INT J CURR SCI 2013, E 35-40
RESEARCH ARTICLE
ISSN 2250-1770
Antibacterial properties of secondary metabolites from the endophytic
marine algal bacterial population against chicken meat microbial pathogen
Vinodhkumar Ta*, Lubobi Ferdinand Shamalaa, SS Maithilia, G. Ramanathanb and S. Sudhakara
aDepartment
of Microbiology, AVS College of Arts and Science, Salem-636 106, India
cDepartment
of Microbiology, V.H.N.S.N. College, Virudhunagar-626 001, India
*Corresponding author: [email protected]; Phone: +91- 9965464010
Abstract
The world’s oceans comprise the largest part of the biosphere and contain the most ancient and diverse forms
of life. The seaweed genus, Amphiroa is a group of marine algae belonging to the class Florideophyceae has come to
attention for more pharmacological products and have received comparatively less bioassay attention. From this
study endophytic bacteria were isolated from seaweeds along the East Coast of India, Palk Strait to find out the
potential of antibacterial activity of Total Heterotrophic bacterial(THB). Based on the morphological characters, 20
different strains were isolated. The effect of bioactive compound from chosen heterotrophic bacteria strains were
assayed for antibacterial effect through Minimum inhibitory concentration and Minimum Bactericidal concentration
and tested for the antimicrobial sensitivity against Vibrio, Streptococci sp and Yersinia sp by cross streak assay. Of
them, 8 strains were showed sensitivity against pathogenic bacteria. The isolated endosymbiotic strains which shown
sensitivity against three pathogenic bacteria were subjected for the Minimum Inhibitory Concentration (MIC) assay
by following standard methodology. It shows that the strain no SW4, SW20 has MIC value of 125 µg against Vibrio
sp the SW16 SW20 and SW4 showed MIC value of 250 µg against Vibrio sp, Streptococci sp and Yersinia pestis,
the SW16 showed MIC value of 250 µg against Vibrio sp, and Streptococci sp and Yersinia pestis but the MIC value
was found lower by 125 µg against Streptococci sp. At a MIC value of 125 µg/ml, the strain SW4 and SW20 showed
inhibitory activity against Vibrio sp. At a MIC value of 250 µg/ml, the strain SW4 and SW16 showed inhibitory
activity against Yersinia pestis.
Keywords: seaweeds; THB strains; antimicrobial activity; Amphiroa anceps
Received: 08th January; Revised: 16th February; Accepted: 14th March; © IJCS New Liberty Group 2013
Introduction
Life originated in the sea and has sustained itself
(ESF Marine Board Feasibility Study Group Report,
to the present day. The world’s oceans comprise the
2001). The marine environment is a rich source of both
largest part of the biosphere and contain the most
biological and chemical diversity and has been the
ancient and diverse forms of life. The marine biotopes
source of unique chemical compounds with potential
contain an unmatched metabolic and organism diversity
for
industrial
development
as
pharmaceuticals,
Vinodhkumar et al., 2013
cosmetics, nutritional supplements, molecular probes,
mangrove
fine chemicals and agrochemicals. In recent years, a
microorganisms in the plant host is in the most cases
significant number of novel metabolites with potent
beneficial for plant. Secondary metabolites produced by
pharmacological properties have been discovered from
endophytes provide a variety of fitness enhancements
marine organisms. Although there are only a few
such as increased resistance to herbivore, parasitism,
marine-derived products currently on the market,
drought as well as growth enhancements. Endophytes
several robust new compounds derived from marine
usually produce the enzymes necessary for the
natural products are now in the clinical pipeline, with
colonization of plant tissues. It was demonstrated that
more clinical development.
most endophytes are able to utilize, at least in-vitro,
Seaweeds refer to any large marine benthic algae
that
are
multicellular,
of
endophytic
most plant cell components. Most of investigated
endophytes utilize xylan and pectin, show lipolytic
differentiated from most algae that are of microscopic
activity and produce non-specific peroxidase and
size. They are rich and varied source of bioactive
laccases (Leuchtmann et al., 1992), chitinase (Li et al.,
natural products and have been studied as potential
2004) and gluconase (Moy et al., 2002). Endophytes
biocidal and pharmaceutical agents. These plants form
represent a huge diversity of microbial adaptations that
an important renewable resource in the marine
have
environment and have been a part of human civilization
environments, and their diversity and specialized
from time immemorial. In addition, there are a number
habituation make them an exciting field of study in the
of seaweeds with economic potential. It will be of great
search new medicines. The hunt of new drug is
significance if these species could be the major role
particularly important in view of the fact that so many
players in drug development. They grow in the
diseases are developing immunity to some of the
intertidal, shallow and deep sea areas up to 180 meter
current treatment. Contamination of poultry meat with
depth and also in estuaries and black waters on the
food borne pathogens remains an important public
solid substrate such as rocks, dead corals, pebbles,
health issue, because it can lead to illness if there is
shells and other plant materials. They form one of the
malpractice in handling, cooking or post cooking
important living resources grouped under three
storage of the product. In developed countries, food
divisions
algae),
borne illness causes human suffering and loss of
Phaeophyceae (brown algae) and Rhodophyceae (red
productivity, and adds significantly to the cost of food
algae). They are abundant on hard substrates and
production and healthcare. It is also a possible cause of
commonly extending to depths of 30-40 m. About 624
mortality, which is even more of a problem in
species have been reported in India with a potential of
developing regions, where the health status of many
77,000 tons (wet weight) per annum. The red seaweeds
individuals is already compromise. Numerically, the
contribute 27.0, brown 0.2 and others 72.8%. About
most important agents are Salmonalle, Staphylococcus,
206 species of algae have been reported from the
Vibrio and Campylobacter sp.
Chlorophyceae
and
Presence
thus
namely,
macrothallic,
environment.
(green
developed
www.currentsciencejournal.info
in
special
and
sequestered
Vinodhkumar et al., 2013
The discovery, development and clinical use of
antibiotics
during
the
20th
century
pathogens were isolated by serial dilution method. The
decreased
chosen isolates were identified using standard staining
substantially the morbidity and mortality from bacterial
and biochemical test. (Bergey’s manual-Holt et al.,
infections. However, since the 1980’s the introduction
1994)
of new agents for clinical use has declined reflecting
Antibacterial sensitivity assay
both the challenge of identifying new drug classes to
The antagonistic activity was tested by following
antibacterial drug discovery by the pharmaceutical
cross streak assay method (Sivakumar et al., 2005)
industry .Marine bacteria being a heterotrophy with
using 20 different THB strains against chicken meat
simple cell multiplication process, which can be
associated pathogen viz, Vibrio, Streptococcus, and
cultivated in large amounts inexpensively. This has
Yersinia pestis. Single streak of the isolated strains
prompted the present study, to assess the possible
was done on sterile Muller Hinton Agar plates
utilization of associated bacteria as resources, to meet
followed by overnight culture of chicken
the sufficient supply of desired metabolites. The
associated
present investigation was initiated to screen the
perpendicular to the original streak of isolates and
antibacterial efficacy of marine algae associated
incubated at 37±2°C. Bacterial strains showed
bacterial population against the meat pathogens
maximum inhibitory effect against tested pathogens
Materials and Methods
were subjected for mass cultivation in broth and was
Description of the Study area
filtered by using Millipore filter. Filtrate was mixed
pathogens
were
meat
streaked
at
Thondi is situated in the Palk Strait region of
with equal volume of ethyl acetate (v/v) in separating
Tamil Nadu. The study area lies in the latitude of
funnel and shaken well and then allowed to stand
99°44”N and 79 10’ 45” E. Amphiroa anceps species
without any disturbance for 15 minutes. After that, the
of seaweeds were collected during month of (January
lower aqueous phase was discarded and the upper
2012) for the isolation of endophytic THB strains
solvent phase was concentrated in a vacuum
Isolation of Endophytic Organisms
evaporator at room temperature for 24 hrs to obtain
The 1 gm of fresh sea weeds species were
powder form of crude extract and stored in a
aseptically weighed, washed thrice with sterilized
refrigerator for further analysis (Kang et al., 2007).
distilled water and were plated with the Zobell marine
For
agar medium. The bacterial species enumerated on the
concentration of extracts (31, 62, 125, 250, 500, 1000,
Zobell marine 2216e (Hi-media) agar medium were
1500 and 2000 µg) was prepared with Dimethyl
counted and the total number of bacterial counts was
sulphoxide (DMSO) and mixed with 0.5 ml of nutrient
expressed as Colony Forming Unit (CFU).
broth. 50 µl of bacterial inoculums serves as positive
Isolation of chicken meat associated pathogens
control. Nutrient Broth alone served as negative
MIC
determination
0.5
ml
of
various
The fresh chicken meat samples were collected in
control. Whole setups in duplicates were incubated at
a sterile container. The chicken meat associated
37°C for 48 hrs. The MIC was the lowest
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Vinodhkumar et al., 2013
concentration of the extract that did not permit any
effective concentration of sensitivity (MIC and MBC)
visible growth after 24 hrs of incubation and it was
by the isolated bacterial strains against Yersinia pestis
examined on the basis of turbidity. To avoid the
particularly the isolated strains of SW4 and SW16
possibility of misinterpretations due to the turbidity of
against Vibrio sp and Streptococci sp.
insoluble compounds, the minimum bactericidal
Fig. 1. Marine algae Amphiroa anceps
concentration (MBC) was determined by sub culturing
the MIC dilutions on to the sterile agar plates. The
lowest concentration of the extracts which inhibits the
growth of tested bacteria are observed and tabulated.
Results
About 20 bacterial strains were isolated as
endosymbiont from the marine algae Amphiroa anceps
and all of them have been tested for the antimicrobial
sensitivity against Vibrio, Streptococci and Yersinia sp
Discussion
by cross streak assay. Of them, eight strains were
Life originated in the sea and has sustained itself
shown sensitivity against three pathogenic bacteria.
to the present day. The Marine ecosystem is a rich
The isolated endosymbiotic strains which shown
source biological diversity. This has been exploding in
sensitivity against three pathogenic bacteria were
the discovery
subjected for the Minimum Inhibitory Concentration
potential
(MIC) assay by following standard methodology. It
pharmaceuticals. In recent years, a significant number
shows that the strain no SW4, SW20 has MIC value of
of novel
125 µg against Vibrio sp the SW16 SW20 and SW4
pharmacological properties have been discovered from
showed MIC value of 250 µg against Vibrio sp,
marine organisms. The world’s oceans comprise the
Streptococci sp and Yersinia pestis, the SW16 showed
largest part of the biosphere and contain the most
MIC value of 250 µg against Vibrio sp, and
ancient and diverse forms of life. The endo symbiotic
Streptococci sp and Yersinia pestis.
heterotrophic bacteria have been isolated from sea
The MIC value was found lower by 125 µg
against Streptococci sp.
of
for
unique
industrial
chemicals
having
development
secondary metabolites
with
as
potential
weed species. Several studies have indicated that
Surprisingly, the strain no
consumption of poultry meat has been associated with
SW4 showed MBC value of 500 µg to 2 of the
incidence of outbreaks of food borne illnesses
pathogenic bacteria viz., Vibrio sp and Yersinia pestis.
(Lunden et al., 2003; Prakash et al., 2005). Reduction
However, the other strains did not showed any MIC
of initial bacterial load in meat is of prime importance
and MBC values as mentioned in the tables. The
in an attempt to improve the shelf-life of the product
presence and absence of bacterial turbidity and the
(Lillard et al., 1984).
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Vinodhkumar et al., 2013
Table 1. Antibacterial efficacy of marine algal
sponges are produced by functional enzyme clusters,
associated bacteria against chicken meat associated
originated
pathogens through cross streak assay. SW – Seaweed;
microorganisms. A wide range of chemical and
+ - Presence of Activity; - - Absence of Activity
functional diversity has been observed among
Endophytic
Chicken meat bacterial pathogen
Bacterial Strain
from
the
sponges
associated
bioactive compounds. Of the various classes of
compounds,
polyketides,
alkaloids,
fatty
acids,
peptides and terpenes are the most abundant ones.
Strain no
Vibrio
sp
Streptococci Yersinia
sp
sp
Sw13
-
+
+
Sw14
+
-
+
Sw4
+
+
-
Strain
Vibrio sp
Sw5
-
+
-
no.
MIC MBC
MIC
MBC
MIC
MBC
Sw6
-
+
-
SW4
125
250
500
500
125
500
Sw16
+
+
+
SW5
-
-
-
-
-
-
Sw18
-
+
+
SW6
-
-
-
-
-
-
SW13
-
-
-
-
-
-
Sw20
-
+
SW14
-
-
-
-
-
-
SW16
250
500
125
250
250
250
SW18
-
-
-
-
-
-
SW20
125
250
250
500
500
-
Table 2. Minimum Inhibitory Concentration (MIC)
and Minimum Bactericidal Concentration (MBC) (µg
ml-1) of the extracts from endophytic bacteria against
chicken meat associated pathogen. SW – Seaweed
Streptococci sp
Yersinia sp
Hence, the present study was undertaken to produce
valuable secondary metabolites from the marine algae
against chicken meat associated pathogens. Based on
the morphological characters, 20 strains were isolated
Majority of them show antimicrobial, antitumor and
and all of them have been tested for the antimicrobial
anticancer properties. Tritetracontane, Nonadecane,
sensitivity against Vibrio, Streptococci sp and Yersinia
Docazane, Indolirine, Trifluroaceticacid, n-octadecyl
sp by cross streak assay. Generally, the endophytic
ester are highly abundant and have been shown to be
bacteria isolated from seaweeds showed maximum
multifunctional. Triterpenes attract attention because
sensitivity against several human bacterial pathogens.
of their biological activities of endosymbionts. Hence,
Moreover,
bioactive compounds from
steps have been undertaken to find out the reason for
endophytic bacteria showed maximum sensitivity with
the maximum activity of endophytic bacteria from
minimum concentration than the bioactive compounds
seaweeds and complete structural elucidation of the
from epiphytic bacteria of other biological origin. The
potential metabolites will also been focused in future.
earliest marine bio active compounds were isolated
Acknowledgment
the
from endosymbionts of sponges. The bioactive
The authors are thankful to the authorities of
secondary metabolites isolated from endosymbionts of
AVS College of Arts And Science, Salem and
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Vinodhkumar et al., 2013
V.H.N.S.N. College, Virudhunagar, Tamil Nadu, India
Kang, Cheong H, Ryu CM, Kim JF (2007). Two
for providing required facilities to complete this work.
bacterial endophytes eliciting both plant growth
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