KADA NARAYANA MURTHY, R. MOHANRAJU, P. KARTHICK, CH.

RAMESH
ANDSUMANTHANARAYANA
Department of Ocean Studies and Marine Biology, Pondicherry University
Brookshabad Campus, Port Blair - 744 J J 2, Andaman & Nicobar Islands, India

Study was made to isolate Vibrios from green, brown and red algae and a total of22 isolates
were obtained from the selected seaweeds plated on TeSS medium. Among these, 17 were
sucrose positive (Yellow- Vibrio cholerae like organisms) and 5 were sucrose negative (Green-
V.parahaemolyticus like organisms). All the isolates showed positive results for oxidase,
string and catalase tests.

and aquaculture settings worldwide. Studies

Bacteria in the Genus 'Vibrio' are one
of the ubiquitously distributed organisms
belonging to the family Vibrionaceae that
inhabit in both brackish water and marine
environment. These microorganisms are in
association with various marine fauna and flora
acting both as symbionts and pathogens.
They are gram negative rod shaped bacteria
which show positive reaction to oxidase and
catalase properties with a fermentative ability.
They are motile bacteria with polar flagella,
mostly prefer sodium chloride as an important
growth requirement and show sensitivity to
the Vibrio static agent 0/129. Sawabe et al.
(2007) defmed Vibrios as groups of strains that
share >95% gene sequence similarity and
>99.4% Amino Acid Identity (AAI) based on
multi locus sequence analysis inferring the
evolutionary history of Vibrios. Vibrios are
copious in the aquatic environments including
estuaries, marine coastal waters and sediments
conducted by various researchers on both
culturable and non-culturable bacteria showed
that the Vibrios are associated with various
marine fauna and flora communities such as
planktons, fish, molluscs, corals, sponges,
crustaceans, seagrasses and seaweeds
(Thompson et aI., 2004).
Seaweeds are marine macro algae
which serve as a platform for a wide diversity
of bacterial associations like mutualistic,
commensal and parasitic, over obligate and
facultative both of endo and ectophytic
interactions (Reiman, 2008). These macro algae
are very important communities which are one
of the major sources of primary productivity
in the coastal environments. Besides primary
productivity, they are well known for their role
as dietary component in many countries used
as food and also has several applications in
the extraction of chemicals like agar, alginates
and carrageenan. Seaweeds are also being
investigated for their medicinal properties
showing antibiotic activity towards clinical
and fish pathogens.
Fischer in 1889 first reported bacterial
association on seaweeds and isolated a large
number marine bacteria from planktons and
seaweeds during planktonic expedition of
Humboldt Foundation across the Atlantic
Ocean. Hans Gazer in 1906 also found a large
number of bacteria from seaweeds during
German South polar expedition in the South
Atlantic and Antarctic Ocean (Hollants et al.,
2012). They had concluded that the large
number of bacteria is not only the impact of
high organic influx of dead seaweeds but also
because of the symbiotic association of
bacteria with living macro algae. The detailed
study on the description of epiphytic bacteria
was made by Harold and Stanier in 1955 and
observed the bacteria Leucothrix mucor
continuously as an algal epiphyte (Hollants
et al., 2012). After this, several researchers
had isolated bacteria from the seaweeds which
had given a clear picture with respect to the
seaweed bacterial associations.
Marine macro-algae have a (APW-pH 8.5, Hi media) and incubated at 37"C
specificity for the bacterial association which
differs from the seawater communities
(Longford et aI., 2007, Lachnit et al., 2009).
Burke et al. (2011) found highly variable
bacterial species composition among the local
individuals of Viva australis by 16S r RNA
screening which showed that each individual
hosts a different pattern of species
composition. The composition of seaweed
associated bacteria can change over seasons,
life span and different parts of the thallus due
to biotic and abiotic factors (Bengtsson et aI.,
2010). Studies on the diversity of epiphytic
Vibrios from the seaweeds are very few.
Studies by Gallardo et al. (2004) had shown
that Vibrios account for nearly 20% of the
total gram negative bacteria isolated from
the seaweed Monostroma undulatum.
Mahrnud et al. (2006, 2007 and 2008) isolated
Vibrio parahaemolyticus and V. vulnificus
from seaweeds in Japan. Except these few
studies there is not much research on the
isolation and characterization ofVibrios from
the seaweeds. Hence, a preliminary study was
made on the epiphytic Vibrios from various
seaweeds of Andamans. The present study
focuses on the initial screening of Vibrio like
isolates from the seaweeds.
Materials and Methods
In the present study, 8 species of
commonly occurring seaweeds belonging to
Chlorophyceae (4), Phaeophyceae (2) and
Rhodophyceae (2) were screened for vibrio
like organisms (Table-I). Seaweeds were
collected from the intertidal region during low
tide in sterile polythene bags and transported
to the laboratory. Small piece of each seaweed
sample was placed in sterile petridishes and
washed thrice with a spray of sterile filtered
seawater to remove the unattached bacteria.
Each piece was transferred to a labelled vial
containing 30 ml of Alkaline Peptone Water
for 6 - 8 h for enrichment of epiphytic Vibrios.
100 IIIof inoculum was pipetted out from each
vial and transferred to sterile petridish to which
18 - 20 ml of TCBS medium (Thiosulphate
Citrate Bile-salts Sucrose, Merck) was added
and incubated at 37°C for 24 h (Farmer and
Hickman, 1992). Sucrose positive colonies
(Yellow, Vibrio cholerae like organisms) and
Sucrose negative (Green, V.parahaemolyticus
like organisms) were picked up and streaked
on to fresh TCBS plates and incubated at
37°C for 24 h. Isolated colonies from each plate
were restreaked onto fresh marine agar (Hi
media) plates and incubated. These isolates
were used to screen Vibrios by Gram's stain,
oxidase (1% N, N, N, N-tetramethyl-
parapheny lene-d iam ine-d ihydrochloride,
Sigma - oxidase reagent), string (0.5%
 Table-I. List of the Vibrios isolatedfrom the negative which formed green co loured
seaweeds colonies, as they were sucrose non -fermenting
bacteria. On subjecting to Oxidase, String and
Catalase tests, it was confirmed that all these
Chlorophyceae isolates were Vibrios. Among the 17 sucrose
positive isolates, only 4 showed growth when
1. Caulerpa lentillifera HCLl, HCL2, HCLJ
streaked on 0.5% NA confirming them to be
2. Caulerpa racemosa CCSW3a V.cholerae (Farmer and Hickman, 1992). Further
3. Dictyosphaeria sp. KSWla, CCSW3a TSI test showed that all 5 sucrose negative
4. Viva lactuca HULl, HUL2, HULJ, isolates showed KIA reaction (Alkaline slant
HUL4,HUL5 over acidic butt) on TSI slant confirming them
Phaeophyceae to be V.parahaemolyticus (Table-2).
5. Sargassum
Seaweeds are potential sources of
turbinaroides
various complex compounds like xylopyranose
6. Turbinaria ornata and glucopyranose in green algae, mannitol
Rhodophyceae and laminarin in brown algae, D-galactose and
agar in red algae (Date et aI., 2012) besides
7. Graci/aria corticata HGCl, HGC2, HGC3
HGC4, HGC5, HGC6 alginates and carrageenan. Michel et al (2006)
reviewed the bio-potential of various marine
8. Acanthophora spicifera PAS 1, PAS3
epiphytic bacteria isolated from red algae
towards the secretion of enzymes like agarases
deoxycholate, Sigma - String reagent) and and carragenases. Penesyan et al. (2009) and
catalase tests (3% HP) (Farmer and Hickman, Sujith et al. (2012) observed the antagonistic
1992). Sucrose positive (yellow) isolates were potential of bacteria isolated from seaweeds
streaked on 0.5% Nutrient agar (NA, Hi media) towards selected human pathogens.
for preliminary confirmation of Vibrio Penesyan et al. (2009) highlighted the concept
cholerae and Sucrose negative (green) that the surfaces of marine eukaryotes like
isolates were stabbed into Triple Sugar Iron seaweeds provide a unique habitat for the
(TSI, BD) slants for the preliminary bacteria to colonize and this interaction which
confirmation of Vibrio parahaemolyticus. All is mediated by the chemical signalling between
the isolates were stabbed in 1% nutrient agar host and the bacteria supports the microbial
with 0.8% agar, sealed and stored at room diversity and their function. Studies show that
temperature. Working cultures were made in epiphytic bacteria have a good potential of
slants and stored at 4 °C for further tests. enzymes that can be used industrially for bio-
processing. Many new drugs can be
Results and Discussion developed from the seaweed associated
Twenty two Vibrio like isolates were bacteria due to the antagonistic antibacterial
found from various seaweeds (Table-I). activity towards human pathogens.
Among these, 17 were sucrose positive which Studies by Gallardo et al (2004)
formed yellow co loured colonies on TCBS showed .that seaweed acts as a platform for
plate due to the production of acid which were various bacterial genera like Vibrio,
identified with the pH indicator Bromothymol Escherichia, Pseudomonas, Aeromonas and
blue and Thymol blue and 5 were sucrose Staphylococcus and observed that Vibrio
 HCLl
HCL2
HCL3
CCSW3a
KSWla
CCSW3a
HULl KIA
HUL2 KIA
HUL3 KIA
HUL4 KIA
HUL5
B4112a
D4112a
D6112a
HGCI
HGC2
HGC3
HGC4
HGC5
HGC6
PASI
PAS3

TCBS - Thiosulphate Citrate Bile salts Sucrose agar; MA - Marine Agar; GS - Gram's Staining;
OT - Oxidase Test; ST - String Test; CT - Catalase Test; 0.5% NA - Nutrient agar with 0.5% NaCI;
TSI - Triple Sugar Iron; Y -Yellow; G - Green; KIA -Alkaline slant over a~idic butt

accounts for 20% of the total gram negative
bacteria isolated from the seaweed
Monostroma undulatum. Mahmud et al.
(2006, 2007 and 2008) isolated Vibrio
parahaemolyticus and Vibrio vulnificus from
seaweeds in Japan and found that the Vibrios
were the dominant group. The present study
was focused on isolating Vibrio from different
seaweeds of Andaman and 22 Vibrio like
organisms had been isolated based on the
growth on TCBS, 0.5% NA and TSI along with
preliminary tests like oxidase, string and
catalase tests to confirm them till genus level.
A good number of Vibrio like isolates was
observed from the seaweeds after enrichment
in alkaline peptone water. But the diversity
based on the morphological features seems
to be less which shows that seaweed
selectively colonizes only few species of these
bacteria.
 Increase in the dumping of human
wastes into the coastal waters paves a way
for the increase in the concentration of human
pathogens upon the marine macro organisms
like seaweeds by replacing the useful bacteria
on the surfaces leading to diseases caused
on consumption or by degrading the quality
of seaweed by-products. Further studies are
necessary on the identification of isolates till
species level and identifying the presence of
virulence genes among the isolates and
strains. The impact ofthese identified bacteria
on the quality ofthe seaweed derived products
will be a potential outcome of the present
preliminary work.
Acknowledgements
We thank the Head of the Department
and University Authorities for providing the
facilities to carry out this work.
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