International Journal of Food Microbiology 173 (2014) 1420

Contents lists available at ScienceDirect

International Journal of Food Microbiology

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

Seasonal dynamics and diversity of bacteria in retail oyster tissues

Dapeng Wang , Qian Zhang, Yan Cui, Xianming Shi
MOST-USDA Joint Research Center for Food Safety & Bor Luh Food Safety Center, School of Agriculture and Biology & State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University,
Shanghai 200240, PR China

a r t i c l e

i n f o

Article history:
Received 14 October 2013
Received in revised form 6 December 2013
Accepted 8 December 2013
Available online 19 December 2013
Keywords:
Retail oyster
Tissues
DGGE
Seasonal dynamics
Bacterial diversity

a b s t r a c t
Oysters are one of the important vehicles for the transfer of foodborne pathogens. It was reported that bacteria
could be bio-accumulated mainly in the gills and digestive glands. In articially treated oysters, bacterial
communities have been investigated by culture-independent methods after harvest. However, little information
is available on the seasonal dynamics of bacterial accumulation in retail oyster tissues. In this study, retail oysters
were collected from local market in different seasons. The seasonal dynamics and diversity of bacteria in oyster
tissues, including the gills, digestive glands and residual tissues, were analyzed by denaturing gradient gel
electrophoresis (DGGE). It was interesting that the highest bacterial diversity appeared in the Fall season, not
in summer. Our results indicated that Proteobacteria was the predominant member (23/46) in oyster tissues.
Our results also suggested that bacterial diversity in gills was higher than that in digestive glands and other tissues. In addition, not all the bacteria collected from surrounding water by gills were transferred to digestive
glands. On the other hand, few bacteria were found in oyster tissues except in the gills. Therefore, the gills
could be the best candidate target tissue for monitoring of pathogenic bacteria either to human or to oyster.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Oysters are lter feeders. Because of this character, oysters bioaccumulate almost all kinds of particles, which exist in surrounding
water, including bacteria and viruses. Some of them can cause infections
with serious consequences either in human consumers or in oysters
(Green, Barnes, 2010; Romero et al., 2002; Wang and Shi, 2011). Therefore, most literatures focused on the detection of those pathogens in
oysters (Green and Barnes, 2010; Liu et al., 2012; Nordstrom et al.,
2007).
The microora present in oysters depends on the environmental
conditions (Kenneth et al., 2009; Prapaiwong et al., 2009).After harvest,
bacterial communities, including pathogenic bacteria, in oyster would
be changed dramatically even during refrigeration (Chen et al., 2013;
Fernandez-Piquer et al., 2012; Gooch et al., 2002). It was reported that
the total aerobic bacteria counts were over 107 CFU/g in autumn after
1 week storage at 4 C (Prapaiwong et al., 2009). Therefore, the dynamics of bacterial diversity was a concerned issue.
In general, culture-based assay was standard for detection of bacteria in oyster, but, it was not good enough to evaluate the bacterial diversity in oysters (Broekaert et al., 2011; Fernandez-Piquer et al., 2012). It
was reported that less bacteria could be cultured using the currently
available methods (Kenneth et al., 2009; Romero et al., 2002). Therefore,
culture-independent methods were widely used to reveal the bacterial

Corresponding author at: 800 Dongchuan Rd. Minhang, Shanghai 200240, PR China,
Tel.: +86 21 34206613; fax: +86 21 34206616.
E-mail addresses: dapengwang@163.com, norovirus@163.com (D. Wang).
0168-1605/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.008

communities in oyster tissues. In this eld, many culture-independent

methods were applied to evaluate the bacterial diversity, including
denaturing gradient gel electrophoresis (DGGE), uorescent in situ hybridization (FISH),terminal restriction fragment length polymorphism
(T-RFLP) and automated ribosomal intergenic spacer analysis (ARISA)
(Chen et al., 2013; Fernandez-Piquer et al., 2012; Hernandez-Zarate,
Olmos-Soto, 2006; Zurel et al., 2011). In all these methods, DGGE was
perhaps the most commonly used in the culture-independent ngerprinting techniques (Broekaert et al., 2011; Chen et al., 2013), and it
was widely used for detection of microbes in food (Broekaert et al.,
2011; Ercolini, 2004; Kenneth et al., 2009; Li et al., 2012).
Recently, tissues from oysters were investigated to look for the bacterial diversities, such as live oyster (Azandegbe et al., 2012; FernandezPiquer et al., 2012; Kenneth et al., 2009), digestive glands (Green and
Barnes, 2010; King et al., 2012), and the gills (Chen et al., 2013;
Hernandez-Zarate et al., 2006; Zurel et al., 2011). Generally, the oysters
were collected from aquatic farms and analyzed after different articial
treatments, including different storage temperature, high-pressure,
quick-frozen, and infection with parasites as mentioned above. Those
data boarded our knowledge on the bacterial diversity in oyster tissues.
However, it was reported that oysters collected directly in the wild and
laboratory-based might have different bacterial communities (Kenneth
et al., 2009; Thompson et al., 2005; Trabal et al., 2012). So far, less information was available on bacterial diversities in retail oysters.
To address this issue, this study investigated the seasonal dynamics
and diversities of bacteria in retail oysters during transport and storage
processes. This study was designed to: (1) investigate the changes of
communities in different seasons; (2) reveal the diversities of bacterial
communities in different retail oyster tissues. The results would provide

D. Wang et al. / International Journal of Food Microbiology 173 (2014) 1420

more information and would be valuable as an analog of proling on the

diversities of bacteria in retail oysters.
2. Material and methods
2.1. Oyster sample treatments
Oysters (Crassostrea gigas) were collected randomly from Jiangyang
market in Shanghai in 2011. The oysters were collected for analysis after
shipping to the market. Oysters were stored with ice in boxes. The information of samples was listed in Table 1 and treated as previously
described (Wanget al., 2010a, 2010b). Briey, oysters (n = 20) were
collected randomly and opened with sterilized knives. Different tissues
were dissected, including the gills, digestive glands (stomach, gut and
digestive diverticula) and residual tissues (mantle and adductor
muscle). Each tissue (50.0 g) was mixed with 200 ml (1:4) sterile physiological saline, and homogenized in sterile grinders. All tissues were
grounded at 12,000 rpm for 1 min. After treatment, all supernatants
(0.5 ml) were stored in 20% (nal concentration) sterile glycerol at
80 C until further use.
2.2. Extraction of genomic DNA
The pre-treated supernatants (0.2 mL) were vortexed and centrifuged at 10,000 rpm (Eppendorf 5814D, Hamburg, Germany) for
5 min. The precipitates were washed twice with sterile physiological
salt (1.0 mL) and resuspended in 0.1 mL sterile physiological salt.
Genomic DNA was extracted with Allmag Blood Genomic DNA kit
(Allrun, Shanghai, China) according to the manufacturer's protocol.
Finally, genomic DNA eluted with 50 L elution buffer was stored at
80 C until further use.

15

60 s, primer annealing at 55 C for 45 s, and primer extension at

72 C for 60 s; then nal extension at 72 C for 10 min. The PCR
products were visualized in a 1.5% agarose gel. All PCR reactions
were repeated separately for three times.
To avoid bias, triplicate production of PCR was pooled, which amplied from each sample. Then, DNA was recovered with DNA Gel Extraction
Kit (Omega Bio-Tek, Inc., USA) according to the manufacturer's protocol.
2.4. Denaturing gradient gel electrophoresis
DGGE analysis was performed on Dcode universal mutation detection system (Bio-Rad, USA) as previously described (Muyzer et al.,
1993) with minor modications. Briey, 10.0 L puried PCR product
was loaded to an 8% (wt/vol) polyacrylamide gel in TAE buffer. Optimal
electrophoresis experiments were performed at 60 C by using gels
containing a linear 35%55% denaturant gradient (100% corresponding
to 7 mol/L urea and 40% acrylamide). Electrophoresis was performed
at a constant voltage of 150 V for 5 h.
Following electrophoresis, the gels were preceded for silver staining.
The solutions used were 1.0% (vol/vol) ethanol plus 0.5% (vol/vol) acetic
acid for xation for 15 min; wash with ltered water (Milli-Q, Millipore)
for 2 times; 0.2% silver nitrate (wt/vol) for staining for 15 min, freshly
prepared stain solution containing 0.15% (vol/vol) formaldehyde; wash
with ltered water for 2 times; 1.5% NaOH for developing for 57 min,
freshly prepared developing solution containing 0.5% (vol/vol) formaldehyde; nally, 10.0% (vol/vol) ethanol and 0.5% (vol/vol) acetic acid to
stop the development. The gels were digitalized and analyzed with the
software package Quantity One Ver. 4.6.2 program (Bio-Rad, USA). The
virtual DGGE image was performed to create a virtual DGGE image and
recorded the bands in each lane.
2.5. Second amplication and sequencing

2.3. Amplication of 16S rRNA genes

The V3-region of 16S rRNA genes was amplied by Polymerase
Chain Reaction (PCR) in a PTC-200 thermocycler (MJ research, CA,
USA). The bacterial DNA from different tissues was amplied using
GC338F and 518R primers. The PCR mix consisted of 2.0 L of genomic
DNA, 2.0 U TaqE (Fermentas, Fisher Scientic, USA), 5.0 L PCR buffer
(with Mg2+), 3.2 L of 2.5 mmol/L dNTPs, 1.0 L of 20.0 mol/L primers
(GC338F: 5- CCTACGGGAGGCAGCAG 3 and 518R: 5- ATTACCGCGG
CTGCTGG-3), the GC clamp sequence is 5-CGCCCGGGGCGCG CCCCGG
GGCGGGGCGGGGGCGCGGGGGG-3 (Muyzer et al., 1993) and deionized water to bring up to a total reaction volume of 50.0 L. The cycling conditions were as follows: initial denaturation at 94 C for
5 min; 30 cycles consisting of template denaturation at 94 C for

The bands were excised from DGGE gel and eluted in ultra pure
water. DNA was recovered with Poly-Gel Extraction Kit (Omega
Bio-Tek, Inc., USA) according to the manufacturer's protocol. The
V3-region genes were amplied again using 338F and 518R primers
(Muyzer et al., 1993). The PCR mix consisted of 2.0 L of recovered
DNA, 2.0 U TaqE (Fermentas, Fisher Scientic, USA), 5.0 L PCR buffer
(with Mg2+), 3.2 L of 2.5 mmol/L dNTPs, 1.0 L of 20.0 mol/L primers
and deionized water to bring up to a total reaction volume of 50.0 L.
The cycling conditions were as follows: initial denaturation at 94 C
for 4 min; 30 cycles consisting of template denaturation at 94 C for
30 s, primer annealing at 55 C for 30 s, and primer extension at 72 C
for 30 s; then nal extension at 72 C for 10 min. The PCR products
were visualized in a 1.5% agarose gel.

Table 1
Sample information and relative species diversity (H), evenness (E), and richness (S) estimates from analysis of the DGGE band proles of retail oyster samples.
Sample

ShannonWiener Index (H)

Evenness (E)

Richness (S)

Seasons (Month)

Tissues

I
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV

3.019
2.924
2.815
3.407
2.982
2.904
2.986
2.886
2.657
2.907
2.874
2.886
2.923
2.650
2.992

0.938
0.960
0.925
0.943
0.951
0.926
0.952
0.920
0.938
0.915
0.904
0.920
0.932
0.917
0.929

25
21
21
37
23
23
23
23
17
24
24
23
23
18
25

Spring (Apr.)
Early Summer (Jun.)
Later Summer (Aug.)
Fall (Nov.)
Winter (Jan.)
Spring (Apr.)
Early Summer (Jun.)
Later Summer (Aug.)
Fall (Nov.)
Winter (Jan.)
Spring (Apr.)
Early Summer (Jun.)
Later Summer (Aug.)
Fall (Nov.)
Winter (Jan.)

Gills

Digestive glands

Residual tissues

16

D. Wang et al. / International Journal of Food Microbiology 173 (2014) 1420

The isolated amplicons were recovered with DNA Gel Extraction Kit
(Omega Bio-Tek, Inc., USA) and cloned into the pMD18-T (TaKaRa, Dalian,
China) according to the protocol provided by the kit. Following incubation, the ligation products were transformed into Escherichia coli DH5
competent cells and plated onto Luria-Bertani (LB, OXIOD, Ltd, England)
agar plates with 50 g/mL ampicillin (Sigma, MO, USA), X-gal and isopropyl -D-1-thiogalactopyranoside. After16 h incubation at 37 C, the 3
white clones were selected randomly, and sequenced by Beijing Yiming
Fuxing Biotechnology Co.
2.6. Statistical and phylogenetic analysis
The ShannonWiener index and Evenness (equitability) were calculated using the equations from Krebs (Krebs., 1989). The richness was
estimated as described by Oguntoyinbo (Oguntoyinbo et al., 2011).
Sequences were initially compared to the available database using
BLAST (Basic Local Alignment Search Tool) to determine their approximate phylogenetic afliations and orientation. Sequences were aligned
with reference 16S rRNA sequences from GenBank using the computer
software program MEGA Ver. 5.0 (www.megasoftware.net). Phylogenetic trees were constructed by the neighbor-joining distance method
(bootstrap =1000).
2.7. Nucleotide sequence accession numbers
Nucleotide sequences obtained in this study were submitted to the
NCBI GenBank database with the accession numbers of KF633394
KF633439.
3. Results
3.1. DGGE analysis of V3 regions
The V3 regions of 16S rRNA from different species amplied from
puried genomic DNA by PCR were separated in DGGE proles. Total

Digestive glands

Gills
I

II

III

IV

VI VII VIII IX

Residual tissues
X

XI

XII XIII XIV XV

of 46 different bands were observed in the DGGE gel (Fig. 1). All of the
bands were recovered and sequenced after second round PCR. The differences and changes in bacterial communities in oyster tissues were
clearly observed (Figs. 1 and 2). There was signicant difference
among the three parts of tissues (Figs. 1 and 2).
When the bands were analyzed according to different tissues, the results indicated that almost all bacteria were present in the gills; some
were transferred from the gills to digestive glands (Fig. 2).In Fall season,
bands 27 and 28 were obvious in digestive glands and residual tissues,
while they were not obvious in the gills (Fig. 1).Meanwhile, not all
bacteria, which were bio-accumulated by the gills, could be transferred
to other tissues (Fig. 2). On the other hand, many bands were only
shown in the gills, such as, bands 4, 5, 9 (Fig. 2).
The bacterial diversity in the gills was highest compared with other
tissues in every season (Figs. 1 and 2). In the Fall season, there were 31
bands present in the gills (lane 4 in Fig. 1), while only 14 bands were
observed in residual tissues (lane 14 in Fig. 1). In digestive glands, the
bacterial diversity seemed to be the same in different seasons except
in the Fall season (Fig. 1).
From Fig. 2, 22 bands always appeared in the different tissues. Most
of them (12/22) were grouped into Proteobacteria (Supplementary
Table 1). The bacterial diversity in the digestive glands (25 bands) was
the lowest in all tissues samples. The highest diversity was in the gills
(45 bands). There were some bands that appeared in two parts of
tissues.
According to time course, the bacterial diversity in Fall season was
the highest in the whole year with 36 bands detected in DGGE
(Fig. 3). Summer followed with 31 bands (Fig. 3), and 28 bands in
early summer and 26 bands in later summer respectively (Supplementary Table 1).On the other hand, in winter, there were only 23 bands
appearing in different tissues (Fig. 3).
From Fig. 3, 16 bands always appeared in the different seasons. Most
of them (9/16) were also grouped into Proteobacteria (Supplementary
Table 1). In addition, all of the 16 bands always appeared in the different
tissues (Figs. 2 and 3).The bacterial diversity in the winter season (23
bands) was the lowest in all oyster samples. The highest diversity was
in the Fall season (36 bands). There were a couple of bands that
appeared in two or three seasons.
According to the ShannonWiener index, Evenness index and richness, the bacterial diversity in gills obtained in Fall was the highest.
Same richness in different tissues was found between early summer
and later summer (Table 1). In the same tissue, not much difference
was observed between spring and winter according to the richness
index. However, in Fall season, much difference in richness was
shown between the gills and other tissues (Table 1). On the other
hand, bacterial diversities in richness showed little difference between
digestive glands and residual tissues in Fall season (Table 1).

3.2. Phylogenetic analysis

Fig. 1. The DGGE prole of the tissue samples from retail oyster. Lanes corresponding to
different tissue samples from different seasons are indicated by Roman numerals at the
top (I, VI, XI: Spring; II, III, VII,VIII, XII, XIII: Summer; IV, IX, XIV: Fall; V, X, XV: Winter).
The bands indicated by numbers were puried, re-amplied and subjected to sequencing.

After sequencing, a phylogenetic tree was drawn using MEGA Ver.

5.0 software. Phylogenetic analysis of 16S rRNA clone was shown in
Fig. 4. The results indicated the bacterial communities in retail oyster
samples were composed of Proteobacteria, Actinobacteria, Bacteroidetes,
Fusobacteria, Acidobateria, Firmicutes, Nitrospirae and Verrucomicrobia
(Fig. 4).
From Fig. 3, members of the Proteobacteria (23/46) were dominant in
oyster samples. Most of them were clustered into Vibrio sp. (12/23). This
result demonstrated that Vibrio sp. was predominated in the oyster tissues. In particular, 8 sequences classied into Vibrio were only retrieved
from gills, while 6 of them were discovered only in Fall sample (Figs. 2
and 4). The second predominated phylum was Bacterodetes wherein 7
sequences were assigned (Figs. 2 and 4). Except Proteobacteria, most sequences classied into other phyla were related to uncultured sequences (Fig. 4 and Supplementary Table 2).

D. Wang et al. / International Journal of Food Microbiology 173 (2014) 1420

17

Digestive glands
Total bands: 25

28

22, 35
1, 2, 3, 11, 14,
15, 16, 19, 20,
21, 23, 26, 27,
30, 32, 33, 36,
37, 40, 41, 43,
44

4, 5, 9, 10, 13,
17, 18, 24, 25,
29, 34, 38, 39,
42, 45, 46

Total bands: 22

6, 7, 8, 12, 31
Gills
Total bands: 45

Residual tissues
Total bands: 28

Fig. 2. The bands were analyzed according the different tissues, which are numbered in the DGGE prole. 146: Band number labeled in DGGE gel.

Spring
Total bands: 26

Summer
Total bands: 31

8, 39

5,18, 29
9, 22,
31, 42

3, 36, 43,
44

1, 2, 14, 15, 16, 20,

21, 23, 26, 27, 30,
32, 33, 37, 40, 41

6, 7

Total bands: 16

4, 7, 10, 11
12, 13, 17, 19, 24
25, 28, 34, 38, 45, 46
Fall
Total bands: 36

35
Winter
Total bands: 23

Fig. 3. The bands were analyzed according to different seasons, are which numbered in the DGGE prole. 146: Band number labeled in DGGE gel.

18

D. Wang et al. / International Journal of Food Microbiology 173 (2014) 1420

Fig. 4. Phylogenetic tree showing the afliations of 16S rRNA partial sequences retrieved from this study with selected reference sequences. The clones were shown as the band number
from the DGGE results. The tree was constructed by neighbor-joining method. Bootstrap values are based on 1000 replicates and shown at the nodes with more than 50% bootstrap
support. The scale bar represents 5% sequence divergence.

4. Discussion
In this study, the main ndings were as follows: (1) bacterial diversity is high in retail oysters; (2) the highest diversity of bacteria was
observed in Fall samples; (3) the gills was a promising candidate for
monitoring the bacterial pathogens in retail oysters.
Shanghai is located in the Yangtze River estuary. The salinity of
seawater is too low to raise the shellshes. Almost all shellshes sold
in the market were transported from other coastal provinces. It takes
one or a couple of days to harvest and transport the shellshes to the
local markets in Shanghai. Before they were sold, shellshes would be
stored in market for a couple of hours to several days, which depended
on the demand of customers. During the storage and transportation process, bacterial communities may shift (Chen et al., 2013; Fernandez-

Piquer et al., 2012). In addition, bacterial community in oyster was evaluated after different treatments (Azandegbe et al., 2012; Chen et al.,
2013; Fernandez-Piquer et al., 2012). In this situation, we wondered
what kind of prole would exist in retail shellshes. To address this
issue, the oyster was used as a model, and the community structure in
its tissues was evaluated by DGGE through the whole year. For this
point, understanding the seasonal dynamics and diversity of bacteria
in the retail oyster will help us to improve the safety of oysters as food.
Dynamics of pathogenic bacteria in oyster was also investigated
during different seasons by culture-based assay (Parveen et al., 2008).
The investigators focused on the cultivable bacteria and evaluated the
dynamics of them. But, the bacterial diversity was different between
culture-dependent and culture-independent assays (Kenneth et al.,
2009; Kisand and Wikner, 2003). From those results including this

D. Wang et al. / International Journal of Food Microbiology 173 (2014) 1420

study, more 16S rRNA genes were also amplied from uncultivable bacteria (Fernandez-Piquer et al., 2012; Green and Barnes, 2010). Although
bacterial communities could be evaluated by culture-dependent
methods, it was only a partial view from the whole picture of bacterial
communities in oysters (Broekaert et al., 2011; Kenneth et al., 2009).
It was suggested that the culture-dependent methods were not sufcient to investigate the seasonal dynamics and diversity of bacteria in
oysters, especially in retail oysters. Therefore, the culture-independent
method was a better way to evaluate the seasonal dynamics and the
structure of the bacteria in oyster.
In previous reports, the gills and digestive glands were the main
tissues, which bio-accumulated pathogens including bacteria and viruses (Wang et al., 2008a, 2008b, 2010a). We expected in these main
tissues, the changes of bacterial diversity were the key point. In this
study, these seasonal dynamics and diversity of bacteria were investigated focusing on the main tissues. As we expected, the gills harbored a relatively diverse assemblage of phylotypes in different
seasons (Figs. 1 and 2). In addition, the DGGE prole demonstrated
that 31 bands were found in the gills, while 14 bands in the digestive
glands and residual tissues respectively in the Fall season (Fig. 2). Our
result was similar with other reports, which was investigated by different
approaches (Fernandez-Piquer et al., 2012; Hernandez-Zarate and
Olmos-Soto, 2006).
It was described that two kinds of bacteria were found in oyster including autochthonous and allochthonous. The former may supply nutrient factors to keep oyster alive or defend its pathogens (Pujalteet al.,
1999), while the latter pass through with water (Romero et al., 2002).
In this study, there were 16 bands always detected in the samples in all
seasons (Fig. 3). It means that 16 species were relatively permanent associated with oyster tissues. From our data, the diversity of bacteria in the
gills was more complex than the autochthonous. It suggested that almost
all allochthonous were in the gills, which further conrmed the previous
report (Zurel et al., 2011). In other words, the allochthonous, such as
foodborne pathogens, were mainly present in the gills, not in other tissues. On the other hand, from our results, the bacterial diversity in the
gills was different from all other tissues (Figs. 1 and 2). Bacterial structures
in the gills appeared to be highly diverse (Figs. 1 and 2). Similar results
were reported that the bacterial communities in the gills were signicantly different from those in guts by ARISA (Zurel et al., 2011) and FISH
(Hernandez-Zarate and Olmos-Soto, 2006).Therefore, the gills were
more suitable to be the target tissues for detection of foodborne pathogens from a public health standpoint. It was also a target tissue to monitor
for pathogens in oysters.
Because of loading capacity, 15 samples were loaded in the DGGE gel
in this study. The main tissues, the gills and digestive glands, were
studied to evaluate the bacterial and seasonal dynamics in retail oyster.
In general, the population of bacteria in oyster was related to the environmental temperature (Gonzalez-Acosta et al., 2006; Zurel et al.,
2011). Therefore, in this study, the samples were collected twice
(early summer and later summer) to make sure of the bacterial diversity
in retail oyster tissues. To our surprise, the bacterial diversity in summer
was not the highest in different seasons as we expected.
Different bacterial diversity in oyster depended on the local aquatic
environment (Kenneth et al., 2009; King et al., 2012). Higher bacterial
abundance was associated with higher water temperature (DePaola
et al., 2003; Parveen et al., 2008).From the results of DGGE and phylogenetic analysis in this study, which exceeded our expectation, the highest
bacterial diversity in retail oyster was in Fall, not in summer (Fig. 1). In
Fall, the temperature was not the highest in the whole year, but the rain
was much less than that in summer. Although there was less rain in
spring and winter either, it was too cold for bacteria to grow. We inferred that the rain season (in summer) was one of the main reasons
to limit the concentration and diversity of bacteria in aquatic farm. In
this case, the rain affected the bacterial structures in oyster tissues. In
general, oyster will be harvested in Fall in China, and largest quantity
of oyster was consumed compared to other seasons. According to our

19

results, it is a critical season for government to monitor the health risk

during the oyster harvest season.
In this study, 46 different 16S rRNA coding sequences were recovered and cloned (Figs. 1 and 4). In previous reports, 18 or 25 sequences
were observed in the gills or individual oyster by DGGE after articial
treatment (Chen et al., 2013; Kenneth et al., 2009). More than 73 different genera-related clones in individual oysters were reported by T-RFLP
(Fernandez-Piquer et al., 2012). All of these data demonstrated that
high diversity was present in oysters. In addition, some of those
sequences were from uncultivable bacteria (Fernandez-Piquer et al.,
2012; Green and Barnes, 2010). In the last decades, all kinds of molecular assays were developed to rapidly detect these pathogens in oyster,
such as PCR and real-time PCR (Liu et al., 2012; Nordstrom et al.,
2007). Most methods were focused on those common pathogens, such
as Vibrio parahaemolyticus and Vibrio vulnicus. However, it was hard
to determine whether those uncultivable bacteria were pathogens or
not. Therefore, to know those bacteria well, new media should be developed to get the pure cultures.
In fact, members of the genus Vibrio are natural inhabitants of marine
environments that are typically associated with oysters (Prapaiwong
et al., 2009). The data demonstrated that the Proteobacteria predominated
(23/46) in bacterial communities of retail oyster, which was an indicator
to risk analysis. It was similar with other reports by different cultureindependent methods (Fernandez-Piquer et al., 2012; Hernandez-Zarate
et al., 2006). Additionally, the seasonal variation detected in terms of diversity and density of total bacterial community demonstrated the need
for a careful monitoring of oyster throughout the year. In the future,
foodborne pathogenic bacteria or virus should be investigated to better
understand the dynamics of these pathogens in oyster in each season.
Conict of interest
None declared.
Acknowledgments
This work was jointly supported by the grant No. 31000063 from
the National Natural Science Foundation of China, the grant No.
2012AA101601 from the Ministry of Science and Technology of China
and grant from the School of Agriculture and Biology (NQN201008). We
also thank Dr. Yanhong Liu from the Eastern Regional Research Center
for critical reading of this manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijfoodmicro.2013.12.008.
References
Azandegbe, A., Poly, F., Andrieux-Loyer, F., Kerouel, R., Philippon, X., Nicolas, J.L., 2012.
Inuence of oyster culture on biogeochemistry and bacterial community structure
at the sediment-water interface. FEMS Microbiol. Ecol. 82, 102117.
Broekaert, K., Heyndrickx, M., Herman, L., Devlieghere, F., Vlaemynck, G., 2011. Seafood
quality analysis: molecular identication of dominant microbiota after ice storage
on several general growth media. Food Microbiol. 28, 11621169.
Chen, H., Liu, Z., Wang, M., Chen, S., Chen, T., 2013. Characterisation of the spoilage bacterial microbiota in oyster gills during storage at different temperatures. J. Sci. Food
Agric. 93, 37483754.
DePaola, A., Nordstrom, J.L., Bowers, J.C., Wells, J.G., Cook, D.W., 2003. Seasonal abundance
of total and pathogenic Vibrio parahaemolyticus in Alabama oysters. Appl. Environ.
Microbiol. 69, 15211526.
Ercolini, D., 2004. PCR-DGGE ngerprinting: novel strategies for detection of microbes in
food. J. Microbiol. Methods 56, 297314.
Fernandez-Piquer, J., Bowman, J.P., Ross, T., Tamplin, M.L., 2012. Molecular analysis of the
bacterial communities in the live Pacic oyster (Crassostrea gigas) and the inuence
of postharvest temperature on its structure. J. Appl. Microbiol. 112, 11341143.
Gonzalez-Acosta, B., Bashan, Y., Hernandez-Saavedra, N.Y., Ascencio, F., Cruz-Aguero, G.,
2006. Seasonal seawater temperature as the major determinant for populations of
culturable bacteria in the sediments of an intact mangrove in an arid region. FEMS
Microbiol. Ecol. 55, 311321.

20

D. Wang et al. / International Journal of Food Microbiology 173 (2014) 1420

Gooch, J.A., DePaola, A., Bowers, J., Marshall, D.L., 2002. Growth and survival of Vibrio
parahaemolyticus in postharvest American oysters. J. Food Prot. 65, 970974.
Green, T.J., Barnes, A.C., 2010. Bacterial diversity of the digestive gland of Sydney rock oysters, Saccostrea glomerata infected with the paramyxean parasite. Marteilia sydneyi.
J. Appl. Microbiol. 109, 613622.
Hernandez-Zarate, G., Olmos-Soto, J., 2006. Identication of bacterial diversity in the
oyster Crassostrea gigas by uorescent in situ hybridization and polymerase chain
reaction. J. Appl. Microbiol. 100, 664672.
Kenneth, J., La Valley, S.J., Gomez-Chiarri, Marta, Dealteris, Joseph, Rice, Michael, 2009. Bacterial community proling of the eastern oyster (Crassostrea virginica): comparison of
culture-dependent and culture-independent outcomes. J. Shellsh Res. 28, 827835.
King, G.M., Judd, C., Kuske, C.R., Smith, C., 2012. Analysis of stomach and gut microbiomes
of the eastern oyster (Crassostrea virginica) from coastal Louisiana, USA. PLoS One 7,
e51475.
Kisand, V., Wikner, J., 2003. Combining culture-dependent and -independent methodologies for estimation of richness of estuarine bacterioplankton consuming riverine
dissolved organic matter. Appl. Environ. Microbiol. 69, 36073616.
Krebs, C.J., 1989. Ecological Methodology. Harper Collins Publishers, New York.
Li, S., Sun, L., Wu, H., Hu, Z., Liu, W., Li, Y., Wen, X., 2012. The intestinal microbial diversity
in mud crab (Scylla paramamosain) as determined by PCR-DGGE and clone library
analysis. J. Appl. Microbiol. 113, 13411351.
Liu, B., He, X., Chen, W., Yu, S., Shi, C., Zhou, X., Chen, J., Wang, D., Shi, X., 2012. Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from
seafood. Protein Cell 3, 204212.
Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Proling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain
reaction-amplied genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695700.
Nordstrom, J.L., Vickery, M.C., Blackstone, G.M., Murray, S.L., DePaola, A., 2007. Development of a multiplex real-time PCR assay with an internal amplication control for
the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters.
Appl. Environ. Microbiol. 73, 58405847.
Oguntoyinbo, F.A., Tourlomousis, P., Gasson, M.J., Narbad, A., 2011. Analysis of bacterial
communities of traditional fermented West African cereal foods using culture
independent methods. Int. J. Food Microbiol. 145, 205210.

Parveen, S., Hettiarachchi, K.A., Bowers, J.C., Jones, J.L., Tamplin, M.L., McKay, R., Beatty, W.,
Brohawn, K., Dasilva, L.V., Depaola, A., 2008. Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters. Int. J. Food
Microbiol. 128, 354361.
Prapaiwong, N., Wallace, R.K., Arias, C.R., 2009. Bacterial loads and microbial composition
in high pressure treated oysters during storage. Int. J. Food Microbiol. 131, 145150.
Pujalte, M.J., Ortigosa, M., Macian, M.C., Garay, E., 1999. Aerobic and facultative anaerobic
heterotrophic bacteria associated to Mediterranean oysters and seawater. Int.
Microbiol. 2, 259266.
Romero, J., Garca-Varela, M., Laclette, J.P., Espejo, R.T., 2002. Bacterial 16S rRNA gene
analysis revealed that bacteria related to Arcobacter spp. constitute an abundant
and common component of the oyster microbiota (Tiostrea chilensis). Microb. Ecol.
44, 365371.
Thompson, J.R., Marcelino, L.A., Polz, M.F., 2005. Diversity, sources, and detection of
human bacterial pathogens in the marine environment. In: Belkin, C. (Ed.), Oceans
and Health: Pathogens in the Marine Environment. Springer, New York, pp. 2968.
Trabal, N., Mazon-Suastegui, J.M., Vazquez-Juarez, R., Asencio-Valle, F., MoralesBojorquez, E., Romero, J., 2012. Molecular analysis of bacterial microbiota associated
with oysters (Crassostrea gigas and Crassostrea corteziensis) in different growth
phases at two cultivation sites. Microb. Ecol. 64, 555569.
Wang, D., Shi, X., 2011. Distribution and detection of pathogens in shellsha review. Wei
Sheng Wu Xue Bao 51, 13041309.
Wang, D., Wu, Q., Kou, X., Yao, L., Zhang, J., 2008a. Distribution of norovirus in oyster
tissues. J. Appl. Microbiol. 105, 19661972.
Wang, D., Wu, Q., Yao, L., Wei, M., Kou, X., Zhang, J., 2008b. New target tissue for foodborne virus detection in oysters. Lett. Appl. Microbiol. 47, 405409.
Wang, D., Yu, S., Chen, W., Zhang, D., Shi, X., 2010a. Enumeration of Vibrio parahaemolyticus
in oyster tissues following articial contamination and depuration. Lett. Appl. Microbiol.
51, 104108.
Wang, D., Zhang, D., Chen, W., Yu, S., Shi, X., 2010b. Retention of Vibrio parahaemolyticus
in oyster tissues after chlorine dioxide treatment. Int. J. Food Microbiol. 137, 7680.
Zurel, D., Benayahu, Y., Or, A., Kovacs, A., Gophna, U., 2011. Composition and dynamics of
the gill microbiota of an invasive Indo-Pacic oyster in the eastern Mediterranean
Sea. Environ. Microbiol. 1 13, 14671476.