RESEARCH ARTICLE
Phylogenetic characterization of culturable bacterial diversity
associated with the mucus and tissue of the coral Acropora digitifera
from the Gulf of Mannar
Paramasivam Nithyanand & Shunmugiah Karutha Pandian
Department of Biotechnology, Alagappa University, Tamil Nadu, India
Correspondence: Shunmugiah Karutha
Pandian, Department of Biotechnology,
Alagappa University, Karaikudi, Tamil Nadu
630 003, India. Tel.: 191 45 65 22 52 15; fax:
191 45 65 22 52 02; e-mail:
[email protected]
Received 20 February 2009; revised 28 May
2009; accepted 28 May 2009.
Final version published online 8 July 2009.
DOI:10.1111/j.1574-6941.2009.00723.x
MICROBIOLOGY ECOLOGY
Editor: Riks Laanbroek
Keywords
coral-associated bacteria; Acropora digitifera;
actinomycetes; antibacterial activity; Gulf of
Mannar.
Abstract
Corals, considered the rainforests of the oceans, harbour an abundance of different
bacterial populations throughout the coral structure. In the present study we
attempted to characterize the cultivable bacterial population associated within the
mucus and tissue of the coral Acropora digitifera from the Gulf of Mannar.
16S rRNA gene was amplified from the cultured mucus and tissue isolates.
Amplified ribosomal DNA restriction analysis, performed with a combination of
restriction enzymes to determine the polymorphic groups of bacteria, generated 19
distinct groups in the coral mucus and 17 distinct groups in the coral tissue.
Phylogenetic analyses based on the full-length sequences of 16S rRNA gene
sequences showed that the majority of bacterial isolates belonged to the group
Firmicutes, followed by Gammaproteobacteria and Actinobacteria. On investigating
their antimicrobial activity, mucus isolates showed about 25% activity and tissue
isolates showed 48% activity. This study revealed the presence of actinomycetes in
both the coral mucus and the coral tissue, which had high activity against
pathogens. This study, for the first time, demonstrates that actinomycetes existing
within corals also have potential antibacterial activity. This has been overlooked so
far, and indicates that, in addition to mucus, bacteria within the tissue of corals
might defend the coral host against pathogens.
Introduction
Corals act as host organisms (holobiont) to a multitude of
diverse bacterial population (Rohwer et al., 2001, 2002;
Wegley et al., 2007). These bacteria localize in the surface
mucus layer, the coral tissue and the calcium carbonate
skeleton of the corals (Rosenberg et al., 2007), and each of
these habitats harbour different bacterial species (Koren &
Rosenberg, 2006). Although the amounts of bacteria found
in the coral mucus are similar to those found in the coral
tissue, the abundance of bacterial species in the coral mucus
is different from the abundance of bacterial species present
in the coral tissue (Bourne & Munn, 2005; Koren &
Rosenberg, 2006).
The cornucopial amount of bacteria in the coral mucus
layer has been estimated at 105–106 CFU mL1, which
is 100–1000-fold higher than the surrounding seawater
(Rosenberg et al., 2007). On investigating the roles of coral-
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c
associated microorganisms it has been proposed that bacteria associated with corals are mostly heterotrophic; they use
the complex carbon compounds in corals and aid in carbon
and nitrogen fixation (Wegley et al., 2007). The carbohydrate-rich mucus is exploited as a medium for microbial
growth. Mucus also keeps the coral surface clean of sediment, and is an energy carrier and particle trap in the reef
ecosystem. When shed, this mucus provides a major nutrient source for the reef environment (Wild et al., 2004).
The carbon source utilization pattern by the coral mucus
bacteria is coral specific and the utilization pattern differs
among various species of corals. As each species of coral has
mucus that is biochemically unique, the differences in the
composition of the surface mucus produced by specific
corals results in different populations of associated microorganisms (Ritchie & Smith, 1995).
The coral tissue-associated bacteria are also extremely
diverse. The presence of a physiologically favourable niche
FEMS Microbiol Ecol 69 (2009) 384–394
385
Culturable bacterial diversity of A. digitifera
for bacterial growth within the coral tissue and the trapping
of bacteria at the coral–seawater interface are two factors
contributing to the diverse bacterial communities associated
with the corals (Klaus et al., 2005).
Interactions between epibiotic marine bacteria and the
host organism are known to play a significant role in the
marine ecosystem, but this association has received little
attention. There is a growing recognition of the importance
of bacterial symbiosis; bacteria may be the true producers of
many bioactive compounds isolated from corals, sponges,
ascidians and other marine invertebrates (Fenical, 1993). It
is hypothesized that the coral holobiont harbours a particular group of bacteria that may protect the coral from
pathogens through filling entry niches and/or producing
antibiotics (Rohwer et al., 2002).The coral mucus acts as a
medium for secreted allochemicals with antimicrobial properties (Brown & Bythell, 2005). Extracts from soft corals,
gorgonian corals and certain scleractinian coral species have
antimicrobial properties (Kim, 1994; Slattery et al., 1995;
Kelman et al., 2006). However, the origin of these allochemicals is unknown. Bacterial symbionts have been shown to be
responsible for the production of secondary metabolites
previously attributed to the host organism (Elyakov et al.,
1991). A previous study reports that 30% of bacteria isolated
from coral species have antibiotic capabilities (Castillo et al.,
2001) and hence bacteria might be responsible for the
antimicrobial properties exhibited by the corals. Ritchie
(2006) showed that mucus from healthy Acropora palmata
harbours bacteria capable of producing antibiotics, implicating a microbial contribution to the protective properties
of the coral mucus. Very recently, it has been demonstrated
that bacteria with antibacterial activity exist on the coral
surface (Shnit-Orland & Kushmaro, 2009), thereby eliciting
a probiotic effect on microbial communities associated with
the coral holobiont (Nissimov et al., 2009).
The marine environment is a virtually untapped source of
novel actinomycete diversity (Stach et al., 2003; Bull et al.,
2005) and of new metabolites (Magarvey et al., 2004; Jensen
et al., 2005). As marine environmental conditions are
extremely different from terrestrial ones, it is surmised that
marine actinomycetes have different characteristics from
those of their terrestrial counterparts and, therefore, might
produce different types of bioactive compounds. Novel
actinomycete groups have been found in sponges and novel
bioactive metabolites have been obtained from actinomycetes isolated from sponges (Webster et al., 2001; Kim et al.,
2005). Reports on actinomycetes associated with corals are
very scanty and only a few reports (Lee et al., 1999; Lampert
et al., 2006, 2008) discuss the actinomycetes associated with
corals.
In the present study, we have used biochemical as well as
molecular tools to characterize the culturable bacterial
communities associated with the mucus and tissue of the
FEMS Microbiol Ecol 69 (2009) 384–394
coral Acropora digitifera from the Gulf of Mannar. In
addition, we characterize the antibacterial potential of the
bacterial community, including actinomycetes associated
with corals.
Materials and methods
Sample collection
Mucus and tissue samples of four healthy individuals of the
coral A. digitifera were collected from Hare Island (9112 0 N,
7915 0 E). Hare Island spreads over an area of 120 ha. The
coral-surface mucus layer was swabbed using sterile cotton
swabs (Guppy & Bythell, 2006). Mucus samples of c. 1-cm2
coral surface area were taken with these swabs. The swabs
were immediately placed in sterile polypropylene tubes. The
coral tissue samples were collected by removing c. 2 2 cm
of the coral tissue from the coral (Rohwer et al., 2001). At
the surface, the coral tissue sample was washed twice with
sterile seawater to remove loosely attached bacteria and was
immediately placed in a plastic bag, which was then placed
on ice. Seawater samples were collected with 50-mL sterile
tubes that were opened under water adjacent to the same
corals. Sediment samples were collected from right below
the corals. All samples were transported to the laboratory
(within about 4 h) kept ice cold and were plated for isolation
of bacteria.
Isolation of bacteria from coral samples
The mucus swab samples were transferred to sterile tubes
with 1-mL autoclave-sterilized seawater, in a sterile hood.
The bacteria from the cotton swabs were suspended in
seawater by vigorous vortexing. Bacteria were isolated using
standard serial dilution and plating techniques in triplicate
on Zobell Marine Agar (Himedia Laboratories, Mumbai,
India) (Guppy & Bythell, 2006). To isolate the bacteria from
the coral tissue, coral tissue pieces measuring c. 2 2 cm
were airbrushed with sterile seawater, which is referred to
below as tissue slurry. Tissue slurry of 100 mL was serially
diluted and plated on Zobell Marine Agar. All plates were
incubated at 27 1C, corresponding to the temperature of the
ambient seawater for 7–12 days (Rohwer et al., 2001).
Bacterial counts that represent the number of culturable
bacteria were recorded as CFUs and expressed as CFU cm2
of coral surface area for mucus and tissue. Culturable
bacteria from seawater and sediment were recorded as
CFU mL1 (of seawater) and g1 (of sediment), respectively.
Biochemical identification of coral-associated
bacteria
All the isolated bacteria were identified by performing
various biochemical tests according to Bergey’s manual.
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386
The coral bacterial isolates were subjected to various
morphological and biochemical tests (see Supporting
Information, Tables S3 and S4). Carbohydrate tests were
performed using the HiCarbohydrate kit (Himedia Laboratories; Cat. No. KB009). The sensitivity of mucus- and
tissue-associated bacteria to antibiotics (10 U penicillin-G,
30 mg chloramphenicol, 30 mg novobiocin, 30 mg tetracycline, 100 mg piperacillin, 10 mg penicillin and 2 mg clindamycin), each applied to a paper disc, was determined after
incubation for 24–48 h at 30 1C on Zobell Marine agar.
Genomic DNA extraction from coral-associated
bacteria
The culture grown on Zobell Marine broth overnight at 27 1C
was centrifuged at 4600 g for 3 min. Bacterial genomic DNA
was isolated according to Babu et al. (2009). The pellet was
resuspended in 400 mL of Sucrose TE. Lysozyme was added to
a final concentration of 8 mg mL1 and incubated for 1 h at
37 1C. To the tube, 100 mL of 0.5 M EDTA (pH 8.0), 60 mL of
10% SDS and 3 mL of proteinase K from 20 mg mL1 were
added and incubated at 55 1C overnight. The supernatant was
extracted twice with phenol : chloroform (1 : 1) and once with
chloroform : isoamylalcohol (24 : 1) and ethanol precipitated.
The DNA pellet was resuspended in sterile distilled water.
Amplification of 16S rRNA gene
Bacterial 16S rRNA gene was amplified from the extracted
genomic DNA using the following universal eubacterial
16S rRNA gene primers: forward primer 5 0 -AGAGTTT
GATCCTGGCTCAG-3 0 (Escherichia coli positions 8–27)
and reverse primer 5 0 -ACGGCTACCTTGTTACGACTT-3 0
(E. coli positions 1494–1513). PCR was performed in a
50-mL reaction mixture containing 2 mL (10 ng) of DNA as
the template, each primer at a concentration of 0.5 mM,
1.5 mM MgCl2 and each deoxynucleoside triphosphate at a
concentration of 50 mM, as well as 1 U of Taq polymerase and
buffer as recommended by the manufacturer (MBI Fermentas). After the initial denaturation for 3 min at 95 1C, 40 cycles
consisting of denaturation at 95 1C for 1 min, annealing at
55 1C for 1 min and extension at 72 1C for 2 min, and a final
extension step of 5 min at 72 1C were carried out (Mastercycler Personal, Eppendorf, Germany). The amplification of
16S rRNA gene was confirmed by running the amplification
product in 1% agarose gel in 1 Tris-acetate-EDTA.
P. Nithyanand & S.K. Pandian
of the PCR product was digested with HinfI at 37 1C for 3 h.
Digested DNA samples were analyzed in 2% agarose gel.
Cloning and sequencing of 16S rRNA gene
The amplified product (c. 1500 bp) was purified using
GFXTM PCR DNA and Gel Band Purification Kit (Amersham Biosciences) according to the manufacturer’s instructions. The 16S rRNA gene amplicon was cloned in pTZ57R/
T vector according to the manufacturer’s instructions (InsT/
AcloneTM PCR Product Cloning Kit #K1214, MBI Fermentas). Full-length sequencing of the rRNA gene (about
1500 bp) for all the coral-associated bacterial isolates was
carried out in Macrogen (Seoul, Korea).
Nucleotide sequence analysis
The full-length sequences obtained were matched with
previously published sequences available in NCBI using
BLAST (Altschul et al., 1997). Multiple sequence analysis was
carried out using CLUSTALX (Thompson et al., 1997), and a
further neighbor-joining plot (Perrière & Gouy, 1996) and
PHYLODRAW (Choi et al., 2000) were used to construct the
phylogenetic tree. To validate the reproducibility of the
branching pattern, a bootstrap analysis was performed.
Screening coral-associated bacteria for
antibacterial activity
All mucus- and tissue-associated bacteria were screened for
antibacterial activity using a primary screening method. The
bacterial isolates were grown in Zobell broth at room
temperature. Overnight cultures were centrifuged, and the
cells were resuspended in Zobell medium and grown as a
lawn on Zobell agar. Plugs, 11 mm in diameter, were
stanched out with a cork borer and placed with the bacterial
side down onto agar plates that had been seeded with a
1 : 200 dilution of an overnight culture of the pathogenic
strains Staphylococcus aureus (ATCC 11632), Pseudomonas
aeruginosa (ATCC 10145), Aeromonas hydrophila (ATCC
7966), Vibrio parahaemolyticus (ATCC 27519) and Vibrio
vulnificus (ATCC 29307). Following overnight incubation at
30 1C, the plates were inspected for the formation of
inhibition zones around the agar plugs (Pabel et al., 2003).
The strains that showed antibacterial activity in primary
screening were further screened by culturing the strains in
Zobell Marine broth by the shake flask method at 28 1C for
1 week. Cell-free culture supernatant was used to determine
antibacterial activity.
Amplified ribosomal DNA restriction
analysis (ARDRA)
Antimicrobial activity test
With the objective of determining bacterial diversity, all the
16S rRNA gene amplicons representing various isolates were
subjected to ARDRA. To examine the ARDRA profile, 10 mL
Antimicrobial activity was assayed by the disc diffusion
susceptibility test according to the recommendations of the
National Committee for Clinical Laboratory standards
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FEMS Microbiol Ecol 69 (2009) 384–394
387
Culturable bacterial diversity of A. digitifera
(NCCLS, 2000). The disc diffusion test was performed on
Müller–Hinton agar (MHA) (Himedia Laboratories) for the
bacterial pathogens. Freshly grown colonies of the pathogens were used to inoculate 25 mL of Müller–Hinton broth
(Himedia Laboratories) in a shaking water bath for 4–6 h
until a turbidity of 0.5 McFarland (1 108 CFU mL1) was
reached. Final inocula were adjusted to 5 105 CFU mL1.
The inoculum (100 mL) from the final inocula was applied to
each agar plate and uniformly spread over the surface with a
sterilized cotton swab. Absorption of excess moisture was
allowed to occur for 10 min before application of dried
paper discs with a diameter of 6 mm containing 20 mL each
of culture supernatant. The paper discs were deposited onto
MHA plates and the plates were incubated at 37 1C and the
zones of inhibition were measured after 24 h.
Screening of coral actinomycetes for
antimicrobial activity
Each actinomycete isolate was grown as an c. 2-cm colony for
10–14 days on Petri plates with specific combining ability.
Bacteria, on the other hand, were streaked about 1–1.5 cm
from the edge of the colony being tested (Zin et al., 2007).
Well-characterized clinical microbial strains S. aureus (ATCC
11632), P. aeruginosa (ATCC 10145), A. hydrophila (ATCC
7966), V. parahaemolyticus (ATCC 27519) and V. vulnificus
(ATCC 29307) were used as the indicator microorganisms for
antibacterial activity assay. Most of these organisms were
selected because they represent a wide range of pathogens of
human as well as representative Gram-positive and Gramnegative bacteria. Growth of the test organisms was evaluated
after 24, 48 and 72 h, and recorded as growth, inhibition and
no growth as compared with a control plate containing no
actinomycetes colonies. Secondary screening was performed
by agar well-diffusion assay (Harald et al., 2007) to confirm
the antibacterial activity of five actinomycetes strains. The
cell-free supernatant obtained from a 7-day-old culture of
actinomycetes isolates, cultivated in International Streptomyces Project (ISP2) broths (Himedia Laboratories) was
tested against the bacterial pathogens.
Cultivation of actinomycetes for
antimicrobial assay
Each of the six actinomycete strains isolated from corals was
transferred aseptically into 250-mL Erlenmeyer baffled
flasks with cotton plugs, containing 50 mL ISP2 medium
(Himedia Laboratories), incubated for 3–5 days at 28 1C
with agitation in a rotary shaker at 250 r.p.m.
Extraction of crude metabolites
After 3 days of incubation, the culture broth was filtrated
through a press to separate mycelium and supernatant. The
FEMS Microbiol Ecol 69 (2009) 384–394
supernatant was extracted twice with ethyl acetate, chloroform and n-butanol (2 100 mL). The solvent extracts were
combined and evaporated to dryness under reduced pressure to yield crude extracts, and each crude extract obtained
was weighed. The crude extracts were dissolved in methanol : chloroform (1 : 1, v/v) and used for antibacterial
screening (Zin et al., 2007).
Antimicrobial activity test
Antimicrobial activity was assayed by the disc diffusion
susceptibility test according to the recommendations of the
National Committee for Clinical Laboratory standards
(NCCLS, 2000) as mentioned above. Absorption of excess
moisture was allowed to occur for 10 min before application
of dried paper discs with a diameter of 6 mm containing
20 mL each of organic and aqueous extracts. The paper discs
were deposited on MHA plates and the plates were incubated
at 37 1C and the zones of inhibition were measured after 24 h.
Ethyl acetate, chloroform and n-butanol were applied to
paper discs as negative controls for each experiment.
Results
Isolation and enumeration of bacteria
The culturable bacterial count in the mucus was
7.5 106 3 105 CFU cm2. The enumeration of culturable bacteria from the coral tissue is presented as CFU cm2 of
coral surface (Koren & Rosenberg, 2006). The culturable
bacterial count in the coral tissue was 1.8 106
7 104 CFU cm2 of coral surface. In comparison, there
were 2.4 104 3 103 CFU mL1 and 2.6 105 2 103
CFU g1 culturable bacteria in seawater and the sediment
adjacent to the corals, respectively.
A total of 49 bacterial strains were isolated from both
mucus and tissue of the coral A. digitifera. Twenty-four
isolates were obtained from mucus and 25 isolates from the
coral tissue. The strains were characterized using biochemical and molecular methods.
ARDRA showed the presence of different polymorphic
groups of bacteria in the coral mucus and the coral tissue.
ARDRA analysis revealed 17 and 19 polymorphic groups
from 24 isolates of the coral mucus and 25 isolates of the
coral tissue. Among the 17 ARDRA groups of the coral
mucus found, 13 polymorphic patterns for HinfI and RsaI,
and 17 polymorphic patterns for HaeIII (see Fig. S1) were
observed. Similarly, among the 19 ARDRA groups of the
coral tissue, 15 polymorphic patterns for HinfI and RsaI,
and 16 polymorphic patterns for HaeIII (see Fig. S2) were
observed. Three ARDRA groups (groups 12, 17 and 19)
from the coral mucus and five ARDRA groups (groups 8, 9,
12, 16 and 17) from the coral tissue appeared more than
once.
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388
All the strains were identified by 16S rRNA gene sequencing. Sequence analysis of the coral mucus strains revealed
that 25% belonged to the Gammaproteobacteria, 4% to the
Actinobacteria, 67% the to Firmicutes and 4% to the
Cytophaga–Flavobacter group (see Fig. S4). In contrast,
sequence analysis of the coral tissue strains revealed that
25% belonged to the Gammaproteobacteria, 20% to the
Actinobateria and 55% to the Bacillales group (see Fig. S4).
All the isolates were closely related to previously described
bacterial species, with an average identity of 99% of the
1500 bp of the 16S rRNA gene sequenced. In the present
study, the phylum Firmicutes was dominated by the family
Bacillaceae; both the mucus and the tissue had the same
amount of members of this family (Fig. 1). Apart from the
family Bacillaceae, the phylum Firmicutes also contained
members of the family Staphylococcaceae and Enterococcaceae. The phylum Gammaproteobacteria consisted of the
families of Vibrionaceae, Halomonadaceae and Enterobacteriaceae. Vibrionaceae members were present in high numbers
in the coral mucus when compared with the coral tissue,
where the predominant bacteria were V. parahaemolyticus
and Vibrio natrigens. This is the first report of V. natrigens in
a coral. The Enterobacteriaceae family was represented by the
presence of Providencia rettgeri, which belongs to the coliform group, and it was present in both the coral mucus and
the coral tissue. Halomonas salaria of the Halomonadaceae
family was also present in both the mucus and the tissue of
the coral A. digitifera. There were higher numbers of
Actinobacteria members in the coral tissue than in the coral
mucus, consisting of three different families, with the
bacterial species Brachybacterium paraconglomeratum belonging to family Dermabacteraceae, Brevibacterium linensis
to family Brevibacteriaceae and Kocuria flavus and Kocuria
rosea to family Micrococcaceae.
Phylogenetic analysis
Phylogenetic analysis of the bacteria isolated from the coral
mucus and the coral tissue revealed the presence of three
P. Nithyanand & S.K. Pandian
major groups of bacterial domain, the Gram-positive Actinobacteria (high G1C), Firmicutes (low G1C) and the Gramnegative Gammaproteobacteria. Representatives of the
Firmicutes were the most abundant in both the coral mucus
and the coral tissue. Phylogenetic analysis of the coral mucus
strains showed that 16 strains are clustered within the
Firmicutes group (Fig. 2) belonging to several Bacillus sp.
with 98–100% similarity between them (see Table S1). Three
strains (CM6, CM18 and CM26) from the Firmicutes group
fall under the family Staphylococcae with 99% similarity to
Staphylococcus arlettae (GenBank accession number
AB009933.1) and Staphylococcus sciuri (GenBank accession
number AJ421446.1). Only one strain, CM22, among the
mucus isolates clustered with Actinobacteria; BLAST analysis
revealed that this strain is a close relative with 99% similarity
to the strain Brachybacterium sp. I20-12 (EU181223.1)
isolated from deep-sea sediments.
BLAST analysis showed that the bacterial strains CM2,
CM3, CM7, CM-11, CM12 and CM27 were members of
the Gammaproteobacteria. It is noteworthy that the strain
CM27 had 99% similarity with H. salaria (AM229316.1), a
novel halophilic species isolated from saline waters of Korea.
The strain CM9 belonged to the Bacterioides group, having
99% similarity with an endophytic bacteria Myroides odoratimimus (EU331413.1) (Table S1).
Phylogenetic analysis of the coral tissue strains showed
that the groups Firmicutes and Actinobacteria had a cluster
of 13 and five strains, respectively (Fig. 2). One of the strains
(CT21) belonged to the Lactobacillales member Vagococcus
carniphilus (AY669387.1). Gram-negative strains of the coral
tissue had 99% similarity to their respective Gammaproteobacteria members: H. salaria (AM229316.1), V. parahaemolyticus (EF467290.1) and P. rettgeri (AM040492.1) (see Table
S2).
There was a larger Actinomycetes population in the coral
tissue than in the coral mucus. BLAST analysis of the strains
CT-6 and CT-24 revealed that the strains are close relatives,
with 99% similarity to Brachybacterium sp. I20-12
(EU181223.1). Strain CT-8 is a close relative, with 99%
similarity to Brevibacterium sp. CO63 (DQ643065.1). Both
strains CT-9 and CT-22 fall into the genus Kocuria, having
99% similarity to K. flavus HO-9041 (EF602041.1) and
K. rosea (DQ060382.1), respectively (see Table S2).
Biochemical characterization of the cultured
bacteria
Fig. 1. Distribution of bacterial isolates (at the order and family level) in
the coral mucus and the coral tissue.
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The bacterial strains isolated in this study had different
biochemical profiles (Tables S4 and S5). In concordance
with an earlier report (Lampert et al., 2006) our results show
the isolates that are close relatives according to the phylogenetic tree exhibited different biochemical profiles and antibiotic sensitivity. This phenotypic variation, even among
FEMS Microbiol Ecol 69 (2009) 384–394
389
Culturable bacterial diversity of A. digitifera
Fig. 2. Neighbour-joining phylogenetic tree from analysis of 16S rRNA gene sequence of bacterial isolates from the mucus and the tissue of the coral
Acropora digitifera. The numbers are the percentages indicating the levels of bootstrap support, based on a neighbor-joining analysis of 1000 resampled
data sets. The scale bar represents 0.1 substitutions per nucleotide position.
closely related isolates, is attributed to the fact that overreliance on a small number of subjectively chosen properties
often leads to misclassification and the properties looked
for, are themselves inadequate for identification (Stackebrandt & Goodfellow, 1991). Hence 16S rRNA gene sequencing is the widely used method for identifying bacterial
isolates. The sensitivity of mucus- and tissue-associated
bacteria to various antibiotics showed that most of the
isolates were sensitive to chloramphenicol and clindamycin,
and were resistant to tetracycline (see Table S5).
Screening coral-associated bacteria for
antibacterial activity
All 49 bacterial strains isolated from mucus (n = 24) and
tissue (n = 25) were screened for antibacterial activity.
Of the 49 isolates, 18 (36.7%) showed antibacterial activity
against different pathogens. Of the 24 isolates screened
from mucus, five (25%) exhibited antibacterial activity,
and of the 25 isolates screened from the coral tissue,
12 (48%) showed antibacterial activity (Table 1). Both
mucus- and tissue-associated bacteria showed antibacterial activity against Gram-positive and Gram-negative
bacteria.
FEMS Microbiol Ecol 69 (2009) 384–394
Screening coral actinomycetes for
antimicrobial activity
In the primary screening, actinomycetes strains were
screened for their antibacterial activity against test pathogens using the cross-streak method. Five (10%) actinomycetes strains (CM 22, CT 6, CT 8, CT 9 and CT 22) showed
antibacterial activity against the bacterial pathogens
S. aureus, P. aeruginosa, A. hydrophila, V. vulnificus and
V. parahaemolyticus. The growth inhibition halos caused by
the actinomycetes cultures were measured in millimeters.
Secondary screening was performed by agar well-diffusion
assay to confirm the antibacterial activity of five actinomycetes strains. The cell-free supernatant obtained from 7-dayold culture of actinomycetes isolates cultivated in ISP2 broth,
was tested against the bacterial pathogens. All five actinomycetes strains showed a growth inhibitory zone against the
bacterial pathogens. Among the five actinomycetes two
strains (CM 22 and CT 9) showed enhanced activity and
hence these two strains were selected for further studies.
Extraction of antibacterial compound
Three solvents were used (n-butanol, chloroform and ethyl
acetate) for the extraction of antibacterial compound from
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c
390
P. Nithyanand & S.K. Pandian
Table 1. Antibacterial activity of coral-associated bacteria against pathogens
Zone of inhibition (mm) against various pathogens
Sample
Strain
Coral mucus
CM2
CM8
CM11
CM19
CM21
CM22
CT3
CT4
CT6
CT7
CT8
CT9
CT10
CT11
CT14
CT21
CT25
Coral tissue
Staphylococcus
aureus
8
Pseudomonas
aeruginosa
–
6
6
9
7
10
–
12
14
9
18
17
–
9
–
9
–
–
12
8
8
5
10
18
12
13
12
5
12
–
9
–
–
9
4
9
12
6
10
9
6
11
9
6
12
3
8
9
Aeromonas
hydrophila
–
–
Vibrio
parahaemolyticus
Vibrio
vulnificus
9
11
9
6
10
11
8
13
15
9
20
14
8
9
–
12
–
5
7
8
9
12
16
6
10
10
12
15
8
6
8
–
14
8
–, no activity.
the cell-free supernatant of actinomycetes isolates. Each
actinomycete was grown in culture and then the filtered
culture fluid was extracted with one of the three solvents.
The dried organic solvent extract was then subjected to
bioassay testing to determine whether any biological activity
could be successfully extracted from the culture fluid. In
each case, some biological activity was extractable with each
organic solvent, but chloroform extracted the greatest
amount of material with the greatest activity. As an example,
when the extractable solids of actinomycetes CM22 and
CM27 representing each organic solvent were subjected to
the plate assay, the chloroform extract showed the greatest
biological activity, followed closely by the n-butanol extract
(see Fig. S3). The extracts of the other actinomycetes showed
a similar profile (data not shown). It appears that the
bioactive component(s) mostly have a lipophilic profile
given their organic solvent preference (see Table S6).
Discussion
The culturable heterotrophic bacterial community of both
the mucus and tissue of the coral A. digitifera is composed
mainly of the bacterial groups Gammaproteobacteria, Firmicutes, Actinobacteria and members of the Cytophaga–Flavobacter/Flexibacter–Bacteroides group (see Fig. S4). When
comparing the distribution of bacterial species in the coral
mucus and the coral tissue we observed that some bacterial
species were specific to the coral mucus or the coral tissue,
and some bacterial species were commonly present in both
(Fig. 3).
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Published by Blackwell Publishing Ltd. All rights reserved
c
Fig. 3. Venn diagram showing the distribution of bacterial isolates (at
the species level) in the coral mucus and the coral tissue. The numbers in
parentheses indicate the number of isolates.
A culture-dependent study (Lampert et al., 2006)
reported the isolation of Actinobacteria for the first time from
the mucus of the coral Fungia scutaria. The present study
reports for the first time, the isolation of Actinobacteria from
both the coral mucus and the coral tissue using a culturedependent approach. Our results indicate that actinobacterial
genera are generally present in corals and we believe that
members of Actinobacteria are distributed both in the coral
mucus and in the coral tissue. A study on sponge-associated
bacteria from the Great Barrier Reef (Webster et al., 2001)
reports that among several media used, Marine Agar 2216
FEMS Microbiol Ecol 69 (2009) 384–394
391
Culturable bacterial diversity of A. digitifera
gave the highest number of morphotypes, resulting in the
isolation of several novel Actinobacteria members. Hence, in
the present study, Zobell Marine agar was used (nearly
equivalent in composition to that of Marine agar 2216) for
the isolation of heterotrophic bacteria, including Actinobacteria from the mucus and the tissue of the coral A. digitifera.
Apart from Firmicutes and Gammaproteobacteria, Actinobacteria were also found on both the mucus and the tissue of
the coral, exhibiting different polymorphic ARDRA ribotype groups (see Table S7, Figs S1 and S2).
The Actinobacteria members isolated from the coral
A. digitifera were different from the Actinobacteria members
present in the coral F. scutaria (see Fig. S5). The actinomycetes strains B. paraconglomeratum and B. linensis are
reported here for the first time in corals.
Gammaproteobacteria were dominated by V. parahaemolyticus and V. natrigens in the coral A. digitifera. Vibrios are
often associated with disease in corals (Rosenberg et al.,
2007). It is really intriguing to note the presence of vibrios in
healthy corals because it is also reported that some vibrios
may establish mutualistic partnerships with corals by providing nutrients and secondary metabolites (e.g. bacteriocins) to their hosts (Ritchie, 2006). This further supports the
statement of Shnit-Orland & Kushmaro (2009) that coralassociated bacteria are ubiquitous, as the same species of
bacteria are present in different species of corals that are also
geographically distinct. The same authors further state that
vibrios associated with the coral mucus produce antibacterial compounds against several pathogens, thereby protecting
the coral host against pathogens.
Firmicutes were the largest bacterial group in the coral
A. digitifera and were dominated by Bacillus genera. Bacillus
sp. may play a protective role in the coral host, as several
Bacillus sp. present in the mucus of corals exhibit antibacterial activity against pathogens (Shnit-Orland & Kushmaro,
2009). A few bacilli of marine origin have been reported to
produce unusual metabolites, different from those isolated
from terrestrial bacteria (Jensen & Fenical, 1994). Although
marine microorganisms have only recently become a target
for natural product drug discovery, it has become increasingly clear that Gram-positive strains are a rich source of
new structures that possess promising antimicrobial and
anticancer activities (Bernan et al., 2004; Blunt et al., 2006;
Kwon et al., 2006). Within the Firmicutes, strains of the
genus Bacillus, in particular, are common producers of
antimicrobial compounds. Approximately, 800 metabolites
with antibiotic activity, including the important group of
peptide antibiotics such as bacitracin, gramicidin and polymyxin B, are produced by various Bacillus sp. (Wiese et al.,
2009). A recent study shows that novel bacterial phylotypes
belonging to the Gram-positive group can be isolated using
low-nutrient media (Gontang et al., 2007). Devising such
intelligent strategies for culturing coral-associated bacteria
FEMS Microbiol Ecol 69 (2009) 384–394
might open a new avenue for natural product discovery; the
cultured novel strains can be subjected to taxonomic characterization, and their physiology, ecology and biotechnological potential explored.
On investigating the antibacterial activity of coral-associated bacteria, 37% of isolates were found to exhibit
antibacterial activity against different pathogens, which
concurs with a previous report stating that between 25%
and 70% of cultivable bacteria from the coral mucus display
antibacterial activity (Shnit-Orland & Kushmaro, 2009).
There were more bacterial isolates displaying antibacterial
activity (48%) in tissue than in mucus isolates (25%). The
presence of a larger number of bacteria with antibacterial
activity within the coral tissue than in the coral mucus can
be attributed to the fact that the there is greater competition
for a given niche in tissue than in the coral mucus. Bacillus
sp. and Vibrio sp. were the predominant genera in both the
mucus and tissue showing antibacterial activity. This agrees
with the report of Shnit-Orland & Kushmaro (2009) who
showed several Bacillus sp. and Vibrio sp. from mucus
exhibiting antibacterial activity. We found that several
species of Bacillus such as Bacillus pumilis, Bacillus firmus,
Bacillus horikishii and Bacillus endophyticus, and members of
the Vibrio genera such as V. parahaemolyticus and V. natrigens showed antibacterial activity against different pathogens. In concordance with Nissimov et al. (2009), both
mucus- and tissue-associated bacteria varied in strength
and spectra of activity against pathogenic bacteria and we
presume that the microbial interactions among mucus- and
tissue-associated bacteria might also be diverse. Both mucus- and tissue-associated bacteria showed antibacterial
activity against Gram-positive and Gram-negative bacteria,
which shows that the associated bacteria have a broad
spectrum of activity.
Of the 49 isolates screened, five (10%) actinomycetes
isolates exhibited antibacterial activity against various
pathogens. There was a greater number of actinomycetes in
tissue than in the coral mucus. Actinomycetes present in the
tissue were quite diverse, and we therefore envisage that the
tissue-associated actinomycetes might produce a diverse
array of antibacterial compounds. Contrary to our study,
Shnit-Orland & Kushmaro (2009) report that Actinobacteria
members, namely, Micrococcus sp. and Arthrobacter sp.,
isolated from three different corals did not show any
antibacterial activity against any of the tested pathogens.
Actinomycetales and Bacillales are responsible for almost
50% of the bioactive microbial metabolites discovered to
date, including many well-known antibiotics (Berdy, 2005).
Although bioactive compounds from marine actinomycetes
are being discovered, there are few reports of coral-associated actinomycetes that produce bioactive compounds.
The actinobacterial compound thiocoraline was isolated
from a soft coral (Lombo et al., 2006), but this is the first
2009 Federation of European Microbiological Societies
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c
392
study to report antibacterial activity in scleractinian-associated Actinobacteria. The isolated actinomycetes showed
antibacterial activity against both Gram-positive and Gramnegative pathogens. As the results of the extractable solids of
the actinomycetes show that the chloroform extract showed
the greatest biological activity, closely followed by the nbutanol extract, it appears that the bioactive components
have a mostly lipophilic profile given their organic solvent
preference (see Table S6). Several studies have reported the
isolation of novel marine actinomycetes (Jensen et al., 2005;
Lampert et al., 2006) producing bioactive compounds. Our
study suggests that coral actinomycetes might also be a
repository for many bioactive compounds and secondary
metabolites similar to sponges, and might also have a
symbiotic association with the coral hosts. As it has been
shown earlier that mucus from healthy coral harbours
bacteria capable of producing antibiotics (Ritchie, 2006),
we envisage that the coral mucus could be targeted for
isolation of actinomycetes with bioactive properties. Actinomycetes strains isolated in this study, such as Kocuria sp.,
Brachybacteruim sp. and Brevibacterium sp., have already
been reported to produce bioactive compounds; further
support for our proposal that coral-associated actinomycetes needs to be explored for bioactive compounds.
Acknowledgements
This work was supported by the Department of Biotechnology, Government of India; Grant no. BT/PR3987/AAQ/03/
198/2003. The authors gratefully acknowledge the computational and bioinformatics facility provided by the Alagappa
University Bioinformatics Infrastructure Facility (funded by
the Department of Biotechnology, Government of India;
Grant no. BT/BI/04/2001). In addition, the authors thank
their colleagues K. Balamurugan and B. Vignesh Kumar for
their help in epifluorescent microscopy. The financial
support provided to P.N. by the Department of Biotechnology, Government of India (Grant no. BT/PR3987/AAQ/03/
198/2003) in the form of a Research Fellowship is gratefully
acknowledged.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. ARDRA pattern dendrogram illustrating the relationship (% similarity) between different bacterial strains
isolated from the coral mucus.
Fig. S2. ARDRA pattern dendrogram illustrating the relationship (% similarity) between different bacterial strains
isolated from the coral tissue.
Fig. S3. Antibacterial activity of Actinobacteria members
CM22 and CT24 chloroform extracts against (a) Aeromonas
hydrophila, (b) Vibrio parahaemolyticus and (c) Vibrio
vulnificus.
Fig. S4. Comparative illustration of distribution of bacterial
groups (phylum) in the coral mucus and the coral tissue of
the Gulf of Mannar coral Acropora digitifera.
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394
Fig. S5. Radial phylogenetic tree showing the relationship
between Actinobacteria members isolated from Fungia scutaria (Fun) (Lampert et al., 2006) and Acropora digitifera
(CM, CT) (this study).
Table S1. 16S rRNA gene sequencing analysis of bacteria
isolated from the mucus of the coral Acropora digitifera
based on BLAST analysis.
Table S2. 16S rRNA gene sequencing analysis of bacteria
isolated from the tissue of the coral Acropora digitifera based
on BLAST analysis.
Table S3. Biochemical profile of the coral mucus isolates.
Table S4. Biochemical profile of the coral tissue isolates.
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P. Nithyanand & S.K. Pandian
Table S5. Antibiotic sensitivity profile of coral-associated
bacteria to various commercial antibiotics.
Table S6. Zone of inhibition (mm) of some test pathogens
used by representatives of coral-associated actinomycetes.
Table S7. Bacterial strains representative of ARDRA ribotype groups isolated from mucus and tissue of the coral
Acropora digitifera.
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
FEMS Microbiol Ecol 69 (2009) 384–394