ABSTRACT

The present investigation was undertaken in order to isolate bacteria from

eighteen different water samples collected from three different sectors of
‘Chilika’ lagoon of India and to study the resistance against ten different
antibiotics viz., norfloxacin, tetracycline, ciprofloxacin, neomycin, nalidixic
acid, ofloxacin, chloramphenicol, nitrofurantoin, streptomycin and
amoxicillin as well as their serological implications. Four different pathogenic
bacteria species viz., Shigella dysenteriae, Streptococcus lactis, Bacillus cereus
and Klebsiella pneumoniae were isolated which showed a wide range of
sensitivity to norfloxacin, tetracycline, ciprofloxacin, ofloxacin and
nitrofurantoin. S. dysenteriae was sensitive to streptomycin where as other
isolates were found to be resistant. Agarose gel electrophoresis failed to
reveal plasmid DNA band indicating that the observed resistance was perhaps
encoded by nucleotide sequences harboured on the chromosomal DNA.
Bacterial isolates were used as antigen for the production of polyclonal
antibodies in rabbits. All the isolates exhibited strong antigenic character with
specific serological relationship which can be implicated towards
development of novel and pharmaceutically effective anti- bacterial products.

Keywords: ‘Chilika’, bacterial isolates, antibiotic resistance, serological test

INTRODUCTION
‘Chilika’ is the largest lagoon in the subcontinent which is situated along the east
coast of India between latitude 19o 28’ and 19o 54’ N, and longitude 85o 05’
and 85o 38’E. This is a shallow, brackish-water lake formed due to the silting
action of the Mahanadi River, which drains into the northern end of the lake,
and the northerly currents in the Bay of Bengal, which have formed a sandbar
along the eastern shore (Arya and Lakhotia, 2006). It is the largest brackish water
wetland complex in Asia, declared as a Ramsar site under the convention on
“Wetlands of International Importance”. This lagoon is presently under threat
from both natural and anthropogenic pressures (Nayak et al., 2004). The marine
realm constitutes the major habitat of the biosphere covering 73 % of the earth
surface which provides the largest inhabitable space for living organisms,
particularly microbes. Marine microbes flourish not only in the surface water of
the sea, but also in the lower and abyssal depths from coastal to the offshore
regions and from the general oceanic to the specialized niches like blue waters
of coral reefs to black smokers of hot thermal vents at the sea floor. As the water
being served as major solvent, it plays an important role in the pharmaceutical
industries. It is considered as the primary source of contamination for
pharmaceutical products (Tamara et al., 1998). Microbes present in the marine
waters represent a potential source for commercially important bioactive
compounds and their bioremediation capabilities are also remarkable. There is
an increasing demand of biodiversity from natural resources for development
of therapeutic drugs. Antibiotics are broadly used as chemotherapeutic agents
which, albeit in a very low quantity, can inhibit the pathogenic activity of
microorganisms. The potential contribution of marine organisms to the
discovery of new bioactive molecules is increasingly challenging (Sponga et al.,
1999; Skulberg, 2000). The microorganisms have become a significant attraction
as natural source of bioactive molecules with a broad range of biological
activities, such as antibiotics, antivirals, antitumorals, antioxidant and anti-
inflammatory (Okami, 1982; Kamei et al., 1987; Nunez et al., 2006; Uzair et al.,
2009; Shankar et al., 2010). Evidence of phycochemical and pharmacological
studies on microbes is available in the literature (Zeeshan et al., 2010; Odeyemi
et al., 2010; Bragadeeswaran et al., 2010. Previously, there were a number of
researchers that had worked on ‘Chilika’ regarding hydrological
characterization, water quality variation, antimicrobial activity and
physiochemical variation (Rao et al., 1981; Nayak et al., 2004; Patra et al., 2009,
Patra et al., 2010). Unfortunately progress made on pharmaceutical studies of
marine microorganism from ‘Chilika’ is inadequate. This underpinned the
present study with a view to investigating the physiological, biochemical, and
serological characterization of bacteria isolated from marine waters of ‘Chilika’
aiming at their exploitation for pharmaceutical benefits.

A Brackish water ecosystem is a type of aquatic ecosystem that is made up of

shallow, partially enclosed areas. When freshwater joins seawater, brackish
water is found. Brackish water ecosystems have a salt level of 5 to 35 parts per
million. Estuaries, salt marshes, mangrove swamps, and forests are just a few
examples. The River Thames, which runs through London, is an example of a
traditional river estuary.

Brackish Water Ecosystems - Features

The term brackish water refers to waters that are mildly salty, as opposed to
salty seawater on the one hand and non-salty freshwater found in freshwater
ecosystems on the other hand. Salinity varies widely in brackish water
ecosystems because it is formed by mixing seawater carried in by tides with
fresh water from rivers or rainwater. They are distinguished by variable salinity
gradients, tides, temperature variations, siltation, and nutrient availability.
Aquatic brackish water organisms have evolved survival strategies to cope with
the changing physical circumstances in brackish water environments.

Brackish Water Ecosystem- Flora

Mangroves, marsh grasses, and colonial green algae are all common estuary
flora. Few animals actively consume them, although a vast fraction is digested
as debris. Bacteria are thought to be one of the regulating factors for plant and
animal distribution, unlike temperature and nutrient concentrations.
Phytoplankton, mudflat algae, and epiphytic plant occurring on marginal marsh
vegetation can be found in the brackish water ecosystems. The tall grasses that
line the coastline are one of the most noticeable features of these habitats.
Smooth cordgrass is one of these grasses.

Brackish Water Ecosystem - Animal Adaptations

Organisms in brackish water face two significant challenges: adapting to salinity
variations and maintaining their position. In addition to a wide range of salinity
tolerance, animals have evolved physiological adaptations and systems to aid
internal consistency in the face of variable external salt concentrations. For
example, although the edible blue mussel and lugworm lack an osmoregulatory
mechanism, they can adjust to a certain medium dilution since their tissues can
function at low salt concentrations.

Brackish Water Ecosystem - Fauna

Detritus feeders including oysters, clams, lobsters, and crabs are the most well-
known estuarine animals. Various insect larvae, annelid worms, and molluscs
reach the estuary from freshwater, and most nearshore marine zooplankton, as
well as several types of bigger animals, can be found partway into the estuary.
Most importantly, brackish water ecosystems provide a breeding place for a
diverse range of marine species, including shrimp, crabs, and fish.

Fig. 1. Shigella dysenteria

Fig. 2. pneumoniae Klebsiella

Fig. 3. Bacillus cereus

Fig. 4. Streptococcus lactis

Most of the people live indoor, houses, schools, colleges, hospitals and hospitals
etc., where they are exposed to many environmental conditions that effect their
health. Microorganisms are normally present in both indoor and outdoor
environments. The quantity of microorganisms in a particular area depends
upon the presence of water and other nutrients sources in that particular
environment where they can develop extensively. Usually microorganisms enter
into building through doors, windows, air conditioners and also by people
entering from outside. The type of species and amount of organism present
depends on the viscocity, temperature, lighting and food available in that
particular environment. Among the microorganisms present in the indoor
environment, some species of microorganisms if present beyond the limit can
cause serious health problems. So, the isolation, identification and
measurement of different types of microorganism especially in indoor
environment has become a very hot ho topic at present and it has attracted
everyone’s attention in this field. Among the indoor microorganisms; some may
be pathogenic and could secrete toxic metabolites which can cause allergy and
even serious diseases. Environmental pathogens are defined as microorganisms
that normally spend a substantial part of their lifecycle human hosts, but when
introduced to humans cause diseases with measurable frequency. They are
borne in the water, soil, air, food and other elements of our surroundings, and
they affect almost every individual on the planet. The adverse effect on human
health and productivity cannot be controlled without first obtaining through
understanding of their environmental niche, their incidence, and the
epidemiology of the disease they cause. To achieve this understandings,
surveillance of the environment to determine the numbers and distribution of
environmental pathogens and other human pathogens is their ability to survive
and thrive outside the host. Their outside occurrence in the environment makes
them difficult to monitor and control so, proper care and sanitation should be
maintained in the college premises. The environment significantly influences
multiple factors in the chain of infection. Although microbiologically
contaminated surfaces can serve as reservoirs for pathogens, these surfaces
generally are directly connected with transmission of infections the transmission
of microorganism from environmental surface to students is largely via hand
contact with the surface. Although hand hygiene is important to minimize the
impact of this transfer, cleaning and disinfecting environmental surfaces
appropriately is fundamental in reducing their potential contribution to the
incidence of healthcare associated infection.
REVIEW OF LITERATURE

EMILY Mattock Ariel J Blocker reported that Shigella is the major cause of
bacillary dysentery world-wide. It is divided into four species, named S. flexneri,
S. sonnei, S. dysenteriae, and S. boydii, which are distinct genomically and in
their ability to cause disease. Shigellosis, the clinical presentation of Shigella
infection, is characterized by watery diarrhea, abdominal cramps, and fever.
Shigella's ability to cause disease has been attributed to virulence factors, which
are encoded on chromosomal pathogenicity islands and the virulence plasmid.

Gregory P Keefe reported that Streptococcus agalatiae continues to be major

cause of subclinical mastitis in dairy cattle and a source of economic loss for the
industry. Veterans are often asked to provide information on herd level control
and eradication of S. agalactiaemastitis . the review collects and collates
relevant publication on the subject. The literature search was concluded in 1993
on the Agricola database. Articles related to S. agalactiae epidermology,
pathogen identification techniques, milk quality consequences, and control,
prevention, and therapy were included.

Racha Majed, Christine faille reported that bacillus cereus displays a high
diversity of lifestyles and ecological niches and include beneficial as well as
pathogenic strain. These strains were widespread in the environment , are found
on insert as well as on living surfaces and contaminate persistently the
production lines of the food industry.

LS tzouvelekis, A Marogiannakis reported that a review of the literature

reviewed high failure rates in the case s of monotherapy with these drugs, while
monotherapy with either a crabapenem or an amino glycoside appeared to be
more effective.
Elisabeth A.M. Westerbeek, Anemonevanden Berg reported that this study is
to review the normal development of the intestinal microflora of pretem infants
and and the factor influencing the development. Pretem infants have an
increased intestinal permeability, which may lead to bacterial translocation to
systemic organ and tissues. In combination with immaturity of the immune
system the risk to systemic infections might be increased. Especially potential
pathogenic bacteria are able to translocate. the intestinal microflora of the
breast fed infants, dominated by the bifidobacterial and lactobacillim, is thought
to suppress the growth of potentially pathogenic bacteria. Many attempts have
been made to stimulate the presence of bifiiodobacteria and lactobacilli with
changes in the diet and ingredients like prebiotics and probiotics.

Jose G . Dorea Carmen, m. Donangelo reported that mercury and lead are toxic
metals are widespread in the environment with bioaccumulative features that
raises public health concerns . both metals are equally dispersed in the human
food chain but but exposure and risk of toxicity during early human
development are modulated by the diet and nutritional status. Understanding
how Hg and Pb occur and interact with the nutrients is fundamental to establish
guidelines for diminishing exposure and the risk of toxicity. the risk of fetal
infant exposure to Hg can be influenced risk of fetal infant exposure to Hg can
be influenced risk of fetal infant exposure to Hg can be influenced by maternal
amalgam filing and fish consuming, whereas the risk to exposure to Pb is
complex: maternal absorption depends on nutrient interactions ( Ca and P) and
maternal body Pb accumulation responds to all factors known to interact to Hg
to Pb is more important during fetal development than breast feeding
Moreover, these metals (especially Pb) are frequently higher in infants formulas
which do not carry the nutritional and physiological advantages and protection
of breastfeeding.
OBJECTIVE

1. The pathogenic bacteria from brackish water of chilika project in an

application of the principles and particles of classic microbiological
investigation .
2. These four species of pathogenic bacteria produce enzymes like
chitinases, lipases, proteases, amylases, cellulases and pectinases which
are very important for biotechnology sector.
3. These four species of pathogenic bacteria produce enzyme which are
biodegradable and could prove useful in the biotechnology sector the
species are have been named after chilika lagoon and a locality near the
lagoon .
MATERIALS AND METHOD

Isolation and characterization of bacterial strains

The total study area is comprised of 18 sampling stations covering three

different sectors of the ‘Chilika’ lagoon i.e. Central, Southern and Outer
Channelduring the period from January to March 2008. The exact sampling
locations were fixed by using Global Positioning System (GPS). All the water
samples were collected in the morning hours between 7:00 A.M-11:00 A.M.
During sample collection necessary precautions had been taken to collect
undisturbed water samples in the lake. Samples were collected in pre-sterilized
polypropylene bottles (500 mL). Physical parameters like temperature and pH
of the samples were determined on the spot of collection. Then the water
samples were kept in the icebox and transferred to laboratory for further
analysis and the samples were preserved at 4°C before isolation and
identification in the laboratory. The experiments were carried out within 4
months of collection. Chemicals used for preparation of reagents in the present
investigation were of analytical reagent grade and for preparation of solutions
double-distilled water was used. One milliliter of water sample was subjected
through serial dilution to obtain 10-8 dilution. After that 1mL of 10-8 diluted
sample was mixed with 10 mL of sterilized Nutrient broth medium and then it
was incubated at 37 °C in a BOD incubator for 24 h. After incubation a loop-full
of microbial culture were picked up from different colonies and streaked
separately on the different agar-gelled sterilized media and the plates were
incubated at 37 °C for 24 h for isolation of pure culture. The colony
characteristics such as colour, appearance, and shape of the isolate were
recorded. Further, morphological, and biochemical tests were carried out in the
laboratory by following standard microbiological methods described by
Cappuccino and Sherman (2002) for identification and characterization of
isolated microorganism. The isolated strains were also characterized through
different physiological factors such as pH, temperature and salinity to observe
their tolerance capacity in the stressed environmental conditions.

Collection and preservation of pre-immunized serum

For production of antisera 3-4 month-old white rabbit (2.5 -3 kg) were
selected and kept in separate cage and labeled properly. The pre-
immunized sera of the rabbit used for the test were collected before
7 days of immunization with different a ntigens to confirm that the
rabbit did not contain any antisera. The sterilized tube containing fresh
drawn blood was kept at room temperature for 1-2 h for clot
formation. The clots was stirred with glass rod and was kept overnight
in refrigerator to allow the clot contraction. The plasma were
centrifuged (3000 rpm, 15 min). The straw-coloured serum was
withdrawn into a sterilized centrifuge tube which was labeled
properly. For preservation of serum, it was added with 0.02 % sodium
azide (NaNH2) to give a final concentration of 1:5000 and kept in a
deep freeze at -20°C for further use.
 Fig. 5. Location map of ‘Chilika’ lagoon in the State of Odisha of India

Fig. 6. Revealing different ecological sectors as sampling sites of Chilika

Serological test
Preparation of whole cell antigen:

The antigen was prepared from the freshly grown bacterial cultures. The
bacteria were cultured on NSA medium in test tube at 27°C for 24 h. The test
tube was filled with PBS or saline water. The culture was scraped with
sterilized inoculation needle. The scraped bacteria with saline water were
transferred to the centrifuge tube. The bacterial growth was mixed
thoroughly with the help of a vortex mixture to make it completely
homogenized. The whole cell of bacteria was centrifuged (3000 rpm, 30 min,
room temperature). The supernatant was discarded and the pellet was re-
suspended with saline water and centrifuged. The process was repeated
three times to make the bacteria free from any foreign material. After the last
centrifugation bacteria were re-suspended in normal saline/butler saline,
collected in sterilized specimen tube, labeled properly, preserved and stored
in deep freeze at - 20 °C for further use.

Preparation of antigen for agglutination test:

The antigens were prepared by suspending live bacteria in buffer saline as
described earlier except that the antigen repared was more concentrated having
an optical density of 0.5 using a Gallen Kamp colorimeter at 520 mm.

Preparation of antigen for agglutination test:

The antigens were prepared by suspending live bacteria in buffer saline as
described earlier except that the antigen prepared was more concentrated
having an optical density of 0.5 using a Gallen Kamp colorimeter at 520 mm.
Antibiotic sensitivity test
Antibiotic sensitivity test of the identified bacteria were repeated 3 times for
each strain using 10 different antibiotics namely norfloxacin, tetracycline,
ciprofloxacin, neomycin, nalidixic acid, ofloxacin, chloramphenicol,
nitrofurantoin, streptomycin and amoxicillin (Hi- Media, Mumbai, India) by disc
diffusion method (Bauer et al.,1966). Antibiotic activity was measured in terms
of zone inhibition (mm diameter).

Plasmid DNA isolation and agarose gel electrophoresis

The alkaline-lysismini-prep method (Wizard Plus Minipreps DNA
Purification Systems, Promega, USA) was used for plasmid extraction from all
the four bacterial isolates. Overnight-grown bacterial cultures (3-5 mL) were
pelleted by centrifugation (10,000 x g, 10 min), supernatant decanted and pellet
re-suspended in 300 µL of Cell Re-suspension Solution (50 mM Tris, pH 7.5; 10
mM EDTA; 100 µg/mL RNase A). The re-suspended cells were transferred to 1.5
mL micro-centrifuged tube to which 300 µL of Cell Lysis Solution (0.2 M NaOH,
1 % SDS) was added and samples thoroughly mixed. To the cleared lysate 300
µL of Neutralization Solution (1.32 M potassium acetate, pH 4.8) was added
and mixed properly. The lysate was centrifuged (10,000 x g, 5 min) and plasmid
purification was carried out using the vacuum manifold (Promega Vac-Man,
USA). The purified samples were electrophoresed using 0.7 – 1.0 % agarose
gel followed by ethidium bromide (0.5 µg/mL) staining (Sambrook et al., 1989).
Electrophoresis was carried out in 1 x Tris borate-EDTA (TBE) buffer at 120 V
for 3 h or until the loading dye (Genie-Merck, Bangalore, India) neared the
bottom of the gel. Gels were visualized and photographed under GEL DOC XR
SYSTEM (Bio-Rad, USA).
RESULT AND DISCUSSION

To identify the isolates obtained from 3 different sectors of the ‘Chilika’ lagoon
each of them were tested for various morphological, biochemicals,
physiological and serological characters. The results of morphological and
biochemical tests were presented in Table 1.The isolates are closely related to
Shigella dysenteriae, Streptococcus lactis, Bacillus cereus, Klebsiella
pneumoniae (Table 1 and Table 2). In nutrient agar plates Shigella dysenteriae
appeared as thin, even and greyish colonies measuring ca. 3-4 mm diameter.
Streptococcus lactis colonies were thin, even-growths on nutrient agar plates
ranging ca. 2-3 mm diameter. In comparison, Bacillus cereus colonies which
were fermented on nutrient agar plates had abundant, opaque, white waxy
growths measuring ca. 3-4 mm diameter. Klebsiella pneumoniae colonies were
skimy-white, raised growths with ca. 2-4 mm diameter. Each bacterial colony
cultured on nutrient agar medium was then subjected to Gram staining and
thereafter observed under light microscope. The shape and staining property of
the bacteria were recorded. Shigella dysenteriae, Klebsiella pneumoniae and
Bacillus cereus were found to be rod-shaped bacteria whereas
Streptococcus lactis had a spherical (coccus) shape. Streptococcus lactis and
Bacillus cereus were found to be Gram positive bacteria whereas Shigella
dysenteriae and Klebsiella pneumoniae was Gram negative.
In addition to morphological and biochemical characterizations
the isolated strains were also characterized through different
physiological factors such as pH, temperature and salinity to observe their
tolerance capacity in the environmental stress conditions. The identified
bacterial species were tested for their halotolerance
nature on the basis of pH, temperature and salinity. The optimal physiological
conditions of isolated strains are presented in Table 2. It was found that all
isolated strains survived in conditions at a varying pH 8 - 9, temperature regime
40 °C – 45 °C, and salinity ranging 8 – 9 %, thus indicating that these
isolates were halotolerant.

Antibiotic sensitivity tests were performed for each of the four identified
bacterial species. Those cells which were completely inhibited and failed to
resist the action of antibiotics against them became sensitive to the
corresponding antibiotics. But partial inhibition happened when somehow some
mutant cells emerged and they could successively resist the action of the
antibiotics against them (Johnson et al., 1995). In this study Shigella dysenteriae
was the only one that showed sensitivity to streptomycin but the three
remaining strains showed resistance to streptomycin (Table 3). Multi-drug
resistance by Shigella sp. was not uncommon (Lien et al., 1994). Although all the
isolates showed resistance to neomycin and chloramphenicol; they showed a
wide range of sensitivity to norfloxacin, tetracycline, ciprofloxacin, ofloxacin and
nitrofurantoin; albeit intermediate sensitivity to nalidixic acid and amoxicillin.
It was interesting to note that in none of the extracted samples from resistant
strains plasmid DNA was detectable in the agarose gel following electrophoresis
regardless of agarose concentrations in the gel (data not shown). Perhaps, the
observed antibiotic tolerance in such strains isolated from the brackish waters
of the ‘Chilika’ lagoon is due to phosphorylation/acetylation activity of enzymes
encoded by gene loci borne on the chromosomal DNA instead of on plasmids. It
was not uncommon to enteric bacteria among which the multi-drug resistance
regulatory chromosomal locus, mar is widespread as was reported by Cohen et
al., (1993). Constitutive expression of mar-A leading to differential expression of
over 60 chromosomal genes in E. coli has been reported by different
investigators. According to Barbosa and Levy (2000), such kind of expression
possibly conferred multiple antibiotic resistance on E. coli. Another gene namely
emr harboured on E. coli chromosome has been identified that codes for protein
products of membrane translocase family and accounts for multi-drug
resistance.
RESULT AND CONCLUSION

Such strains isolated from the brackish waters of the ‘Chilika’ lagoon is due to
phosphorylation/acetylation activity of enzymes encoded by gene loci borne on
the chromosomal DNA instead of on plasmids. It was not uncommon to enteric
bacteria among which the multi-drug resistance regulatory chromosomal locus,
mar is widespread as was reported by Cohen et al., (1993). Constitutive
expression of mar-A leading to differential expression of over 60 chromosomal
genes in E. coli has been reported by different investigators. According to
Barbosa and Levy (2000), such kind of expression possibly conferred multiple
antibiotic resistance on E. coli. Another gene namely emr harboured on E. coli
chromosome has been identified that codes for protein products of membrane
translocase family and accounts for multi-drug resistance exploiting these
strains aiming at development of novel and specific antibacterial compounds,
antibiotics and enzymes which would offer considerable pharmaceutical value .
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