Abstract: Microbiology relate to the oyster disease outbreak, mortalities, food borne pathogen, spoilage 374 ISJ 13: 374-388, 2016 ISSN 1824-307X REVIEW Microbiological analysis and microbiota in oyster: a review H Chen1,2,3, Z Liu4, Y Shi2, HH Ding2 1Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian 361005, China 2University of Guelph, 50 Stone Road E.,Guelph, ON N1G 2W1, Canada 3Biology Department, Xiamen Ocean Vocational College, Xiamen 361012, P.R. China 4Fisheries Research Institute of Fujian, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen, Fujian 361013, China Accepted November 3, 2016 Abstract Oyster, is a popular shellfish consumed globally. As a bivalve filter-feeding invertebrate mollusk, oyster harbors many microorganisms, which could eventually cause potential health risks of human. Microorganisms were correlated to oyster mortality, shelf life, spoilage, and foodborne pathogenic bacteria. Meanwhile, they could be adjusted by the preservative technologies in order to prolong the shelf life. With the development of molecular biological techniques, such as 16S Polymerase Chain Reaction (PCR), Real-time PCR, Temperature Gradient Gel Electrophoresis (TGGE), Denaturing Gradient Gel Electrophoresis (DGGE), Restriction Fragment Length Polymorphism (RFLP), Fluorescent in situ Hybridization (FISH), etc., microbiological diversity and spoilage mechanism of oyster can be further investigated. The spoilage microbiota belongs to Vibrio, Pseudomonas, Aeromonas, Bacillus, Enterobacteriaceae, Lactic Acid Bacteria (LAB), and Micrococcus, etc., and the main pathogens are Vibrio, Salmonella, Escherichia coli, Listeria, Staphylococcus, Photobacterium, and Shewanella according to current studies. However, little information is available for the spoilage mechanism of entire oyster and different tissues under different preservation conditions. This article reviews the oyster microbiota analysis methods, the impacts of aquaculture and pathogenic bacteria on oyster mortality and food safety, as well as initial and spoilage microbiotas in whole oyster and separated tissues during preservation. Key Words: oyster; microbiota; pathogen; spoilage mechanism; molecular analysis; preservation Background Oyster, a bivalve mollusk, is a nutritious marine food resource that high in protein, vitamin A, vitamin B12 and zinc, but low in calories. Many researchers analyzed various nutritional components from oyster ___________________________________________________________________________ Corresponding author: Huihuang H. Ding University of Guelph 50 Stone Road E, Guelph, ON N1G 2W 1, Canada Email: dingh@uoguelph.ca List of abbreviations: Automated Ribosomal Intergenic Spacer Analysis, ARISA; Denaturing Gradient Gel Electrophoresis, DGGE; Fluorescent in situ Hybridization, FISH; Lactic Acid Bacteria, LAB; Polymerase Chain Reaction, PCR; Restriction Fragment Length Polymorphism, RELP; Temperature Gradient Gel Electrophoresis, TGGE; Terminal Restriction Fragment Length Polymorphism, T-RFLP and verified that they have functional activities (Achour et al., 1997; Shiozaki et al., 2010; Anderson and Beaven, 2001). With the increase of consumption, oyster farming grows fast, and it is the most popular mollusk aquaculture around the world. The top 6 countries contributing to oyster production are China, Japan, Korea, USA, France and Mexico (Heinonen, 2014). Since 1970, the aquaculture of shellfish doubled every decades worldwide, and the demand is still increasing (Dégremont et al., 2015). There are approximately 4 million tons of oysters consumed annually and half of them are eaten raw (Fang et al., 2015). China produces over 2 million tons of oyster per year, which is mainly used to make oyster sauce (Heinonen, 2014). In view of the fast growth of the oyster aquaculture, the impacts of disease and mortality on the yield of oysters attracted prompt attentions by government, farmers, and researchers. However, the research on oyster pathogenic bacteria is challenging due to a wide variety of oysters and aquaculture location worldwide. In previous studies, 375 Vibro aestuarianus and Vibro splendidus were reported to cause the summer mortality of C. gigas oysters in France (Le Roux et al., 2002; Gay et al., 2004; Garnier et al., 2007). Furthermore, the introduction of nonnative oyster may lead to disease outbreak (Beck et al., 2011). The observation of oyster mortality is the main sign of diseases in aquaculture oyster. Preventing contamination and keeping pathogen-free environment is of vital importance in oyster farming (Dégremont et al., 2015). On the other hand, the bacteria from fresh oyster were attracted more attentions because some of the bacteria can bring about the outbreak of human diseases. For example, Vibrio parahaemolyticus is a pathogenic bacterium for oyster, which is also well-documented foodborne bacteria responsible for the outbreaks of shellfish-associated gastroenteritis and diarrhea correlated to seafood consumption in the United States (Dalsgaard, 1998; Liu et al., 2009). Perishable oyster could cause serious foodborne problems in processing and distribution. Microbial activity is mainly responsible for the changes in flavor, texture, and odor (Cao et al., 2009; Prapaiwong et al., 2009a; Montanhini and Neto, 2015). Compared to terrestrial foods, oyster has shorter shelf life due to relatively higher levels of free ammonia nitrogen and high diversity of microbiota (Madigan et al., 2014). The shelf-life of oyster could be affected by many factors, such as extrinsic factor (temperature, atmosphere), intrinsic factors (species, size, age, health and composition) and microbial flora load ( Linton et al., 2003; Cao et al., 2010; Chen et al., 2016). Among those factors, microbiota in oyster plays critical roles on oyster diseases, food safety, and spoilage. This article summarized the oyster microbiota, including the analysis approaches, environmental impacts, pathogenic bacteria, and the microbiota in different oyster tissues. Analysis approaches for oyster microbiota Conventional cultivation method was widely used to analyze the bacterial population, and to isolate them through streak plate method. It plays an important role to obtain the bacterial strains. Cultivation method was used to investigate bacterial microbiota and dominant species in oyster, among which Pseudomonas were accounted for one third of 321 isolates and reported as dominant bacteria (Kueh and Chan, 1985). This method has been widely utilized for oyster microbiota analysis to reveal the bacterial population and community in details ( Colburn et al., 1990; Cao et al., 2009, 2010; Liu et al., 2009; Song et al., 2009; Fang et al., 2015). However, cultivation and following isolation for microbiota analysis was time and resource consuming with poor reproducibility (Cao et al., 2009; Prapaiwong et al., 2009a). The phylogenetic analyses of rRNA genes from laboratory culture and isolates were applied to evaluate the microbiota, of which the efficiency were highly improved in oyster bacterial analysis and many species were identified by sequencing (Prapaiwong et al., 2009a; Green and Barnes, 2010; Lee et al., 2010; Thupila et al., 2011). Conventional cultivation method could result in overestimation or underestimation of the microbiological community, because many bacteria are naturally uncultivable and unsuitable media may lead to biased results (Randazzo et al., 2002; Chen et al., 2013). Molecular approach shows more abundant of bacterial microbiota than cultivation method in oysters (Romero et al., 2002). In the past decades, culture-independent methods of finger print profile were introduced to oyster analysis for bacterial microbiota and diversity, such as TGGE (Fernández et al., 2014) and DGGE (Chen et al., 2013; Wood and Arias, 2015), Terminal Restriction Fragment Length Polymorphism (T-RFLP) (Garnier et al., 2007; Fernandez-Piquer et al., 2012), which revealed that oyster had high diversity in the bacteria. DGGE was widely utilized in the characterization of the bacterial communities from farmed, retailed, and storied oysters. The fingerprints of DGGE gel intuitively reflect the microbiota variation by the band changes, of which the band corresponding to the bacteria (more than 1 %) can be clearly profiled (Chen et al., 2013; Wood and Arias, 2015). However, DGGE method may also subjected to the inaccuracy on bacterial diversity evaluations resulted from DNA extraction, PCR amplification, and sequencing errors from environmental samples (Wintzingerode et al., 1997). This bias was also observed in Wood’s study (Wood and Arias, 2015) when they applied DGGE to reveal the bacteria in oyster, few bands from DGGE couldn’t be amplified and identified. Compared to DGGE, T-RFLP technique is more reproducible and accurate, but more expensive. Both of them provide overview of the bacterial communities and the variation of dominant bacteria in oyster. Real-Time PCR and Multiplex Real-Time PCR were also introduced to identify and track the target bacteria with higher efficiency and accuracy for the bacteria with lower abundant, especially pathogen community (Ward and Bej, 2006; Nordstrom et al., 2007; Kim et al., 2008a). The FISH on the basis of the designed probe were used in different organ microbiota in oyster and the high abundance of the bacteria were observed (Hernández-Zárate and Olmos-Soto, 2006). The ARISA approach also showed the high diversity of oyster gill microbiota effectively (Zurel et al., 2011). However, because of the high cost the new technologies, such as metagenome and transcriptome, were not commonly used in previous oyster microbiota studies. Environmental impacts on oyster microbiota The diversity and community of bacteria in raw oysters were affected by many factors. Oyster is normally eaten by whole body, thus all tissues with its original microbiota are eventually consumed by human. The impacts of aquaculture environmental are of vital importance to original microbiota, because all attached initial bacteria from environment were closely correlated to the microbiota in the growing stages of oyster, harvest, sale, storage, and consumption. These factors include the location of the sea (Cao et al., 2009; King et al., 2012; Madigan et al., 2014; Wood and Arias, 2015), harvest season (Parveen et al., 2008), water temperature (Gonzalez-Acosta et al., 2006; Shen et 376 Table 1 Food borne pathogen in oyster aquaculture, sale and storage Species/ Location Pathogen/ Food borne pathogen* Analysis Method Reference C. virginica oyster/ Mobile Bay, US V. parahaemolyticus* Alkaline phosphatase-labeled DNA probe procedures (Kaufman et al., 2003) Pacific oysters (C. gigas)/ Arcata Bay, US Listeria sp.*, L. monocytogenes* Culture and isolates (Colburn et al., 1990) Ostrea rivularis Salmonella spp. Culture (Fang et al., 2015) Oyster/ Washington, US V. parahaemolyticus* Culture (Liu et al., 2009) Oyster (C. gigas)/ France Vibrio splendidus, Vibrio aestuarianus, Vibrio harveyirelated, Shewanella colwelliana Isolates genotyping by the 16S rRNA and gyvB genes (Saulnier, Decker et al., 2009) Pacific oyster (C.gigas)/ France V. aestuarianus Real-time PCR (Saulnier, De Decker et al., 2009) Commercial oyster/ New Jersey coast, US Shewanella algae, S. putrefaciens, Photobacterium damselae subsp. damselae, 16S rRNA genes sequencing (Richards et al., 2008) Raw oyster V. parahaemolyticus* Real-time PCR (Kim et al., 2008b) Oyster/ Louisiana, US Salmonella*, V. parahaemolyticus* MICRO-IS and API-20E systems (Abeyta et al., 1986) Retailed oyster/ Shanghai,China V. parahaemolyticus* Polymerase chain reaction (PCR) (Yu et al., 2016) Salted oyster (Jeotkal)/ Korea L. monocytogenes*, Staphylococcus aureus*, V. parahaemolyticus* Culture (Song et al., 2009) C. gigas oyster/ Mediterranean, France Escherichia coli* Culture (Derolez et al., 2013) Commercial oyster/ US V. vulnificus*, V. parahaemolyticus*, V. alginolyticus*, A. hydrophila* Culture isolates with 16S rDNA identification (Prapaiwong et al., 2009a) C. virginica oyster/ Dauphin Island, US V. parahaemolyticus*, V. mimicus, V. vulnificus Total Bacteria and Vibrio-Specific Denaturing Gradient Gel Electrophoresis (Wood and Arias, 2015) Oyster tissues V. parahaemolyticus* PCR detection (Wang et al., 2010) Pacific Oyster (C. giga)/ Atlantic coast, France V. aestuarianus, members of the V. splendidus group, V. natriegens, V. parahaemolyticus*, Pseudoalteromonas sp. The dominant colonies were identified by phenotypic and genotypic characters (RFLP) (Garnier et al., 2007) C. virginica oyster/ Mobile Bay, US V. parahaemolyticus* Direct plating method involving an alkaline-phosphatase-labeled DNA probe (Gooch et al., 2002) Oyster L. innocua Isolation and biochemical tests (Colburn et al., 1990) Oyster (Crassostrea belcheri)/ Thailand Salmonella*, V. parahaemolyticus*, V. vulnificus* 16S rRNA gene sequencing (Thupila et al., 2011) Raw oyster/ Korean V. parahaemolyticus* Real-time PCR (Kim et al., 2008a) Pacific oysters (C. gigas) V. parahaemolyticus* Culture (Ma and Su, 2011) http://onlinelibrary.wiley.com/doi/10.1111/jam.12040/full http://onlinelibrary.wiley.com/doi/10.1111/jam.12040/full 377 Zhe oyster (Crassostrea plicatula)/ Zhejiang, China V. parahaemolyticus* Culture (Shen et al., 2009) Alaskan oysters/ US V. parahaemolyticus* Multiplex Real-Time PCR (Nordstrom et al., 2007) Oyster (C. virginica)/ Chesapeake Bay, US V. parahaemolyticus* Quantitative direct-plating method followed by DNA colony hybridization (Parveen et al., 2008) Oyster/ Mandinga Grande Lagoon, US V. parahaemolyticus* Culture (Flores-Primo et al., 2014) Raw oyster/ Alaska, US V. parahaemolyticus* Isolates identified by PCR (McLaughlin et al., 2005) Live oysters Vibrio spp. Multiplex PCR and DNA microarrays (Panicker et al., 2004) Oyster/ Washington, US V. parahaemolyticus* Multiplexed Real-Time PCR (Ward and Bej, 2006) Oyster/ Dauphin Island Bay, Alabama, US V. vulnificus, V. parahaemolyticus* Quantitative PCR (Givens et al., 2014) Raw Pacific oysters V. parahaemolyticus* Culture (Liu et al., 2009) * Food borne pathogen. al., 2009), aquatic environment (La Valley et al., 2009; Shen et al., 2009; Azandégbé et al., 2012; King et al., 2012), and environmental stress (Paillard et al., 2004; Green and Barnes, 2010). The initial bacterial communities from different areas are different. Cruz-Romero (2008b) reported that the initial bacterial communities in raw oyster (C. gigas) from Cork harbor were dominant by Aeromonas, Vibrio, and Pseudomonas. The results were similar to the reported bacterial communities of the oysters from Yellow Sea in China (Cao et al., 2009), in which Pseudomonas, Vibrio were presented as the dominant bacteria. Except Pseudomonas and Flavobacterium, Ortigosa et al. (1995) reported that Alteromonas, Shewanella, Deleya, and Oceanospirillum were detected in the oysters from Mediterranean Coast. Despite the location, the microbiota were different under controlled and natural environments (Colwell and Liston, 1960). In our previous study (Chen et al., 2013), the dominant microbiota in the raw oyster gills were Lactococcus, Lactobacillus, Enterobacter, and Aeromonas. Harvest season was one of the main factors responsible for different varieties of the oyster microbiota (Parveen et al., 2008; Wang et al., 2014b; Roterman et al., 2015), which has been well demonstrated by molecular methods. Prapaiwong et al. (2009a) observed that more Vibrio vulnificus could be isolated from raw oysters living in relatively higher water temperature. In addition, the bacterial communities were correlated to oyster species (Roterman et al., 2015). The water temperature can affect the bacteria loads in oyster. The correlation between seawater and mictobiota in oyster were revealed through isolates and rDNA hybridization with phylogenetic probes, and most isolates unidentified corresponded to α-Proteobacteria (Pujalte et al., 1999). Pathogenic bacteria in oyster The pathogenic bacteria related to oyster diseases and mortality, as well as human pathogens associated with aquaculture oyster were summarized as shown in Table 1. Among main human pathogenic bacteria, Vibrio, Aeromonas, Salmonella, E. coli, Listeria, Staphylococcus, Photobacterium, and Shewanella have been extensively investigated in oyster aquaculture and storage (Table 1). Vibrio and Aeromonas were the main genus of bacterial pathogenic for oyster. The traditional cultivation and identification, 16S PCR sequencing, Real-time PCR, DGGE, RFLP, Multiplex Real-Time PCR, and quantitative PCR were used to investigate the pathogenic bacteria and microbiota in oyster. Real-time PCR and quantitative PCR were regard as the effective way in Vibrio inspection, which were designed to reveal the existence of the target pathogen in oyster (Nordstrom et al., 2007; Kim et al., 2008b; Saulnier et al., 2009). In view of the difficulty of identification, polyphasic approaches have been developed to identify potential pathogens associated with oyster diseases (Paillard et al., 2004). Vibrio species were reported as the main pathogenic species in the oyster leading to 8,000 illnesses per year in the United States (Kaufman et al., 2003), which has been extensive studied regarding oyster diseases and mortality, and food safety (Kaufman et al., 2003; Panicker et al., 2004; Nordstrom et al., 2007; Liu et al., 2009; Saulnier et al., 2009; Yu et al., 2016). The pathogenic bacteria associated with public health are V. vulnificus, V. parahaemolyticus, Vibrio alginolyticus, and Aeromonas hydrophila in raw oysters (Lorca et al., 200; Prapaiwong et al., 2009a). The proliferation of V. vulnificus during storage at temperature abuse conditions (e.g., 7, 13, and 21 °C) makes the oyster unsafe (Lorca et al., 2001). 378 The risk of raw and uncooked oysters resulting in gastroenteritis in consumers has been well described (Kueh and Chan, 1985; Green and Barnes, 2010). Using Vibrio-Specific DGGE and RFLP approaches, the profiles of Vibrio were clearly demonstrated (Garnier et al., 2007; Wood and Arias, 2015). More V. parahaemolyticus have been found in the gills and digestive glands than those in other portions of the oysters (Wang et al., 2010). Prapaiwong et al. (2009a) showed that Shewanella, Vibrio, Psychrobacter and A. hydrophila were also identified in raw oysters, quick frozen oysters, and high pressure processed oysters, whereas V. vulnificus was only detected in raw oysters. The potential risk of V. parahaemolyticus infection might increase, and recently Yu et al. (2016) demonstrated that 33 out of 96 isolates showed resistance to two or more antimicrobial agents in Shanghai, China. Salmonella are regarded as one of the most common human pathogenic bacteria in shellfish; however, they were not detected in oyster either under high pressure treatment or other controlled storage conditions ( Jones et al., 1993; Bej et al., 1994; López-Caballero et al., 2000). E. coli found in raw oyster by culture-dependent DGGE method illustrated that they may have potential hazard for the ingestion of fresh oyster (Chen et al., 2013). Listeria monocytogenes were reported to be associated with foodborne outbreaks (Colburn et al., 1990). L. monocytogenes and Staphylococcus aureus have been presented to be killed using electron beam irradiation in salted oyster (Song et al., 2009). The pathogenic bacteria for oyster can also lead to the death of oysters, which cause big losses in oyster farming and related industry. V. aestuarianus and V. splendidus were reported to be related to the summer mortality of the C. gigas in the sea in France. While in North America, V. tubiashii were found to be associated with the mortalities of hatchery-reared Crassostrea virginica oysters and C. gigas (Saulnier et al., 2009). Garnier et al. (2007) demonstrated similar results in their study as V. aestuarianus was detected in 56 % of isolates while 25% of isolates contains V. splendidus group. Microbiota in different oyster tissues Bacterial microbiota in aquaculture, processing and preservation were studied in the past decades. The predominant bacterial communities were diverse in raw oysters. As list in Table 2, the microbiota in oyster mainly included Pseudomonas, Vibrio, Aeromonas, Moraxella, Shewanella, Flavobacterium, Acinetobacter, Enterobacteriaceae, Table 2 Microbiota and analysis methods for oyster storied at different condition Oyster species/ Location Treatment methods Initial dominant microbiota Spoilage or Survival microbiota Treatment conditions & duration Analyzing method Reference Pacific oyster (C. gigas) Natural flora Pseudomonas, Vibrio, Achromobacter, Flavobacterium, Corynebacterium, Alcaligenes, Micrococcus, Bacillus sp., Enterococci NA NA Culture and isolates (Colwell and Liston, 1960) Pacific oyster (C. gigas)/ Yellow sea, China Refrigeration Pseudomonas*, Vibrionaceae*, Shewanella, Alcaligenes, Enterobacteriaceae, Moraxella, Acinetobacter, Flavobacterium, Corynebacterium, Staphylococcus, Micrococcus, Lactic acid bacteria, Bacillus sp. Pseudomonas*, Vibrionaceae*, Moraxella, Flavobacterium, Micrococcus, Bacillus sp. Storage at 5 ±1 °C for 12d Culture and isolates (Cao, Xue and Liu, 2009) Pacific oyster (C. gigas)/ Yellow sea, China Ozonated water treated Pseudomonas, Vibrionaceae, Shewanella, Alcaligenes, Enterobacteriaceae, Moraxella, Acinetobacter, Flavobacterium, Corynebacterium, Staphylococcus, Micrococcus, Lactic acid bacteria, Bacillus sp. Pseudomonas*, Vibrionaceae*, Enterobacteriaceae, Moraxella, Flavobacterium, Micrococcus, Bacillus sp. Ozonated water (5.0×10-6 g/L for 2 min) Culture and isolates (Cao et al., 2010) 379 Pacific oyster (C. gigas)/ Yellow sea, China Refrigeration Pseudomonas, Vibrionaceae, Shewanella, Alcaligenes, Enterobacteriaceae, Moraxella, Acinetobacter, Flavobacterium, Corynebacterium Staphylococcus, Micrococcus, Lactic acid bacteria, Bacillus sp. Pseudomonas*, Vibrionaceae*, Moraxella, Flavobacterium, Micrococcus, Bacillus sp. Storage at 0 °C Culture and isolates (Cao, Xue, Liu et al., 2009) Pseudomonas, Vibrionaceae, Alcaligenes, Enterobacteriaceae, Moraxella, Flavobacterium, Micrococcus, Lactic acid bacteria, Bacillus sp. Storage at 10 °C C. gigas oyster High hydrostatic pressure Bacillus, Moraxella, Acinetobacter, Pseudomonas, Micrococcus, Coryneforms, Flavobacterium, Cytophaga, Alcaligenes, Agrobacterium Bacillus Control: 300 Mpa for 2 min at 20 °C , 0 d Isolated from agar plates incubated at 7 °C (Linton et al., 2003) Moraxella, Acinetobacter, Flavobacterium, Cytophaga Storage at 2 °C 14 d Bacillus*, Moraxella, Acinetobacter Storage at 2 °C, 28 d C. gigas oyster High hydrostatic pressure Bacillus, Moraxella, Acinetobacter, Micrococcus, Coryneforms, Flavobacterium, Cytophaga, Enterobacteriaceae, Staphylococcus Bacillus, Micrococcus, Alcaligenes, Agrobacterium, Staphylococcus Control: 500 Mpa for 2 min at 20 °C , 0 d Isolated from agar plates incubated at 30 °C (Linton et al., 2003) Bacillus, Moraxella, Acinetobacter, Pseudomonas, Micrococcus, Flavobacterium, Cytophaga, Alcaligenes, Agrobacterium , Staphylococcus Storage at 2 °C, 14 d Moraxella*, Acinetobacter* Storage at 2 °C, 28 d Pacific oyster/Coffin Bay, Australia Refrigeration Prosthecomicrobium, Mycoplasma, Helicobacter, Terasakiella Vibrio, Arcobacter, Pseudoalteromonas Storage at 4 °C, 7 d 16S rRNA pyro- sequencing (Madigan et al., 2014) Sydney rock oysters/ Australia Refrigeration Mycoplasma, Spirochaeta, Haloplasma Pseudoalteromonas, Vibrio, Colwellia Storage at 4 °C, 7 d (Madigan et al., 2014) Pacific oysters (C. gigas) High Pressure Aeromonas, Vibrio, Pseudomonas, Maraxella, Acitenobacter, Micrococcus, Coryneforms, Lactobacillus, Leuconostoc, Enterobacteriaceae, Bacillus Shewanella, putrifaciens, Pseudomonas, fluorescens 260 MPa for 3 min, stored at 2 °C, 14 d API identification system (Cruz-Romer et al., 2008a) Pseudomonas spp.* 500 or 800 MPa for 5 min stored at 2 °C, 14 d Commercial oyster/ US High Pressure Gammaproteobacteria, Alphaproteobacteria, Shewanella, Vibrio, Psychrobacter High pressures of Culture isolates with (Prapaiwong et al., 2009a) 380 Flavobacteria, Bacilli, Actinobacteria, Sphingobacteria 250 to 400 MPa for 1 to 3 min 16S rDNA identificaiton Commercial oyster/ US Quick Frozen NA Shewanella* (in winter); Shewanella*, Vibrio*, and Psychrobacter * (in summer); Psychrobacter* and Vibrio (dominant in fall) Quick Frozen oysters were kept at -20 °C Culture isolates with 16S rDNA identificaiton (Prapaiwong et al., 2009a) Pacific oyster (C. gigas)/ Tasmania Refrigeration Proteobacteria* Spirochaetes, Planctomycetes, Verrucomicrobia, Fusobacteria, Firmicutes, Tenericutes, Cyanobacteria, Bacteroidetes Psychrilyobacter spp.* (phylum Fusobacteria), Fusobacteria, Spirochaetes 4 °C T-RFLP (Fernandez‐Pi quer et al., 2012) Bacteroidetes* 15 °C & 30 °C Oyster (C. plicatula) gill/ Fujian, China Refrigeration L. raffinolactis, Weissella cibaria, Lactococcus sp., Lactococcus lactis subsp. lactis, E. mundtii, E. coli, Aeromonas, Lactococcus garvieae, A. hydrophila subsp. hydrophila Lactococcus*, Lactobacillus*, Weissella confusa, C. difficile 10 °C, 4 & 8 d DGGE (Chen et al., 2013) Lactococcus, Weissella, Enterobacter, Aeromonas 4 °C, 6 & 12 d (Chen et al., 2013) Eastern Oyster (C. virginica)/ Dauphin Island, US Refrigeration V. parahaemolyticus, V. shiloi, V. vulnificus V. diazotrophicus, Listonella anguillarum, V. vulnificus Refrigeration at 6 ± 2 °C Total Bacteria and Vibrio- Specific DGGE (W ood and Arias, 2015) Oysters (Tiostrea Chilensis)/ Chile Room temperature NA Pseudoalteromonas species Room temperature (18 °C) at 4, 25, and 100 h after harvest PCR 16S-23S rDNA (Romero, González et al., 2002) C. gigas oyster/ South Korea Only for raw oyster test Lactobacillus spp., V. alginolyticus, V. proteolyticus NA NA 16S rRNA gene sequencing (Lee et al., 2010) Pacific oysters(C. gigas), Deep Bay, Hong Kong Raw oyster Pseudomonas spp.*, Vibrio, Acinetobacter, Coliforms, Aeromonas spp., Flavohacterium, Cytophaga, Coryneforms, Alcaligenes, Micrococcus NA NA Culture Isolation and identification (Kueh and Chan, 1985) Oysters (C.corteziensis , C. gigas and C. sikamea) Commercial production Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes NA NA Pyro- sequencing approach of the 16S rRNA gene (Trabal et al., 2012) Commercial oysters (C. corteziensis) Different growth phases (post-larvae, juvenile, and adult) ß-Proteobacteria (post-larvae, juvenile, and adult), Spirochaetes (juvenile), Actinobacteria (juvenile) NA Different growth phases PCR, RFLP, TGGE (Fernández et al., 2014) 381 Commercial oysters (C.gigas) -Proteobacteria (post-larvae, juvenile, and adult) β-Proteobacteria (post-larvae, juvenile, and adult) -Proteobacteria (adult), Bacilli (post-larvae, juvenile, and adult ) NA Mangrove oysters/ Gbolokiri creek, Nigeria Depuration of oysters Bacteria: Bacillus spp., Pseudomonas aeruginosa, Proteus spp., Vibrio spp., E. coli, S. aureus, Acinetobacter sp., Micrococcus sp., Corynebacterium sp., Lactobacillus spp. Fungi: Aspergillus niger, A. flavus, A. nidulans, Penicillium spp., Fusarium sp., Rhodotorula sp. Bacteria: Bacillus, Pseudomonas aeruginosa, Proteus spp., Vibrio spp., Streptococcus spp., S. aureus Fungi: ND Brackish water treatment Culture isolated bacterial (Amadi, 2015) Raw oyster Bacteria: Bacillus*, Pseudomonas*, Vibrio, Streptococcus, Proteus, Lactobacillus, Micrococcus, Corynebacterium Fungi: Aspergillus, Penicillium, Fusarium Storage at (30 ± 2°C) ambient temperature for 24 h Oyster (Crassostrea plicatula)/ Fujian, China Gill Lactococcus*, Photobacterium, Weissella, Lactobacillus*, Enterococcus, Enterobacter, Leclercia, Escherichia, Spirochaeta, Aeromonas, Citrobacter Lactobacillus*, Lactococcus* Modified Atmosphere Package DGGE (Chen et al., 2016) * Dominant bacteria; ND: not detected; NA: not available. Photobacterium, Alcaligenes, Micrococcus, Staphylcoccus, Lactococcus, Lactobacillus, Corynebacetrium, and Bacillus Mycoplasma. In addition, fungi of Aspergillus, Penicillium, Fusarium and Rhodotorula were obtained in oyster. The cultivation sites, life stages (e.g. post-larvae at the hatchery, juvenile, and adult) and the oyster species (Crassostrea corteziensis, C. gigas, and Crassostrea sikamea) have an impact on the microbiological communities in oyster ( Trabal et al., 2012; Fernández et al., 2014). In addition to aforementioned microbiota, Shewanella and Photobacterium were identified in spoilage oysters (Richards et al., 2008). Pseudomonas and Vibrionaceae were frequently detected as dominant spoilage bacteria in oyster storage. Cao et al. (2009) studied the C. gigas from Yellow Sea in China, and results showed that Pseudomonas and Vibrionaceae were dominant bacteria in raw oyster which accounted for 22% and 20 % of the total bacteria, respectively. Whereas, Madigan et al. (2014) pointed out that two genera causing the spoilage of Saccostrea glomerata and C. gigas oysters were Pseudoalteromonas and Vibrio. Seasonal difference affects the microbiota in fresh oysters, thus it also determines the dominant micobiotas in spoilage oyster. Psychrobacter appears to be predominant only in fall. Quick frozen oysters primarily contained Shewanella in winter, Shewanella, Vibrio, and Psychrobacter in summer, and Psychrobacter and Vibrio in fall, and most common dominant genera of high pressure treated oyster were Shewanella (15.7 - 23.9 %) and Vibrio (21.4 - 22.6 %) from all seasons (Prapaiwong et al., 2009a). 382 The initial bacterial communities have decisive effect on dominant spoiled bacteria microbiotas in oyster, because spoiled bacteria were demonstrated to be main bacteria detected in fresh oyster in the previous studies. For instance, Cao et al. (2009) found that Pseudomonas and Vibrionaceae in fresh oyster were growing to be dominant bacteria after treatment and chilling storage. In addition, the dominant spoilage bacterial microbiota (e.g., Bacillus, Moraxella and Acinetobacter) after high hydrostatic pressure treatment and storage were also found in fresh oyster (Linton et al., 2003). It is worth noting that not all dominant bacteria in fresh oyster are eventually growing competitive and became dominant spoiled bacteria after storage. Wood and Arias (2015) found that C. virginica oyster were dominated by V. parahaemolyticus (44 %), followed by V. shiloi (21 %) and V. vulnificus (13 %), whereas V. parahaemolyticus was replaced by other nonpathogenic Vibrio species (e.g., Vibrio species, V. diazotrophicus, Listonella anguillarum, V. vulnificus, and unidentified uncultured bacteria) after two weeks storage at 6 ± 2 °C (Amadi (2015) found that the dominant bacteria are Bacillus (20.8 %) and Pseudomonas (16.7 %), whereas those of fungal species are Penicillium species (45.4 %) and Aspergillus flavus (34.1 %). The role of fungi in oyster deterioration and spoilage should be assessed in the future investigation. In oyster, the initial microbiota in different tissues was studied in previous reports as summarized in Table 3. The oyster tissues including gill, stomach, gut, digestive glands and gonads, body fluid, rectal area, crystalline, lower intestine, digestive diverticulum, pallial fluid were detected by culture or molecular approaches. From Table 3, the micriobiotas in different tissues of oyster harvested from different locations were different. Early in 1960, Colwell and Liston (Colwell and Liston, 1960) analyzed microbiota in gill, stomach, and body fluid in the Pacific oysters using cultivation and subsequent biochemical identification. In the past two decades, with the development of molecular analysis techniques for microbiology, the studies in microbiotas from different oyster tissues were gradually increased (Table 3) Table 3 Microbiota in different oyster tissues Tissues Oyster species/ Location Microbiota Analysis methods Reference Glands Sydney rock oysters (Saccostrea glomerata)/ Australia -Proteobacteria, -Proteobacteria, Fusobacteria, Firmicute, Spirochaetes, Chlorophyta, Cyanobacteria, Actinobacteria RFLP (Green and Barnes, 2010) Digestive glands and gonads C. gigas oyster/ Todos Santos Bay, Mexico -Proteobacteria, Gram-positive bacteria with a low G+C FISH (Hernández-Zárate and Olmos-Soto, 2006) Stomach and C. virginica oyster/ Louisiana, US Mycoplasma, Planctomyctes Roche 454 pyrosequencing platform (King et al., 2012) gut Phylotypes closely related to Shewanella and Chloroflexi Gill and C. gigas oyster/ Japanese Pseudomonas,Vibrio, Flavobacterium, Culture and isolates (Colwell and Liston, 1960) stomach Pseudomonas,Vibrio, Achromobacter, Flavobacterium, Micrococcus, Bacillus Body fluid Pseudomonas,Vibrio, Achromobacter, Flavobacterium, Corynebacterium, Micrococcus, Bacillus, Enterococci Culture and isolates Rectum Pseudomonas/Vibrio, Achromobacter, Alcaligenes, Flavobacterium, Micrococcus, Bacillus Culture and isolates Gill C. plicatua oyster/ Fujian, China Lactococcus raffinolactis, Weissella cibaria, Lactococcus sp., Lactococcus lactis subsp. lactis, Enterococcus mundtii, E. coli, Aeromonas aquariorum, Aeromonas jandaei, Lactococcus garvieae, A. hydrophila subsp. hydrophila DGGE (Chen et al., 2013) Gill C. gigas oyster/ Todos Santos Bay, Mexico Cytophaga, Flavobacterium, -Proteobacteria FISH (Hernández-Zárate and Olmos-Soto, 2006) - and -Proteobacterias, Pseudomonas spp. and Bacillus spp. PCR (Hernández-Zárate and Olmos-Soto, 2006) 383 Gill C. pacifica Methanobrevibacter, Corynebacterium, Macrococcus, Streptococcus, Prosthecochloris, Flavobacterium, Sphingomonas, Paracoccus, Maritalea, Nevskia, Schlegelella, Paramoritella, Shewanella, Vibrio, Moraxella, Acinetobacter, Endozocomonas, Spongiobacter Automated ribosomal intergenic spacer analysis (ARISA) (Zurel et al., 2011) C. savignyi Methanobrevibacter, Thalassobacter, Endozoicomonas, Spongiobacter, Acinetobacter, Moraxella, Limnobacter, Schlegelella, Neisseria, Stenotrophomonas, Nevskia, Vibrio, Prosthecochloris, Staphylococcus, Flavobacterium, Eudoria, Corynebacterium, Actinomyces Stomach C. gigas oyster/ Deep Bay, Hong Kong, China Pseudomonas spp., Vibrio, Acinetobacter, Coliforms, Aeromonas spp., Flavohacterium, Cytophaga, Coryneforms, Alcaligenes Culture and isolation (Kueh and Chan, 1985) Crystalline Vibrio, Acinetobacter, Coliforms, Aeromonas spp., Alcaligenes Digestive diverticulum Pseudomonas spp., Vibrio, Acinetobacter, Coliforms, Aeromonas spp., Flavohacterium, Cytophaga,, Coryneforms, Alcaligenes Lower intestine Pseudomonas spp., Vibrio, Acinetobacter, Coliforms, Bacillus Aeromonas spp., Coryneforms, Alcali genes, Micrococcus Gut and Eastern oyster (C. virginica) Bacterial groups include Bacteria (EUB338 I, II, & III), Bacteroidetes (CF319a), and Pseudomonas Group I (Pseudo120) T-RFLP (Pierce et al., 2016) pallial fluid Gills C. gigas oyster/ Shanghai, China Vibrio, Aeromonas, Photobacterium, Pseudoalteromonas, Dokdonella, Microbacterium, Micrococcus, Flavobacterium, Psychrilyobacter, Bacillus, Granulicella, Firmicutes, Verrucomicrobia Culture-independent DGGE (W ang et al., 2014a). Digestive glands Vibrio, Aeromonas, Photobacterium, Pseudoalteromonas, Pseudomonas, Dokdonella, Microbacterium, Micrococcus, Flectobacillus, Flavobacterium, Bacillus, Granulicella, Verrucomicrobia Residual tissues Vibrio, Aeromonas, Photobacterium, Dokdonella, Microbacterium, Micrococcus, Flavobacterium, Fusobacterium, Bacillus, Granulicella, Verrucomicrobia As filter-feeding shellfish, oysters ingest nutrients and microbiology by gills. Thus, the gills of oysters accumulate different types of microorganisms, including Pseudomonas, Vibrio, Flavobacterium, Lactococcus, Aeromonas, Leuconostoc, Lactobacillus, Bacillus, Weissella, Enterobacter, Pseudoalteromonas and Enterococcus, Photobacterium, Dokdonella, Microbacterium, Micrococcus, Psychrilyobacter, Granulicella, Firmicutes,Verrucomicrobia ( Colwell and Liston, 1960; Hernández-Zárate and Olmos-Soto, 2006; Zurel et al., 2011; Chen et al., 2013; Wang et al., 2014a). Colwell and Liston (1960) separated the different part of the Pacific oyster to 384 study the original microbiotas, which showed that Pseudomonas, Vibrio and Flavobacterium were dominant bacteria by traditional cultivation methods. Spoiled micriobiotas of oyster gill under 4 °C, 10 °C , and 20 °C storage could be clearly characterized by DGGE, through which Lactobacillus and Lactococcus were found to be the dominant bacteria at various investigating temperatures (Chen et al., 2013). Other methods, including FISH, Automated Ribosomal Intergenic Spacer Analysis (ARISA), PCR, were also used to investigate the oyster gill microbiotas (Hernández-Zárate and Olmos-Soto, 2006; Zurel et al., 2011). The microbiotas in oyster stomach included Pseudomonas, Vibrio, Achromobacter, Flavobacterium, Micrococcus, Bacillus, Miscellaneous, Acinetobacter, Coliforms, Aeromonas, Flavohacterium, Cytophaga, Coryneforms, and Alcaligenes (Colwell and Liston, 1960; Kueh and Chan, 1985; Hernández-Zárate and Olmos-Soto, 2006). More microbiota information were obtained through Roche 454 pyrosequencing platform by King (King et al., 2012). Kueh and Chan (1985) indicated that the microbiota communities in stomach, crystalline, digestive diverticulum, and lower intestine were different when studying the inner parts of Pacific oysters (C. gigas). Among those microbiotas, Vibrio, Acinetobacter, Coliforms, Aeromonas were detected in all analyzing parts. However, Pseudomonas was previous regarded as main spoilage bacteria found in stomach, digestive diverticulum, and lower intestine (Kueh and Chan, 1985; Cao et al., 2009). The bacteria in the parts of digestive diverticulum and glands were also studied to demonstrate the relationship among the digestive system and original microbiota. RFLP was used and results showed that those microbiota were belonged to -Proteobacteria, -Proteobacteria, Fusobacteria, Firmicute, Spirochaetes, Chlorophyta, Cyanobacteria, Actinobacteria (Green and Barnes, 2010). FISH revealed that -Proteobacteria and Gram-positive bacteria with a low G+C were dominant (Hernández-Zárate and Olmos-Soto, 2006). Kueh and Chan reported that the isolates from glands mainly belong to Pseudomonas, Vibrio, Coliforms, Aeromonas (Kueh and Chan, 1985). Through culture-independent DGGE technology, the dominant communities were clearly profiled (Wang et al., 2014a). These bacteria were considered as the most commonly reported microbiotas in shellfish (Xuyama and Qusi, 1987). The microbiota were complex in whole oyster, because the high diversity in oyster gill, gland, stomach, body fluid, rectal area, and gut were all included in above microbiota studies. Microbiota in oyster preservation Oyster spoilage resulting in quality losses during preservation was investigated by many researchers (Cruz-Romero et al., 2008b; Cao et al., 2010; Xi et al., 2012; Bunruk et al., 2013; Chen et al., 2014). The shelf life and quality changes of raw and treated oysters were well documented. Preservative methods, such as high-pressure treatment (López-Caballero et al., 2000; Prapaiwong et al., 2009b), chitosan coating (Cao et al., 2009), and ozone treatment (Cao et al., 2010; Chen et al., 2014), have been proven to effectively slow down the reproduction of spoilage bacteria. In these studies, the spoilage bacteria were investigated by a culture-dependent method followed by traditional oyster isolate identification. The microbiotas in oyster were mainly affected by the preservation technologies as below. Refrigeration Temperature is the major impact factor for the microbiota in oyster during storage. Different bacterial communities of spoiled oyster under various storage temperatures were summarized in Table 2, which showed that storage temperature affects the dominant bacteria in the oyster microbiota. After stored at 0 °C, 5 °C and 10 °C, Pseudomonas became the major species and took up to 42 % - 66 % of detected microbiotas, and Vibrionaceae was around 20 % (Cao et al., 2009). Abundant Pseudomonas was also found in sampled oysters (Tiostrea chilensis) stored at room temperature (18 °C) (Romero et al., 2002). Except Pseudomonas, Bacillus became dominant bacteria in the oysters if the storage temperature is up to 30 ± 2 °C (Amadi, 2015). At phylum level, Bacteroidetes became the dominant bacteria under 15 °C and 30 °C storage (Fernandez‐Piquer et al., 2012). The spoilage bacteria in the different species of oysters could be different at the same storage temperature. After the storage at 4 °C for 7 days, the spoilage bacteria were Vibrio, Arcobacter for Pacific oysters, and Pseudoalteromonas, while the spoilage bacteria were Pseudoalteromonas, Vibrio and Colwellia for Sydney rock oysters (Madigan et al., 2014). The spoilage bacterial microbiota of Pacific oyster (C. gigas) after 4 °C storage were Psychrilyobacter spp., Fusobacteria, Spirochaetes (Fernandez‐Piquer et al., 2012). Chen et al. (2013) revealed that the main spoilage microbiotas in the gill of oyster were Lactococcus, Lactobacillus, Weissella confusa and C. difficile under 10 °C storage, while the main spoilage microbiota were Lactococcus, Weissella, Enterobacter and Aeromonas under 4 °C storage. Furthermore, the impact of modified atmosphere packaging (MAP) on gill microbiotas suggested that the investigation on the mechanism of oyster spoilage microbiotas during preservation requires to be focused on different tissues as well (Chen et al., 2016). High pressure treatment Cruz-Romero et al. (2008a) demonstrated that the dominant spoilage microbiotas in oyster were Shewanella putrifaciens and Pseudomonas fluorescens after 260 MPa treatment for 3 min and stored at 2 °C for 14 days, while the dominant spoilage bacteria was Pseudomonas spp. after 500 or 800 MPa treatment for 5 min and stored at 2 °C for 14 days. High pressure can inactivate Vibrio effectively in oyster. The Vibrio spp. accounted for 44 % of the microbiotas in untreated oysters, while they were not detected in all high pressure treated oysters after storage at 2 °C for 14 days (Cruz-Romero et al., 2008a). However, Prapaiwong et al. (2009a) demonstrated that the predominant 385 bacteria were Shewanella, Vibrio and Psychrobacter (only in the fall) after treated by high pressures of 250 to 400 MPa for 1 to 3 min, in which Vibrio were survived and became dominant bacteria. High hydrostatic pressures were also utilized in oyster treatment. After 300 Mpa treatment for 2 min, the dominant bacteria were Moraxella, Acinetobacter, Flavobacterium, and Cytophaga after 14d of storage at 2 °C. After 500 Mpa treatment for 2 min, the dominant bacteria were Bacillus (90%), Moraxella, Acinetobacter (10%) after 28 d storage at 2 °C (Linton et al., 2003). Other technologies Other treatments, such as ozonated water treatment, quick frozen and supercritical fluid CO2 pasteurization, were also evaluated. The results of quick frozen treatment of oysters at -20 °C showed that the predominant bacteria were Shewanella in winter, and Shewanella, Vibrio, and Psychrobacter in summer as well as Psychrobacter and Vibrio in fall through 16S rDNA identification (Prapaiwong et al., 2009a). Cao et al. (2010) used ozonated water (5.0×10-6 g/L ozone) to treat oysters for 2 min, and the diversity of initial microbiotas were higher than those of treated oyster, which were dominated by Pseudomonas and Vibrionaceae. As process of cold pasteurization, supercritical fluid CO2 was also proven to reduce oyster-associated bacteria (Meujo et al., 2008; Meujo et al., 2010). MAP was introduced into oyster preservation and was illustrated that appropriate atmosphere composition can inhibit the growth of microbiology and change the bacterial communities in oyster gill (Chen et al., 2016). The mechanism on oyster bacterial spoilage should be further investigated focusing not only on the loads and population of total bacteria counts, but also on the characterization of bacterial microbiotas in whole oyster and different tissues. Prospective Microbiological analysis in oyster is of vital importance as microbiotas are associated with oyster mortalities, shelf life, spoilage, and human diseases. Most studies on oyster preservation were focused on calculating bacterial counts instead of the spoilage bacterial communities during processing or storage. However, the mechanism of oyster bacterial spoilage should be further revealed by discovering the bacterial microbiotas and re-evaluated the spoilage in different oyster species and tissues, instead of focusing on the loads and population of total bacteria counts. Innovative molecular technologies have been introduced to further characterize microbiotas in oyster. These technologies have been reported as effective way for microbiota investigation, which provide more advantages to study microorganism profile than traditional cultivation. Moreover, those high throughput technologies can be used not only on diversity investigation but also on better understanding of dominant microbiota and illustration of spoilage mechanisms. The microbiota in oyster was well revealed on the basis of the present literatures, while applying state-of-the-art technologies such as metagenome and transcriptome will further clarify the functional roles of bacteria and their co-relationship. Aquaculture location and environmental condition, which determine the initial bacteria and affect the proliferation of dominant bacteria and food borne pathogenic bacteria in oyster, should also be emphasized. Furthermore, although the entire oyster microbiota has been well studied to illustrate the dominant spoilage bacteria at the end of shelf life, the spoilage mechanism needs to be characterized by different tissues. As the part of oyster, the gill and gut with complex microbiological diversity are easily resulted in spoilage before unacceptability of entire oyster, which should be paid more attention at the beginning of spoilage. The novel technologies for multi-target pathogens detection can provide potential application to prevent the outbreak of oyster diseases and human foodborne illness. 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