Indian Journal of Geo-Marine Science

Vol. 45(2), February 2016, pp. 304-309

Phenotypic and Molecular characterization of Epiphytic Vibrios from

the marine macro algae of Andaman Islands, India.
Kada Narayana Murthy*, R.Mohanraju, P.Karthick & Chatragadda Ramesh

Department of Ocean Studies and Marine Biology, Pondicherry University, Brookshabad Campus,
Port Blair- 74412, Andaman Islands

*[E.Mail: murthymarino@gmail.com]

Received 25 July 2014; revised 16 September 2014

Biotic and abiotic components in the marine environment supports the growth of several surface associated microbial
communities. Present study focuses on one such microbial community, the Vibrios associated to marine macro algae of
Andaman Islands. A total of 48 strains comprising 10 species of Vibrios were isolated from 10 macro algal species. All the
isolates were confirmed to genus level by both phenotypic and molecular characteristics, up to species level by biochemical
tests. This study showed that Vibrio alginolyticus strains were frequently isolated from almost all the macro algae revealing its
role as a common epiphyte. The present study signifies the abundance of Vibrios on the surface of marine macro algae which
may be attributed to their ecological role in the biogeochemical cycling of nutrients and/ or controlling the surface colonization
of pathogens.

[Key words: Andaman, Epiphytic, Macro algae, Vibrio]

Introduction Vibrios are gram negative rod shaped

Marine environment is a complex
structure where multiple factors contribute their
part in building and strengthening it. Any
substratum whether living or non-living immersed
in it develops a micro environment called biofilm
supporting macro fouling on inanimate structures
or in case of marine organisms, a thin film of
epibiotic bacteria protecting them from macro
fouling1. Marine macro algae provides a
conducive environment for a wide diversity of
bacterial associations like mutualistic, commensal
and parasitic, over obligate and facultative both
endo and ectophytic interactions2. They are
among the major contributors of primary
productivity in the coastal environment and well
known for their role as dietary component in
many countries along with production of
hydrocolloids like agar, agarose, alginates and
carrageenan3.
Marine macro-algae have specificity for
the bacterial associations which differs from the
seawater communities 4,5. The bacterial
composition on macro algae could have seen
changes over seasons, life span and its presence
on various parts of the thallus due to biotic and
abiotic factors6. In some cases the pattern of
bacterial composition varies between two
individuals of same species of macro algae7.
bacteria that are ubiquitously distributed in the
aquatic settings worldwide associated with
various marine faunal and floral communities,
mostly reported to be opportunistic pathogens
causing diseases in humans and marine
organisms8. Besides being pathogens they have a
prominent role in nutrient cycling in aquatic
environments by degrading complex compounds
such as chitin, agar, alginates, polycyclic aromatic
hydrocarbons etc., into smaller, usable entities
and also in providing essential polyunsaturated
fatty acids to the aquatic food web8. Role of
Vibrios in producing antibacterial properties
exhibited to compete with other colonizing groups
in the natural environment were reported9,10.
Vibrios accounts for 20% of the total
gram negative bacteria isolated from the macro
algae11. Earlier studies by several researchers
have reported Vibrios from various macro algae
around the world12,13,14,15,16,17. Even though the
abundance of Vibrios is very prominent on the
macro algal surfaces, their role varies from
symbiotic to parasitic relationship. They are
found to induce morphogenesis, allow zoospore
settlement in macro algae and produce
antimicrobial compounds protecting the macro
algal surface from colonization of harmful
bacteria 15. Vibrios are also reported to cause
diseases in various macro algae and found to
produce various polysaccharide degrading
 MURTHY et al: EPIPHYTIC VIBRIOS FROM THE MARINE MACRO ALGAE OF ANDAMAN ISLANDS
enzymes like agarases, alginases, fucoidanases,
cellulases and pectinases15. This signifies their
role in the biogeochemical cycling of nutrients in
marine ecosystem. The present study focuses on
the phenotypic and molecular characterization of
epiphytic Vibrios from the macro algae of
Andaman Islands.
Materials and Methods
In the present study 10 species of macro
algae comprising 4 green algae, 2 brown algae
and 4 red algae were screened for Vibrio like
organisms. Macro algal samples are collected
from the intertidal regions of Port Blair, South
Andaman and Little Andaman during low tide as
the macro algal distribution is prominent in South
and Little Andaman when compared to the North
Andaman islands18, 19. Samples were immediately
transferred to a sterile polythene cover and
transported to the laboratory. Small portion of
these samples were taken in sterile petridish,
washed thrice with a spray of sterile filtered
seawater to remove the unattached bacteria and
other epiphytes. A surface swab from a small area
of each sample was taken by using sterile cotton
swab and transferred to a labeled vial containing
20 ml of Alkaline Peptone Water (APW-pH 8.5,
Hi media) and incubated for 6 h at 37°C for
enrichment of Vibrios. After the incubation 100
µl of inoculum were plated onto a TCBS agar
plate (Thiosulphate Citrate Bile-salts Sucrose,
Merck) and incubated at 37°C for 24 h 20. Sucrose
positive (Yellow) and Sucrose negative (Green)
colonies were picked and streaked on to a fresh
TCBS plate and incubated at 37°C for 24 h. Each
isolated colony was further purified on Marine
agar plates (MA, Himedia, India) incubated and
pure strains were obtained. All these isolates were
then stabbed in Nutrient agar with 1% NaCl and
0.8% agar, sealed and stored at room temperature.
Working cultures were prepared in slants and
stored at 4° C for further tests.
Phenotypic characterizations of the
isolates were determined according to Bergey’s
Manual based on the colony observation on
TCBS and MA. Gram staining was conducted for
all the isolates following standard protocol. To
identify the isolates to genus level oxidase and
string tests were performed18. Biochemical
characterization was carried out following the
identification key 21 and the results are interpreted
by using the software Identax Bacterial Identifier
Version 1.2 22.
305
The genomic DNA was isolated by
following standard protocol23. 1.5 ml of overnight
broth culture of the isolates were centrifuged at
8000 rpm for 5 min and the cell pellet obtained
was washed twice with Phosphate buffered saline
(PBS), centrifuged again to obtain pellets, which
were dissolved in 200 µl of TE buffer (pH 8.0),
boiled for 10 min, transferred to -20°C for 5 min
for the release of DNA. This was then centrifuged
at 8000 rpm for 5 min and the supernatant
obtained was transferred to a separate vial and
stored at -20°C.
PCR based detection of the Genus Vibrio
All the isolates obtained were identified
to genus level by a PCR based detection method
using rpoA gene as described earlier24. Primers
were purchased from Shrimpex Biotech Services
Pvt. Ltd, Chennai. PCR was carried out in a final
volume of 20 μL containing 12.6 μL of PCR
grade water (Himedia, India), 2 μL 10X PCR
Buffer containing 2 mM MgCl2 (Himedia, India),
0.4 μL of 10 mM dNTP mix (Himedia, India), 1
μL each of 10 μM forward and reverse primers, 1
μL of 1 U/ μL of Taq Polymerase (Himedia,
India) and 2 μL of Template at 50ng
concentration.
Thermocycling conditions adapted were
as follows: initial denaturation at 94°C for 3 min
followed by 35 cycles of 1 min denaturation at
94°C, 1 min annealing at 55°C, and 1 min
extension at 72°C. The final extension was
carried out at 72°C for 5 min. Amplification was
performed in a GeneAmp PCR System 2720
thermal cycler (Applied Biosystems, Foster City,
CA, USA). The PCR products were resolved by
electrophoresis on a 2% agarose gel and stained
with ethidium bromide (10 mg/mL) for 20 min
followed by imaging and analysis of amplicons
was done in a Gel Documentation System
(BIOTOP FluorShot Ver. 1.5).
Results
Forty eight isolates were isolated from 10
species of Macro algae (Table-1). All of them
produced sucrose positive colonies (Yellow) on
TCBS medium, and all were gram negative rods
showing positive for oxidase except for four
isolates and all isolates showed positive for string
tests confirming them to the Genus Vibrio.
Phenotypic characterization found that all the
isolates fall among 10 species of Vibrios (Table-
2).
306 INDIAN J. MAR. SCI., VOL. 45, NO. 2 FEBRUARY 2016

Table – 1: Epiphytic Vibrios isolated from 10 macro algal species

Division Name of the Seaweed Strain Id Division Name of the Seaweed Strain Id
1.HBUL1 25.HAS1
26.HAS2
2.HBUL2 Acanthophora specifera 27.HAS4
3.HBUL3 28.HAS5
4. HBUL4 29.BAS1
5. HBUL5 30.HBA3
Ulva lactuca Asparagopsis taxiformis
6. HBUL6 31.HBA5
7. HBUL7 32.HGC1
8.HBUL8 33.HGC2
9. HBUL9 Red 34.HGC3
10.HBUL10 Algae 35.HGC4
11.HBUL11 Gracilaria corticata 36.HGC5
Green 12.HCS1 37.HGC6
Algae 13.HCS2 38.HGC7
Caulerpa sertularioides 14.HCS3 39.HGC8
15.HCS4 40.HBU1
16.HCS5 Mastophora rosea 41.HBU2
Caulerpa racemosa 17.CCSW3a 42.HBU3
18.KSW1a 43.HBU4
19.BDC1 44.B4112a
Sargassum turbinaroides
20.BDC2 45.B6112a
Dictyosphaeria cavernosa 21.CCSW4a Brown 46.D4112a
22.CCSW4b Algae 47.D6112a
Turbinaria ornata
23.CCSW4c 48.D6112b
24.CCSW4d

Among them three human pathogenic species, V.
alginolyticus, V. harveyi and V.metschnikovii
were also obtained. Vibrio alginolyticus strains
were found to be dominating, which occurred in
about 9 macro algal species used in this study.
Molecular characterization comprising PCR assay
of rpoA gene produced a 242 bp amplicon
confirming all the isolates to the genus Vibrio
(Fig-1).
Discussion
Marine macro algae are potential sources
of various complex compounds like mannitol and
laminarin from brown algae, D-galactose and agar
from red algae, xylopyranose and glucopyranose
from green algae25 besides alginates and
carrageenan. This nutrient rich environment
supports a wide variety of microbial communities.
Surface colonization by microbial communities is
pervasive in the marine environment and every
substratum whether living or non-living is prone
to this phenomenon where macro algal surfaces
are no exemption1, 26. Marine macro algae harbor
a diverse group of bacteria with densities varying
from 102 to 107 cells cm-2 1, 6. Surfaces of marine
eukaryotes like macro algae provide a unique
habitat for the bacteria to colonize and this
interaction mediated by the chemical signalling
between host and the bacteria supports the
microbial diversity and their function27. Macro
algal–bacterial associations or interactions are
diverse ranging from specialists, specific to a
particular host to generalists, found as common
epiphytes on wide variety of hosts26. These
epiphytic bacteria are known to secrete several
secondary metabolites with properties like
antimicrobial activities and hydrolytic enzyme
production27, 28, 29.
Marine macro algae harbors various
bacterial groups like Vibrio, Escherichia,
Pseudomonas, Aeromonas, Staphylococcus spp.
etc. as epiphytes among which Vibrios alone
accounts for 20% of the total gram negative
bacteria11.
 MURTHY et al: EPIPHYTIC VIBRIOS FROM THE MARINE MACRO ALGAE OF ANDAMAN ISLANDS 307
Table-2: Phenotypic characterization of Epiphytic Vibrios

Biochemical Tests VA VB VCA VH VME VN VPE VSP VSU VT

(24)* (1) * (1) * (4) * (4) * (5) * (3) * (4) * (1) * (2) *

TCBS Y Y Y Y Y Y Y Y Y Y
Gram Staining - - - - - - - - - -
Oxidase + + + + - + + + + +
String + + + + + + + + + +
ONPG - + - v - + + + - +
Arginine Dihydrolase - + - - - - - + - +
Lysine Decarboxylase + - + + v - - - - -
Ornithine Decarboxylase + - - + - - - - - -
Citrate + + + v v + + - - +
Urease - - - - - - - - - -
Indole + + + + - + - + - +
Voges- Proskauer + + - - + - - - + -
Gelatinase + + + + + + - + - +
Nitrate + + + + - + + + + +
Aesculin Hydrolysis - + + + + + + + + -
Growth at 0% NaCl - - - - + - - - - -
Growth at 3% NaCl + + + + + + + + + +
Growth at 6% NaCl + + + + + + + + - -
Growth at 8% NaCl + - - + - - + - - -
Growth at 10% NaCl + - - - - - - - - -
Growth at 4o C - - - - - - - - - -
Growth at 20o C + + + + + + + + + -
Growth at 30o C + + + + + + + + + +
Growth at 35o C + + + + + + + + - +
Growth at 40o C + + - + + + - - - +
Resistant Ampicillin 10µg + - + + - - - - + -
Acids from:
Mannitol + + + + + + + + + +
Inositol - - - - - - - - - -
Sorbitol - - - - - - - - - -
Rhamnose - - - - - - - - - -
Sucrose + + - v + + + + + +
Melibiose - + - - - - - - + +
Arabinose - - - - - - - - - -
Lactose - - - - - - - - - -
Mannose - + - + + + + + + +
Salicin - + - - + - - - - -

VA- V.alginolyticus; VB- V.brasiliensis; VCA- V.campbelli; VH- V.harveyi; VME- V.metschnikovii; VN-
V.navarrensis; VPE- V.pelagius I; VSP- V.splendidus I; VSU- V.superstes; VT- V.tubiashi; (+)Positive;
(-)Negative; Y-Yellow; V-variable; *-Number of strains.

Earlier studies several researchers have

Fig. 1- Gel picture showing the positive amplification of the
rpoA gene. Lane-1-5: Vibrio isolates used in this study;
Lane-6: Negative Control (Water); Lane-7: Positive control
(Vibrio cholerae MTCC3905); Lane-8: Positive control
(Vibrio fluvialis NICED IDH02036); Lane-9: 100 bp ladder.
reported the occurrence of Vibrio
parahaemolyticus from Ulva, Fucus, Laminaria,
and Porphyra sp.12,13 and V. vulnificus from
Porphyra, Undaria, Laminaria and Fucus sp. 14 in
Japan; V. rumoiensis and V. rotiferianus from
Fucus vesiculosus & Delesseria sanguine
respectively16 from Baltic sea, Germany; V.
mimicus from Ulva lactuca 30 of Indian coastal
waters and Vibrio spp. 15, 31, 32 from red, green and
brown macro algae. These studies indicate the
abundance of Vibrios in seaweeds.
308 INDIAN J. MAR. SCI., VOL. 45, NO. 2 FEBRUARY 2016
In the present study 48 Vibrio isolates
from 10 species of macro algae were obtained and
were identified by phenotypic characterization
using biochemical tests which fall under 10
species of Vibrios. Among them Vibrio
alginolyticus was found to be dominating in about
9 out of 10 macro algae. Three pathogenic
species20 V. alginolyticus, V. harveyi and V.
metschnikovii were also obtained in this study, yet
a detailed study in endorsing their pathogenicity
by analyzing their virulence factors needs to be
studied. Earlier studies show that these bacteria
cause diseases in humans, finfish and shellfish33.
In humans V. alginolyticus is associated with
wound infections and V. metschnikovii in sporadic
cases along with other pathogens in urine, blood
and wounds20. V. harveyi is associated with
necrosis and luminous vibriosis in finfish and
shellfish33. PCR based detection had shown
positive for the rpoA gene confirming all the
isolates to be Vibrios.
Present study shows the presence of
Vibrios from Ulva lactuca and Gracillaria
corticata which are correlated with the earlier
studies belonging to the same genus of macro
algae12, 15. Vibrio isolates obtained from other
macro algal species used in this study have not
been reported elsewhere. Vibrios associated to the
macro algal surfaces are found to have a
prominent role in biogeochemical nutrient
cycling, inducing morphogenesis and settlement
of zoospores of macro algae15.
There is always an ambiguity with
respect to the biochemical characterization of
environmental strains as their phenotypic
properties vary with the varying environmental
conditions. Thus there is an inevitable need for
molecular typing of these strains to identify them
and confirm their pathogenicity. Our studies on
the above criteria are underway.
Acknowledgements
This research is a part of UGC Major Research
Project. The first two authors thank the University
Grants Commission (UGC), Govt. of India, for
providing Major Research Project (Sanction
Letter No. 42-463/ 2013 (SR), Dt. 22-03-2013)
and the University authorities for providing
facilities.
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