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Antimicrobial activity of Pseudomonas spp. isolated from the marine environment of Karwar coast, West coast of India By Pradeep V. Khajure and J.L.Rathod

Antimicrobial activity of Pseudomonas spp. isolated from the marine environment of Karwar coast, West coast of India By Pradeep V. Khajure and J.L.Rathod

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11/16/2011

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ECOLOGY AND FISHERIES
 
ISSN 0974-6323Vol.
3(1).pp.9-18 2010
ANTIMICROBIAL ACTIVITY OF
 PSEUDOMONAS
SPECIESISOLATED FROM MARINEENVIRONMENT 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 28isolates were subjected to primary screening by perpendicular streak method againstGram-positive (
 Bacillus subtilis
and
Staphylococcus aureus
), Gram-negative(
 E.coli, Klebsiella
species,
Proteus
species and
Salmonella typhi
) test bacteria. Itwas observed that 4 isolates were active against only Gram-negative bacteria, 3against Gram-positive and 8 against both Gram-negative and Gram-positive bacteria.Altogether 15 putative isolates were subjected to secondary screening byAgar well diffusion method to further test the capabilities of primarily screenedorganisms. Selected isolates (10) from the secondary screening belong to the generaPseudomonas (03), Proteus (02), Aeromonas (03) Vibrio (02). Finally species wereselected 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 wereextracted with Ethyl acetate from isolate-inoculated production medium fermentedfor 48 hours at 30
o
C by solvent extraction method. The isolate grew in the presenceof 20 % (w/v) NaCl, antibiotic production being maximum with 5% (w/v) Nacl inthe production medium. Natural seawater stimulated the antibiotic biosynthesis.The absence of catabolic repression during the synthesis of the antimicrobialsubstance was demonstrated by the utilization of glucose by this isolates. Thehighly stable, active principle was purified by Ethyl acetate extraction and thinlayer chromatography (TLC) and each single compound from two species wasfound to posses the broad-spectrum activity. Considering all of the above evidencesand based on comparison with the current literature, the bioactive compoundisolated from Pseudomonas spp. was responsible for Antimicrobial activity.
Key Words
:
Pseudomonas
 
species, Antimicrobial activity, Fermentation.
 
 
Ecology and Fisheries/10______________________________________________________________________________
INTRODUCTION
The Ocean, which is called the ‘Mother of origin of life’, is also the source of structurally uniquenatural products that are mainly accumulated in living organisms. Several of these compoundshow pharmacological activities and are helpful for the invention and discovery of bioactivecompound, primarily for deadly diseases like cancer, acquire immuno-deficiency syndrome, etc.The lives saving drugs are mainly found abundantly in microorganisms, algae and invertebratesand 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 fromthe 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 clinicaltrails, it has proven notoriously difficult to obtain adequate, reliable supplies of these compoundfrom nature. Because of these problems, anew avenue of study focusing on marinemicroorganisms has been gaining considerable attention. At first sight thus, the expectableenormous biodiversity of marine microorganisms might have been the reason for the interest intheir study. Although marine microorganisms are not well defined taxonomically, preliminarystudies 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 invertebrateshas 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. Marineepiphytic bacteria, associated with nutrient rich algal surfaces and invertebrates, have also beenshown to produce antibacterial secondary metabolites, which inhibit the settlement of potentialcompetitors.
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. 14
o
45.46'N. Long. 074
o
02.84'E). Thesamples were brought to the laboratory in aseptic condition. Than the microorganisms werecultivated 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 testorganisms
 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 metabolizedsugar was tested by replacing starch and glucose by an equivalent amount of glucose, maltose andlactose. 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 asshown in fig (1). The effect of composition of the water used for cultivation on growth and
 
 
Ecology and Fisheries/11______________________________________________________________________________
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 theoptimization of the growth
 A.niger 
and
S.aureus
were used as test organisms. The isolates werecultivated on a shaker (200rpm) at 30
o
C.The determinations were done at least thrice and theaverages of the values are reported. For the determination of wet biomass, 1ml of sample wascentrifuged and the weight of the pellet was determined.
Characterization of Isolates:
The potent isolates selected from the secondary screening werecharacterized by morphological and biochemical methods. The results of microscopicexamination were compared with Bergey's manual of Determinative Bacteriology, Ninth edition(2000) and the organism was identified. Various biochemical tests were performed for theidentification of the potent isolates are as follows; Fermentation of sugars, Hydrolysis of starch,Indole production, Methyl red, Vogues Prauskauer, Citrate utilization, Urease test, 2%Peptonewater, 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.al
1986. 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 separatedfrom the aqueous phase. It was evaporated to dryness in water bath at 80°-90°C and the residueobtained were weighed. Thus obtained compound was used to determine antimicrobial activity,minimum inhibitory concentration and to perform bioautography.
Fig(1): Effect of NaCl on Growth and Antimicrobial activity ofPseudomonas spp.
00.511.522.533.544.55
  0  %    N  a  C   l   4  8   h  0  %    N  a  c   l   9  6   h   5  %    N  a  C   l    7  2   h  1  0  %    N  a  C   l   4  8   h  1  0  %    N  a  c   l   9  6   h  1   5  %    N  a  C   l    7  2   h  2  0  %    N  a  C   l   4  8   h  2  0  %    N  a  c   l   9  6   h
   G  r  o  w   t   h   Z  o  n  e  o   f   I  n   h   i   b   i   t   i  o  n
Activity against A.nigerActivity against S.aureusGrowth

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