Marine Pollution Bulletin 94 (2015) 217227

Contents lists available at ScienceDirect

Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Enterococcus species diversity and molecular characterization of

biomarker genes in Enterococcus faecalis in Port Blair Bay, Andaman
and Nicobar Islands, India
Balakrishnan Meena a,,1, Lawrance Anburajan a,1,2, Thadikamala Sathish a, Rangamaran Vijaya Raghavan b,
Dilip Kumar Jha b, Pitchiah Venkateshwaran a, Apurba Kumar Das a, Palaiya Sukumaran Dheenan a,
Nambali Valsalan Vinithkumar a, Gopal Dharani b, Ramalingam Kirubagaran b,
a
Andaman and Nicobar Centre for Ocean Science and Technology, Earth System Sciences Organization-National Institute of Ocean Technology (ESSO-NIOT), Port Blair 744 103,
Andaman and Nicobar Islands, India
b
Marine Biotechnology Division, Ocean Science and Technology for Islands Group, ESSO-NIOT, Ministry of Earth Sciences, Govt. of India, Chennai 600 100, India

a r t i c l e

i n f o

Article history:
Available online 20 March 2015
Keywords:
Enterococcus sp.
Biomarker genes
Box-whisker plots
Cluster analysis
Antibiotic resistance

a b s t r a c t
This study was performed to evaluate the abundance and diversity of Enterococcus sp. and the distribution of biomarker genes in Enterococcus faecalis in Port Blair Bay, Andaman and Nicobar Islands.
The Enterococcus sp. densities at the seven sampling stations were highly inuenced by tidal uctuations
and season. The distributions and diversities of species varied in the inner and outer regions of Port Blair
Bay. Among the 1816 total isolates, the occurrence of fecal Enterococcus was high (1.78  104 CFU/
100 mL) in Phoenix Bay. Moreover, 67.76% of the isolates were identied as Enterococcus, and the most
frequently identied species were E. hirae, E. avium and E. faecalis. Assessments of antibiotic resistance
and biomarker genes revealed the maximum occurrence in the Aberdeen Bay isolates. The most prevalent
biomarker genes observed in the E. faecalis isolates were gelE and asa1, whereas cyl was not found among
the isolates. In silico sequence analysis of biomarker genes of E. faecalis also revealed that they are
evolutionarily well conserved with those of earlier reports. Further, multivariate analysis distinguished
the JB, PB and OS stations from the other stations according to distinctive microbial densities and
compositions. In addition, the Shannon-Wiener diversity indices and box-whisker plots further facilitated
and supported the multivariate results.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Coastal waters generally contain both pathogenic and nonpathogenic microbes derived from runoff, sewage, industrial efuent, agricultural activities, wild life and indigenous microorganisms.
These pathogens can be hazardous to the health of bathers and
consumers when an infective dose colonizes a suitable growth site
of the body and leads to disease (Elmanama et al., 2005; WHO,
Corresponding authors at: Andaman and Nicobar Centre for Ocean Science and
Technology, ESSO-NIOT, Dollygunj, Port Blair 744 103, Andaman and Nicobar
Islands, India. Tel.: +91 96795 58081; fax: +91 3192 225089 (B. Meena). Marine
Biotechnology Division, ESSO-National Institute of Ocean Technology (ESSO-NIOT),
Ministry of Earth Sciences, Government of India, Pallikaranai, Chennai 600 100.
Tel: +91 44 66783418; fax: +91 44 66783423 (R. Kirubagaran).
E-mail addresses: bmeena79@yahoo.com (B. Meena), anburajanl@yahoo.co.in
(L. Anburajan), head.mbt@niot.res.in (R. Kirubagaran).
1
These authors contributed equally to this work.
2
Tel.: +91 96795 50065.
http://dx.doi.org/10.1016/j.marpolbul.2015.02.027
0025-326X/ 2015 Elsevier Ltd. All rights reserved.

1998). Enterococcus bacteria are part of the normal intestinal ora

of animals and humans, and they are released into the environment
directly or via sewage outlets (Farrel et al., 2003). Studies conducted
by the Environmental Protection Agency (EPA) to determine the
correlation between bacterial indicators and digestive system
illness have reported that the most prominent indicators for health
risk from recreational water contact are E. coli and Enterococcus sp.
in fresh and salt waters, respectively (USEPA, 2003).
In the last few decades, Enterococcus sp. has emerged as the
most important causative organism of nosocomial infection and
as a specic threat to public health (Heymann, 2006). The distribution of infectious Enterococcus sp. in the environment via
water may cause an increase in the prevalence of these strains in
humans (Irani et al., 2011). Enterococcus faecalis and Enterococcus
faecium are important opportunistic pathogens (Harwood et al.,
2004). Enterococci are able to grow at a temperature of 45 C and
pH 9.6 in 6.5% NaCl broth and to survive at 60 C for 30 min
(Sherman, 1937). Among the enterococci, E. faecalis can cope with

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unfavorable conditions by maintaining cell viability under starvation for extended periods and become resistant to UV radiation,
heat, and differing/high concentrations of sodium hypochlorite,
hydrogen peroxide, ethanol, and acid (Hartke et al., 1998). Major
factors for the pathogenesis of enterococci include their resistance
to a wide range of antibiotics and virulence factors. Genes encoding
virulence factors include asa1, esp, hyl, gelE, ef0591, aadE, pbp4 and
cylA (Kuzucu et al., 2005).
The coastal waters of the Andaman and Nicobar (A & N) Islands
are pristine compared with those of the highly populated mainland
India (Sahu et al., 2013). The diversity and distribution of
Enterococcus sp. in the environment and coastal waters are not
reective of the pollution rate; however, the population density
and distribution of virulence factors and the pathogenicity of this
indicator organism can dene the microbial pollution rate and
the source of possible contamination in a particular zone. The
objectives of this study were as follows: (i) to determine the seasonal impact on population density and the distribution of
Enterococcus sp. in Port Blair Bay; (ii) to evaluate the species diversity and dominance of Enterococcus sp. among selected stations;
and (iii) to examine the antibiotic susceptibility and distribution
of biomarker genes in E. faecalis during various seasons. This study
is the rst to attempt to correlate the microbial pollution trend

with the diversity of biomarker genes in E. faecalis from Port

Blair Bay, A & N Islands.
2. Methods
2.1. Study area
Port Blair Bay is located in South Andaman Island and extends
from east to west and southwest, opening to the Andaman Sea
on the eastern side (Fig. 1). This bay area is inuenced by the
anthropogenic activities of Port Blair city, where the majority of
the population of the A & N Islands is congregated. In the present
study, seven stations were selected within Port Blair Bay, from
the inner part of the bay to the Open Sea station, including Flat
Bay (FB), Minnie Bay (MB), Junglighat Bay (JB), Haddo Harbor
(HH), Phoenix Bay (PB), Aberdeen Bay (AB) and Open Sea (OS)
(Fig. 1).
2.2. Sample collection and analysis of microbial and physicochemical
characteristics
Sampling was performed quarterly according to seasonal preference (February, May, August and November) from seven

Fig. 1. Sampling stations with drainage points along Port Blair Bay, Andaman and Nicobar Islands.

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B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

stations during low and high tides in 2013. Samples were collected
onboard at 0.3 m below the surface of the water using a GO-FLO
water sampler, and they were then transferred to sterile screwcap bottles according to the sampling procedures described in
the USEPA microbiology methods manual (USEPA, 2003). The samples were mixed thoroughly and ltered through sterile 0.22 lm
membrane lters (Millipore Corporation, USA), which were then
placed on pre-prepared M-Enterococcus agar plates (HiMedia,
India). After incubation overnight at 37 C, bacterial colonies with
dark brown colours were identied as presumptive Enterococcus
species. The results were expressed as colony forming units
(CFU)/100 mL for each water sample.
Temperature and pH were measured onboard using a calibrated
thermometer with 0.1 C accuracy (Brannan, UK) and a pH meter
(Thermo Orion 420 A plus, USA), respectively. The level of total suspended solids (TSS) was determined by ltering 1 L of seawater
through pre-dried and pre-weighed 0.45 lm pore-size lter paper
(Millipore). Dissolved oxygen (DO) at the sampling stations was
measured by Winklers method (Strickland and Parsons, 1968).

The biomarker genes asa1, esp, hyl, gelE, ef0591, aadE, pbp4 and
cylA were PCR-amplied using gene-specic primers (Table 1). PCR
was performed in a 25 lL reaction mixture that contained 50 ng of
genomic DNA, 0.5 lM of each primer, 200 lM of each dNTP (MBI
Fermentas), 1.25 U of Taq DNA polymerase (MBI Fermentas),
1  Taq buffer, 2.0 mM MgCl2 and autoclaved Millipore water.
Amplication was performed in a Mastercycler (Eppendorf,
Germany) under the following conditions: initial denaturation at
94 C for 3 min followed by 30 repeated cycles of 94 C for 30 s,
51 C for 1 min and 72 C for 2 min, and a nal extension at 72 C
for 10 min. PCR amplicons were analyzed on a 1.5% agarose gel
along with a DNA molecular weight marker (MBI Fermentas) and
documented with a gel documentation system (UVP BioSpectrum
Imaging system, USA). Each PCR assay was performed with
E. faecalis ATCC19433 as a positive control. Positive amplicons as
determined by size were puried using a MinElute Gel
Purication Kit (Qiagen, Germany).

2.3. Identication of Enterococcus sp.

2.7. Molecular cloning and sequencing of biomarker genes

Presumptive Enterococcus colonies with typical dark brown colour on the membranes were selected for further characterization.
The colonies were cultivated in Trypticase soy broth at 37 C and
identied up to the species level using a biochemical identication
key (Albert and Anicet, 1999). Those isolates that exhibited a positive reaction by pyrrolidonyl arylamidase assay (PYR), esculin
hydrolysis, growth at 45 C, tolerance to 6.5% NaCl and catalase
negative were identied as Enterococcus sp. (American Society for
Microbiology, 2003).

Gel-eluted PCR amplicons of the biomarker genes were cloned

in a T/A cloning vector according to the manufacturers
instructions provided in an InsTAclone PCR Cloning Kit (MBI
Fermentas). pTZ57R/T-biomarker gene constructs were transformed into competent E. coli JM109 (recA1, endA1, gyrA96, thi-1,
hsdR17 (rKmk+), e14(mcrA), supE44, relA1, and D(lac-proAB)/F0
(traD36, proAB+, lac Iq, lacZ DM15), plated on LuriaBertani (LB)
agar containing 100 lg/mL ampicillin, 50 lM isopropyl-b-Dthiogalactoside (IPTG) and 80 lg/mL X-gal and incubated overnight at 37 C. White colonies were selected for PCR amplication
with vector primers M13f-M13r (MBI Fermentas), and clones with
the correct insert as determined by size were sequenced with an
ABI PRISM 377 genetic analyzer (Applied Biosystems).

2.4. Characterization and molecular identication of E. faecalis

Genomic DNA was extracted following the method described by
Ausubel et al. (1994). The universal 16S rRNA eubacterial primers
16S f (50 -ACTCAAAGGAATTGACGG-30 ) and 16S r (50 -TACGGCTA
CCTTGTTACGACTT-30 ) were used for PCR amplication, and the
amplicon was cloned in a T/A cloning vector, according to the
manufacturers instructions provided in the InsTAclone PCR
Cloning Kit (MBI Fermentas, USA). Sequencing was performed
using an ABI PRISM 377 genetic analyzer (Applied Biosystems,
USA) by the dye termination method. The acquired 16S rRNA
sequences were used in a homology search of the available
sequences in GenBank using the BLAST program of NCBI (http://
www.ncbi.nlm.nih.gov) to determine pairwise identities. Multiple
sequence alignments of the sequences were performed using
CLUSTAL-X version 1.81 program, and a phylogenetic tree was constructed with MEGA v5.0 using the neighbor-joining method.
2.5. Antibiotic susceptibility test
The antibiotic susceptibilities of the isolates were assessed
using the disc diffusion method on Muller-Hinton agar (HiMedia,
India), according to the Clinical and Laboratory Standards
Institute recommendations (CLSI, 2009). Antibiotic discs of ampicillin (10 lg), gentamycin (10 lg), clindamycin (2 lg), tetracycline
(30 lg), chloramphenicol (30 lg), streptomycin (30 lg), vancomycin (30 lg) and erythromycin (15 lg) were placed on agar
plates seeded with E. faecalis isolates and incubated overnight at
37 C. The diameter of the antibiotic inhibition zone was measured
in millimeters and recorded as follows, according to CLSI M02-A10:
susceptible (S), intermediate resistant (IR) or resistant (R).
E. faecalis ATCC19433 and Staphylococcus aureus MTCC3160 were
used as positive and negative control strains, respectively.

2.6. PCR detection of biomarker genes

2.8. In silico sequence analysis of biomarker genes

The obtained nucleotide sequences were compared with database sequences using the BLAST program of NCBI (http://www.
ncbi.nlm.nih.gov) and were aligned and clustered using CLUSTALX program. The output alignments were imported into GeneDoc
program (http://www.psc.edu/biomed/genedoc/) and BioEdit
version 7.05 (www.mbio.ncsu.edu/BioEdit/) to calculate the percent identities among the nucleotide and amino acid sequences.
A phylogenetic tree was constructed using MEGA version 5.0 by
the neighbor-joining method.
Table 1
Gene-specic primers for the biomarker genes.
Gene

Primer sequences 50 -30

References

asaI

GCACGCTATTACGAACTATGA
TAAGAAAGAACATCACCACGA
AGATTTCATCTTTGATTCTTGG
AATTGATTCTTTAGCATCTGG
ACAGAAGAGCTGCAGGAAATG
GACTGACGTCCAAGTTTCCAA
TATGACAATGCTTTTTGGGAT
AGATGCACCCGAAATAATATA
ACTCGGGGATTGATAGGC
GCTGCTAAAGCTGCGCTT
AGAGGGACGATCAGATGA
ATTCCAATTGACGATTCA
ATGGAACGAAGCAATCGT
CTTTGATATTGGCTGTT
TGATTTGCTGGTTACGGTGAC
CGCTATGTTCTCTTGCTTTTG

This study

esp
hyl
gelE
cylA
ef0591
pbp4
aadE

This study
This study
This study
This study
This study
This study
This study

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B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

2.9. Statistical analysis

Multivariate analyses were carried out using PRIMER (Clarke
and Warwick, 1994; Clarke and Gorley, 2006). Non-parametric
agglomerative hierarchical cluster analysis (AHCA) was performed
to classify the assemblages/clusters (Clarke and Warwick, 1994).
Non-metric multidimensional scaling (MDS) ordinations based on
Bray-Curtis similarities of species abundance data were produced
to provide a visual representation in a two-dimensional plot of
the relative similarities in species composition and abundance at
the different sampling stations. Further, SPSS software (version
18.0) was used for box-whisker plots, and the diversity index of
Enterococcus sp. in Port Blair Bay at the different stations was
analyzed using PAST program (Ryan et al., 1995).
3. Results
3.1. Evaluation of physicochemical parameters at sampling stations
The atmospheric and surface water temperatures varied from
27.1 C to 32.2 C and from 28.2 C to 30.3 C, respectively. The
salinity and pH ranged from 29.75 to 33.08 PSU and from 7.87 to
8.38 units, respectively. The relative humidity of the ambient
atmosphere ranged from 50% to 80%. The concentration of total
suspended solids (TSS) widely uctuated from 9.3 to 53.9 mg/L
and the DO ranged from 4.82 to 7.38 mg/L. The annual rainfall during the sampling seasons of this study amounted to approximately
3405 mm and was prolonged from May to December of 2013.
Maximum precipitation was recorded during south-west monsoon
(May to October) and north-east monsoon (November to
December).
3.2. Enterococcus population
The results clearly indicate the impact of tidal inuences and
seasonal climatic changes on the Enterococcus count at the seven
stations in Port Blair Bay during the sampling period (Fig. 2). A trend
of increasing Enterococcus counts was observed in the bays, and a
reduction in number was observed toward the open sea.
However, the Enterococcus counts were substantially increased at
the PB, JB and AB stations. Throughout the seasons, the low-tide
samples had a higher Enterococcus count compared with the hightide samples, and the fecal indicator bacterial counts highly uctuated according to the season and sampling site (Fig. 2). The maximal
indicator bacterial density was observed in November of 2013 at PB,
and the minimum density was recorded in February of 2013 at AB.
FB, MB, PB, AB and OS showed the highest indicator bacterial
counts of 1.62  103, 2.00  103, 1.78  104, 6.83  102 and
3.40  101 CFU/100 mL, respectively, during November of 2013
and minimum counts of 6.67  101, 1.67  101, 3.20  102,
1.33  101 and 6.67  100 CFU/100 mL, respectively, during
August of 2013. At JB and HH, the maximum (3.43  103 and
9.83  102 CFU/100 mL, respectively) counts were observed in
May of 2013, and minimum counts were detected (3.67  102
and 3.33  101 CFU/100 mL, respectively) in August of 2013.
Among the sampling sites, the highest Enterococcus counts were
recorded at the PB and JB stations. The Enterococcus count was
found to exceed the USEPA limits at all other study stations, except
OS.
Cluster analysis (Fig. 3a) showed that the stations were grouped
into two major clusters with 40% similarity level based on
Enterococcus sp. density and species composition. The stations OS
and HH formed a single cluster. In terms of bacterial contamination
from anthropogenic sources, the OS station stood apart from the
other stations, as shown by MDS (Fig. 3b). However, it was closest
to HH because both of these stations are located in the outer region

of Port Blair Bay. The low bacterial contamination at this station

can be attributed to frequent water circulation and its position in
the bay compared with the other stations. Further, OS and HH
had similar density counts of 17 and 53, respectively. PB and JB
had higher levels of bacterial contamination and formed a different
cluster, showing over 90% similarity by CA. Aberdeen Bay formed a
cluster with MB, which may have been due to the similarities of
their counts, which were 153 and 150, respectively. Further, FB
formed a separate cluster due to the abundant bacterial count at
that station.
A box-plot of variations in microbial density (Enterococcus sp.)
in Port Blair Bay according to station is presented in Fig. 4a. This
graph shows that the microbial counts of the JB station remained
consistent, irrespective of the tide or season, because of an adjacent major sh landing center as well as sewage discharge lines
located in this region. However, the PB station showed higher
bacterial counts mainly during low tide because of untreated
municipal sewage discharge, whereas tidal ushing reduced the
count during high tide. Further, the OS station, which continuously
receives oceanic water, showed a negligible bacterial count, suggestive of a healthy environment and the absence of contamination
toward the open sea. No conspicuous differences were revealed for
any of the other stations by the boxplot graphs.
3.3. Enterococcus species assemblages
A total of 1816 presumptive Enterococcus sp. were identied at
all sampling stations. Of the 1816 isolates, 69% (1261/1816) were
Enterococcus sp., 30.56% (555/1816) were non-Enterococcus sp.,
and 1.9% (34/1816) were unidentied (Table 2a). E. avium and E.
hirae were predominant throughout the marine ecosystem of
Port Blair Bay. However, E. durans, E. faecium, E. mundii, E. saccharolyticus and E. casseliavus were also frequently recorded at the
different stations. Nevertheless, E. faecalis was the most frequently
identied species at the AB station.
Shannon diversity indices were predicted to study microbial
species richness, and the results are presented in Table 2b.
Species richness (D) was high at JB (18) and MB (18), indicating
the presence of all Enterococcus sp. However, the least richness
was recorded at the OS station (3) due to minimal fecal contamination. Among all of the sampling sites, the highest D value
(0.481) was observed at OS, which may have been due to the dominance of E. hirae over other species at that station. Shannon
Wieners diversity index was high at MB (H0 = 2.361), denoting
the maximum diversity of Enterococcus sp. compared with OS
(H0 = 0.893). A greater amount of species diversity was observed
at MB. However, the individuals were distributed more evenly at
OS (J0 = 0.814), which may have been due to the stable water
environment. Further, PB and JB showed maximum counts of 371
and 347 due to high levels of fecal contamination.
3.4. Species verication of E. faecalis
The 16S rRNA sequences generated in this study were deposited
in GenBank (GenBank accession nos. KJ726742, KJ726743,
KJ726744, and KJ726745). BLAST and phylogenetic analyses
revealed that the deduced nucleotide sequences were highly
homologous (99%) with the reported 16S rRNA sequences of
E. faecalis (KC478511, FJ608830, JQ340031 and HM057977). Based
on phylogenetic analysis, the isolates were identied as E. faecalis.
3.5. Antibiotic susceptibility test
E. faecalis isolates were subjected to an antimicrobial susceptibility test. A total of 50.89% (57/112) of the E. faecalis isolates
were susceptible to ampicillin, and 62.5% (70/112) were resistant

B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

221

Fig. 2. Seasonal and tidal variations in the Enterococcus populations at sampling stations in Port Blair Bay during the year 2013.

to streptomycin. No antibiotic resistance was observed for gentamycin, tetracycline, clindamycin, penicillin G or erythromycin.
However, no antibiotic-resistant E. faecalis isolates were detected
at FB and OS, which are located at the far ends of Port Blair Bay.
A maximum of 97.2% isolates were resistant to ampicillin and
streptomycin at the AB station. At the JB, PB, MB, and HH stations,
small numbers of isolates showed resistance to ampicillin and
streptomycin. Multi-drug-resistant E. faecalis was not detected at

any of the sampling stations. Hence, the waters of Port Blair Bay
are much safer compared with those of mainland India.
3.6. Distribution of biomarker genes
The biomarker genes (asa1, esp, hyl, gelE, ef0591, aadE, pbp4 and
cylA) of E. faecalis encode polynucleotides of 713, 510, 276, 213, 844,
284, 640 and 517 bp in length, respectively (Fig. 5a). Of the 112 E.

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B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

(a)

(b)

Fig. 3. (a) Bray-Curtis cluster analysis of Enterococcus sp. density at different stations in Port Blair Bay as determined by group average linkage. (b) Bray-Curtis similarity
based on Enterococcus sp. abundance.

faecalis isolates, 54.46% possessed the gelE gene, 45.54% had the asaI
gene, 20.54% had the esp gene, 4% had the hyl gene, 16.96% possessed the ef0591 gene, 50.89% had the pbp4 gene and 62.5% had
the aadE gene (Table 3). Among all of the sampling stations, the
maximum number of virulence determinants was found at
Aberdeen Bay. The major virulent gene, cylA, was not detected in
any of the isolates throughout the sampling seasons. The gelE gene
was determined to be the dominant biomarker gene among the isolates. A total of 12 isolates possessed gelE and asa1, 5 had gelE, asa1
and esp, and one was detected with gelE, asa1, esp, and hyl. Among
the Enterococcus sp., biomarker genes were detected only in E. faecalis, and other dominant species, such as E. avium, E. hirae, E. faecium, and E. pseudoavium were not found to possess these genes.
3.7. Sequence analysis of key biomarker genes
In silico sequence analysis of the biomarker genes revealed signicant levels of similarity between the sequences obtained in this
study and those determined by previous studies that were available in the sequence database. The penicillin-binding protein and
Enterococcus surface protein genes showed less signicant divergence from reported sequences from other species (Fig. 5b and
c). Sequence analysis of the aadE and asa1 genes revealed signicant homology with previously reported sequences; hence,

insignicant variation was observed in the phylogenetic tree

(Fig. 5d and f). For the gelE gene, Enterococcus sp. alone formed a
cluster that was separate from other sequences.
4. Discussion
E. coli and enterococci are generally regarded as microbial indicators of water quality (Davis et al., 2005). Several studies of recreational and drinking water have suggested that enterococci are
more relevant indicators than fecal coliforms (Kinzelman et al.,
2003). Port Blair is the capital city of the A & N Islands, and it is also
a major center of anthropogenic activity. An earlier study performed by Nallathambi et al. (2002) examined health indicators
in Port Blair Bay waters. The current study was the rst fecal pollution trend analysis of Port Blair Bay with respect to biomarkers and
antibiotic resistance patterns of E. faecalis isolated during the year
2013. We demonstrated the tidal and seasonal inuences on the
species distribution of Enterococcus isolates using statistical tools.
Our ndings may provide insights into the possible rises in infectious disease and gastrointestinal infections in individuals that
are linked with the seafood chain (Rees, 1993).
Higher Enterococcus counts were found in low-tide samples
compared with high-tide samples during all seasons. At the Open
Sea station, the tidal effect was diminished compared with the

B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

223

(a)

(b)

Fig. 4. (a) Variations in Enterococcus sp. at each station in Port Blair Bay. For each box-plot, the central point represents the median, the box shows the interval between the
25th and 75th percentiles, and the whisker indicates the range. The outlier values are shown as dots. (b) Heat map depicting the abundance of Enterococcus across sampling
locations with single linkage clustering.

other stations of Port Blair Bay, and the Enterococcus counts were
recorded minimum in this station. There are many variables that
affect indicator bacterial densities, including tidal stage, wind
direction and speed, water temperature, season and sample location (Ferretti et al., 2011). Tidal current also plays an important
role in regulating water quality in semi-closed coastal bays
(Dheenan et al., 2014). Sahu et al. (2013) have previously reported
the presence of a higher organic load during low tide compared
with high tide in Port Blair Bay. The maximum Enterococcus count
was recorded during November of 2013, and the minimum count
was observed in August of 2013. Tropical cyclone Lehar produced
heavy rainfall, affecting the South Andaman region during
November of 2013, and it may have been the reason for the
increases in fecal contamination and the Enterococcus population
during this season. Increased Enterococcus counts in coastal waters
after heavy rainfall due to soil resuspension and seasonal inuence
has been previously reported by Janelidze et al. (2011). The maximum Enterococcus counts were recorded at PB and JB at all seasons

in the current study. These two stations receive more sewage output in Port Blair Bay and have been reported to have higher organic
loads by Sahu et al. (2013), which may have been the reason for the
increased counts of indicator bacteria in these regions.
Cluster analysis showed that JB and PB stations formed a single
cluster. These stations were critically affected by anthropogenic
factors, such as domestic waste, untreated municipal sewage and
waste from sh landing centers. Further, the residence period of
tide-inuenced water is higher at JB (a major sh landing center)
(Sahu et al., 2013) and becomes worse due to untreated municipal
sewage outfall and sh trawler activities. Similarly, strong anthropogenic inuences of ferries and sewage outfall were observed at
PB (a major inter-island ferry service providing harbor). The stations OS and HH formed one cluster, and it is known that OS is a
pristine environment compared with other stations (Sahu et al.,
2013) in Port Blair Bay. However, MDS revealed that OS remained
clustered near HH, which is because both of these stations are
located in the outer region of the bay. The low microbial

B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

1
5
5
5
7
8
3

34

0
1
1
0
1
2
0

Table 2b
Diversity of Enterococcus sp. in Port Blair Bay.
Station*

AB

FB

HH

JB

MB

OS

PB

Richness (S)
Individuals
Dominance (D)
Shannon diversity
index (H)
Evenness (J)

16
153
0.177
2.079

15
170
0.226
1.856

10
53
0.189
1.888

18
347
0.175
2.145

18
150
0.143
2.361

3
17
0.481
0.893

17
371
0.19
2.074

0.5

0.426

0.66

0.474

0.588

0.814

0.468

27
23
3
21
10
112
34
21
10
38
19
362
58
338
34
112
1816
Total

1261

273
245
462
106
437
252
41

170
150
347
53
371
153
17

9
11
28
9
19
36
0

2
3
11
1
12
5
0

49
32
87
10
111
38
11

11
8
21
0
13
5
0

60
41
103
17
103
35
3

3
3
6
1
6
0
0

12
5
7
1
11
2
0

1
3
2
0
3
1
0

1
4
5
0
9
2
0

6
5
8
3
8
4
0

12
12
32
5
46
5
0

0
4
2
0
2
2
0

1
4
6
0
8
2
0

0
2
1
0
0
0
0

1
5
7
0
7
3
0

1
2
15
1
5
3
0

AB: Aberdeen Bay; FB: Flat Bay; HH: Haddo Harbor; JB: Junglighat Bay; MB:
Minnie Bay; OS: Open Sea; and PB: Phoenix Bay.

FB
MB
JB
HH
PB
AB
OS

E.
hirae
E.
faecium
E.
faecalis

Enterococcus sp. distribution

Positive
isolates
No. of
isolates
Station

Table 2a
Diversity of Enterococcus sp. during the year 2013.

E.
asini

E.
avium

E.
casseliavus

E.
durans

E.
gallinarum

E.
malodoratus

E.
mundii

E.
pseudoavium

E.
solitarius

E.
rafnosus

E.
cecorum

E.
dispar

E.
saccharolyticus

E.
avescens

Enterococcus
sp.

224

contamination at this station can be attributed to frequent water

circulation and its position in the bay compared with the other stations. Further, OS and HH had similar density counts of 17 and 53,
respectively. However, pollutants in the bay are diluted due to its
large area (nearly 30 km2) and high tidal amplitude (2.4 m), which
rapidly diminish the effects of contaminants (Jha et al., 2014).
Nevertheless, the OS station was found to be a healthy environment due to good water mixing and had the lowest bacterial count
according to the USEPA standards. The bacterial populations in the
bays were higher than those in the open sea because coastal
human habitation depends more on these water bodies for shing
and navigation purposes. Domestic waste is also released directly
into the bay, ultimately diminishing water quality and increasing
the bacterial load (Dunn et al., 2012).
The identied Enterococcus sp. were speciated to determine the
sources and ecology of these organisms in the marine environment.
Of the 1816 isolates, the predominant species identied in the
water samples at all seven study stations were as follows:
E. avium, E. hirae, E. faecalis and E. pseudoavium. Species distribution
analysis elucidated the pollution source of this particular site.
E. faecalis and E. faecium are predominant in the intestinal microora of humans and animals and are considered to be opportunistic
pathogens. E. hirae and E. avium are found in animal microora, and
E. gallinarum, E. casseliavus and E. mundii are associated with
plants and soil (Moore et al., 2008). In this study, the abundance
and distribution of Enterococcus sp. were found to be high at the
JB and PB stations. Biodiversity parameters, such as richness, H
index and population size, were also signicantly elevated at these
stations. E. hirae was the dominant species recorded at these stations, followed by E. gallinarum, E. casseliavus and E. mundii.
These results clearly verify that natural sources, such as plants
and land runoff during heavy rainfall, were the major sources of
pollution at these stations. The prevalences of E. gallinarum,
E. casseliavus and E. mundii at beaches and in runoff samples with
soil, plants and animals have been previously reported by Stern
et al. (1994).
At FB and MB, the dominance and diversity of Enterococcus sp.
widely uctuated during all seasons. These sites are located at
the inner most part of Port Blair Bay and receive freshwater input
and terrestrial runoff from adjacent areas (Sahu et al., 2013). At the
HH and OS stations, the Enterococcus sp. counts and diversity were
lower, and these bays are situated in the outer region of Port Blair
Bay, where depth and mixing are high. The negative richness (J)
value and positive D value indicate that anthropogenic activities
had the least inuence at these stations. At the AB station, the
Enterococcus sp. counts were lower compared with the other inner
bay sites. However, E. faecalis was found to be the dominant
species throughout the seasons, with a low D value observed near
the MB station. The dominance of E. faecalis at this study area
clearly indicates the presence of human fecal pollution. These
results suggest that anthropogenic inuences at the AB station
differed from those at the other bays due to increased recreational
water games and untreated municipal waste outlets. The higher
percentages of E. faecalis and E. faecium in the environmental and

B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

b c

h I

225

l m

(a)

(b)

(c)

(d)

(e)

(f)
Fig. 5. (a) Agarose gel electrophoresis of the PCR amplicons of biomarker genes: lanes a, d, g, h, j, and m; 1 kb DNA ladder; lane b, gelE amplicon (213 bp); lane c, asaI amplicon
(713 bp); lane e, esp amplicon (510 bp); lane f, ef0591 amplicon (844 bp); lane i, aadE amplicon (284 bp); lane k, hyl amplicon (276 bp); and lane l, pbp4 amplicon (640 bp). (b)
Phylogenetic analysis of the major biomarker genes in E. faecalis, including (b) pbp4, (c) esp, (d) aadE, (e) asa1, and (f) gelE.

226

B. Meena et al. / Marine Pollution Bulletin 94 (2015) 217227

Table 3
Prevalence of biomarker genes in E. faecalis during the year 2013 in Port Blair Bay.
Station

FB
MB
JB
HH
PB
AB
OS
Total

No. of
isolates

Biomarker genes
gelE

asaI

esp

hyl

cylA

ef0591

pbp4

aadE

9
11
28
9
19
36
0

0
2
16
1
11
31
0

0
0
13
1
10
27
0

0
0
2
0
4
17
0

0
0
1
0
0
4
0

0
0
0
0
0
0
0

0
0
4
0
1
14
0

4
10
1
7
35

4
17
3
11
35

112

61

51

23

19

57

70

wastewater samples indicated the presence of human and animal

feces.
The misuse or abuse of antibiotics is the primary reason for the
emergence of antibiotic resistance in bacteria. Antibiotics are
excreted from humans and animals due to poor absorption in the
intestines and reduced degradation in the human body, and they
are discharged into sewage. Thus, animal waste plays a major role
in the distribution of antibiotic-resistant bacteria in the environment (Tao et al., 2010). Of the 112 E. faecalis isolates evaluated,
the distribution of biomarker genes and antibiotic resistance were
the greatest at the AB station, indicating that human and animal
fecal contamination must be a pollution source. Among the biomarker genes, gelE was the most dominant virulence factor
detected among the isolates. The presence of gelE in clinical and
environmental E. faecalis isolates has been previously recorded
(Vankerckhoven et al., 2004). In silico sequence analysis of biomarker genes in E. faecalis in this study revealed that they are comparatively well conserved at both the nucleotide and amino acid levels
with those of previous reports. The exact roles of environmental
and clinical E. faecalis isolates in human disease are not clear
(Moellering, 1992).
5. Conclusion
In this study, a seasonal assessment of water quality in terms of
the presence of fecal indicator bacteria and their biomarker genes
was performed at seven sampling stations in Port Blair Bay. Based
on the results, the monitoring of coastal waters for enterococci and
the determination of the prevalences of virulence and antibiotic
resistance factors are highly recommended to combat microbial
pollution and prevent potential waterborne diseases. Except the
Open Sea station, the Enterococcus counts at all six stations were
above the acceptable limit reported by the USEPA. In addition, cluster and PC analyses revealed that the Enterococcus counts and
diversity were high at the JB and PB stations. The assessment of
species diversity of the inner bays revealed that the major pollution sources were land runoff, soil leaching, sewage and biogenous
sources. Anthropogenic perturbations at the AB station were
demonstrated by the maximal E. faecalis counts and prevalence
of biomarker genes. The detection of pathogenicity and virulence
factors in E. faecalis at monitoring stations is considered to be an
effective tool for detecting pollution and its sources, in order to
prevent waterborne diseases. Hence, the presence of antibiotic
resistance and virulent enterococci in coastal waters of populous
nations demands improved surveillance for risk assessment and
preventive strategies for the protection of public health.
Acknowledgements
The authors gratefully acknowledge the nancial support provided by the Earth System Sciences Organization, Ministry of Earth
Sciences, Government of India, New Delhi for conducting the survey

and research. The authors are thankful to Dr. M. A. Atmanand,

Director, ESSO-National Institute of Ocean Technology (NIOT),
Chennai for providing support and encouragement to perform this
research. The authors are profoundly thankful to Dr. M.
Vijayakumaran for his critical comments and suggestions for
improving this manuscript and to Dr. Toms C. Joseph, Senior
Scientist, Central Institute of Fisheries Technology (CIFT), Kochi,
India for performing DNA sequencing and in silico sequence analysis.

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