens 19 045
Methicillin resistant S.aureus 17 Not Determined

Candida albicans 18 0.45

Aspergillus niger 16 0.50

Although various biochemical tests were performed, further the isolates may identify up
to species level by adopting the 16S rRNA sequencing. The minimum inhibitory concentration
(MIC) for the antimicrobial extracted from spp was 5mg/ml. This shows that the antimicrobial
from spp. was more active but there are various factors affecting the activity.
The MIC is not a constant for a given agent, because it is affected by the nature of the test
organism used, the inoculum size, and the composition of the culture medium, the incubation
time, and aeration. For complete characterization of an antibiotic it should be isolated in pure
form as a single component but this is impractical in a screening programme like this. However, a
little effort was made in this approach. According to the TLC separation, the two extracts yielded
components with Rf values similar to the antibacterial compounds as visible on bioautogram. In
addition, the inhibition zones were associated with yellowish green spots, which had been
detected under UV radiation. This may mean that the same compounds are responsible for
antibacterial activity of those isolates.
Although the antimicrobial agents obtained in this study can’t be declared as new
antibiotics, there is the probability of finding new antibiotics in Karwar because of its wide
biodiversity. For proper identification of the antimicrobial extracts it is necessary to obtain in
pure form, which requires a series of purification process and different chemical analysis such as
HPLC, Spectroscopy and other sophisticated techniques. As we know the land of Karwar is
virgin in this field, so lots of works should be done to explore the new antibiotics because any
new antibiotics and its producing organism have been a great demand.
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