STUDY OF BACTERIAL FLORA IN EASTERN OYSTER (Crassostrea virginica) 
TREATED WITH HIGH PRESSURE 
 
 
Except where reference is made to the work of others, the work described in this  
thesis is my own or was done in collaboration with my advisory committee. 
This thesis does not include proprietary or classified information. 
 
 
________________________________________ 
Naparat Prapaiwong 
 
 
 
Certificate of Approval: 
 
 
 
___________________________                                  ___________________________ 
Yolanda J. Brady                                                            Covadonga R. Arias, Chair 
Associate Professor                                                         Associate Professor 
Fisheries and Allied Aquacultures                                  Fisheries and Allied Aquacultures 
 
 
 
___________________________                                  ___________________________ 
Richard K. Wallace                                                        George T. Flowers 
Professor                                                                         Interim Dean 
Fisheries and Allied Aquacultures                                 Graduate School 
 
 
STUDY OF BACTERIAL FLORA IN EASTERN OYSTER (Crassostrea virginica) 
TREATED WITH HIGH PRESSURE 
 
 
Naparat Prapaiwong 
 
 
 
A Thesis 
Submitted to 
the Graduate Faculty of 
Auburn University 
in Partial Fulfillment of the 
Degree of 
Master of Science 
 
 
 
Auburn, Alabama 
May 10, 2008 
 iii
 
 
 
STUDY OF BACTERIAL FLORA IN EASTERN OYSTER (Crassostrea virginica) 
TREATED WITH HIGH PRESSURE 
 
 
Naparat Prapaiwong 
 
 
 
Permission is granted to Auburn University to make copies of this thesis at its discretion, 
upon the request of individuals or institutions and at their expense. 
The author reserves all publication rights. 
 
 
 
 
     
___________________________ 
Signature of Author 
 
     
___________________________ 
Date of Graduation 
 
 
 
 
 
 
 
 iv
 
 
 
VITA 
 
Naparat Prapaiwong, daughter of Paitoon and Somboon Prapaiwong, was born 
on December 2, 1969, in Songkhla, Thailand.  She graduated from Tinsulanonda 
Fisheries College, Songkhla, Thailand with a Diploma of Vocational degree majoring in 
Aquaculture in 1990.  She attended Rajamangala University of Technology, Bangpra 
Campus, Chonburi, Thailand and graduated with a Bachelor of Science degree majoring 
in Fisheries in 1996.  She is a fisheries biologist in The Department of Fisheries, 
Thailand.  She entered Graduate School in the Department of Fisheries and Allied 
Aquacultures at Auburn University in August 2005.  
 
 
 
 
 
 
 
 
 
 
 v
 
 
 
THESIS ABSTRACT 
STUDY OF BACTERIAL FLORA IN EASTERN OYSTER (Crassostrea virginica) 
TREATED WITH HIGH PRESSURE 
 
Naparat Prapaiwong 
 
Master of Science, May 10, 2008 
(B.Sc. Rajamangala University of Technology, Bangpra Campus, 1996) 
 
104 Typed Pages 
Directed by Covadonga R. Arias 
 
Analysis of bacterial communities present in high-pressure treated, quick-frozen, 
and raw oysters was carried out independently during three different seasons: winter; 
summer; and fall of 2006.  Oysters used in all experiments were supplied by Bon Secour 
Fisheries, Inc. Bon Secour, AL.  Determination of bacterial numbers and species 
diversity in each sample was conducted at 0, 7, 14, and 21 days of storage at 4
o
C (high-
pressure treated and raw oysters) and -20
o
C (quick-frozen oysters). 
Results show that the numbers of total bacterial counts in treated oysters were 
significantly lower than in untreated oysters at day 0 in all samplings.  Total numbers of 
aerobic bacteria in high-pressure treated oysters at day 0 were lower than 10
5
 colony 
forming units (CFU)/g in every season whereas quick-frozen oysters maintained their 
 vi
levels between 10
4
 and 10
6
 CFU/g during the 21-day storage period regardless of the 
sampling season.  However, total bacterial counts in quick-frozen oysters during this 
study were statistically different in all seasons (P<0.05).  Season has significant 
influence on variation of total bacterial numbers in both treated and untreated oysters 
(P<0.05).  An increase in the total number of bacterial counts in high-pressure treated 
oysters observed at day 7, 14, and 21 indicating that some bacteria can survive the 
treatment and can be proliferate during storage at 4
o
C.  High-pressure treated oysters 
presented lower total bacterial counts than raw oysters at the time oysters get to market.  
However, the recommended shelf-life for this product (3 weeks) seems to be too long 
based on the number of bacteria present in the oysters after 2 weeks under strict 
refrigeration. 
Sequencing of the 16S rDNA from bacterial isolates revealed seven different 
classes within the bacterial communities in oysters.  The majority of the isolates were 
Gram-negative bacteria, with the Gammaproteobacteria class representing between 56% 
and 92% of all sequences.  The remaining Gram-negative belonged the 
Alphaproteobacteria, Betaproteobacteria, Flavobacteria and Sphingobacteria classes.  
Gram-positive bacteria included two classes: Actinobacteria and Bacilli.  The most 
common bacterial genera found in this study were Shewanella, Vibrio and 
Psychrobacter.  Four species of human pathogenic bacteria were also identified: V. 
vulnificus, V. parahaemolyticus, V. alginolyticus, and Aeromonas hydrophila.  Vibrio 
vulnificus was identified only from untreated (raw) oysters. 
 
 
 vii
 
 
 
ACKNOWLEDGEMENTS 
 
I would like to express my gratitude and obligation to my advisor, Dr. 
Covadonga R. Arias for providing me the opportunity to pursue research and obtain a 
Master degree in the US, as well as her guidance, suggestions and inspiring comments 
throughout my research.  I am grateful for the time and expertise offered by my 
committee members, Dr. Yolanda J. Brady and Dr. Richard K. Wallace.  I would also 
like to thank Mr. Chris Nelson, Vice President Oyster procurement, Bon Secour 
Fisheries, Inc. for supplying all oyster samples used in this study.  
Help and support from administration staff in the Department of Fisheries and 
Allied Aquacultures and friendship and encouragement extended to me by friends in the 
department is also gratefully acknowledged.  I would like to apply my deep appreciation 
to my friends, Suttinee and Suwanit for their assistance, support, and encouragement 
during all aspects of this study.   
Most of all, the greatest thanks to my mother, sisters and brothers for their love, 
support, and encouragement during this study.  
 
 
 
 
 viii
 
 
 
Style manual or journal used: Applied and Environmental Microbiology
 
Computer software used: Microsoft? Office Word 2003; Microsoft? Office Excel 2003; 
SigmaPlot 8.0; SAS 9.1.3; EndNote X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ix
 
 
 
TABLE OF CONTENTS 
 
LIST OF TABLES............................................................................................................ xi 
LIST OF FIGURES ......................................................................................................... xii 
CHAPTER I. LITERATURE REVIEW.............................................................................1 
CHAPTER II. OBJECTIVES...........................................................................................16 
CHAPTER III. ENUMERATION OF BACTERIA IN HIGH-PRESSURE 
TREATED, QUICK-FROZEN AND RAW OYSTERS......................................19 
Abstract.................................................................................................................20 
Introduction...........................................................................................................21 
Materials and Methods .........................................................................................24 
Results...................................................................................................................25 
Discussion.............................................................................................................27 
CHAPTER IV. BACTERIAL COMPOSITION AND SPECIES 
IDENTIFICATION IN COMERCIAL OYSTERS..............................................36 
Abstract.................................................................................................................37 
Introduction...........................................................................................................38 
Materials and Methods .........................................................................................41 
Results...................................................................................................................44 
Discussion.............................................................................................................46 
 x
GENERAL CONCLUSIONS...........................................................................................65 
CUMULATIVE BIBLIOGRAPHY .................................................................................68 
APPENDIX.......................................................................................................................89 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 xi
 
 
 
LIST OF TABLES 
 
Table 1.     Experimental design for oyster sampling and bacterial enumeration.............18 
Table 2.     Winter sampling: total bacterial counts, presumptive Vibrio spp. 
counts, and presumptive V. vulnificus counts isolated from high-
pressure treated, quick-frozen, and raw oysters during a 21-day 
storage period..................................................................................................30 
Table 3.     Summer sampling: total bacterial counts, presumptive Vibrio spp. 
counts, and presumptive V. vulnificus counts isolated from high-
pressure treated, quick-frozen, and raw oysters during a 21-day 
storage period..................................................................................................31 
Table 4.     Fall sampling: total bacterial counts, presumptive Vibrio spp. counts, and 
presumptive V. vulnificus counts isolated from high-pressure treated, 
quick-frozen, and raw oysters during a 21-day storage period.......................32 
Table 5.     Identification of isolates recovered from high-pressure treated oysters.........49 
Table 6.     Identification of isolates recovered from quick-frozen oysters......................53 
Table 7.     Identification of isolates recovered from raw oysters. ...................................56 
Table 8.     Pathogenic bacteria recovered from high-pressure treated, quick-frozen, 
and raw oysters versus total number of sequenced isolates............................61 
 
 xii
 
 
 
LIST OF FIGURES 
 
Figure 1.     Winter sampling: total bacterial counts (CFU/g) from high-pressure 
treated (HP), quick-frozen (QF), and raw (RW) oysters. .............................33 
Figure 2.     Summer sampling: total bacterial counts (CFU/g) from high-pressure 
treated (HP), quick-frozen (QF), and raw (RW) oysters. .............................33 
Figure 3.     Fall sampling: total bacterial counts (CFU/g) from high-pressure 
treated (HP), quick-frozen (QF), and raw (RW) oysters. .............................34 
Figure 4.     Total bacterial counts (CFU/g) from high-pressure treated oysters 
sampled in winter, summer, and fall 2006....................................................34 
Figure 5.     Total bacterial counts (CFU/g) from quick-frozen oysters sampled in 
winter, summer, and fall 2006. .....................................................................35 
Figure 6.     Total bacterial counts (CFU/g) from raw oysters sampled in winter, 
summer, and fall 2006. .................................................................................35 
Figure 7.     Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from high-pressure treated oysters 
sampled in winter 2006.................................................................................62 
Figure 8.     Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from high-pressure treated oysters 
sampled in summer 2006..............................................................................62 
 xiii
Figure 9.     Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from high-pressure treated oysters 
sampled in fall 2006......................................................................................62 
Figure 10.   Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from quick-frozen oysters sampled in 
winter 2006. ..................................................................................................63 
Figure 11.   Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from quick-frozen oysters sampled in 
summer 2006. ...............................................................................................63 
Figure 12.   Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from quick-frozen oysters sampled in 
fall 2006........................................................................................................63 
Figure 13.   Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from raw oysters sampled in winter 2006. ......64 
Figure 14.   Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from raw oysters sampled in summer 2006. ...64 
Figure 15.   Pie diagram illustrating the class-level diversity of 16S rRNA gene 
bacterial sequences isolated from raw oysters sampled in fall 2006............64 
 1
 
 
 
I. LITERATURE REVIEW 
 
Oysters are animals that belong to the order Ostreoida, class Bivalvia, phylum 
Mollusca.  They live in marine or brackish water environments where they provide 
habitat to numerous aquatic species.  Oysters present a pronounced bilateral asymmetry 
typical of their class.  Their internal organs are covered by a fleshy fold of tissue called 
mantle.  A highly calcified shell surrounds their body with strong adductor muscles 
holding the shell closed to protect them from predators.  Oysters are filter feeders that 
obtain food by using their gills to filter plankton, microorganisms, and suspended 
particles from the surrounding water.  They are gonochoric organisms in which male 
and female are in separate individuals, however they may change sex one or more times 
during their life span.  Typically, oysters mature during the first year as males that 
produce sperm for reproduction.  After that period, they spend the next two to three 
years as females that can produce up to 100 million eggs per year (86). 
Oysters inhabit waters with a depth of 2.5 to 8 meters and tolerate temperatures 
between -2 and 32
o
C.  They usually attach to hard substrates encountered during the 
spat stage and live there for their whole life.  Oysters provide habitat for other 
organisms such as anemones, hooked mussels, and barnacles.  In addition, oysters have 
served as a food for humans for many centuries.  Different species can be readily found 
 2
in most of the coastal areas around the world.  Oyster fisheries constitute sizeable 
industries in many countries (86). 
Since oysters are filter feeding organisms, they concentrate microorganisms in 
their body and may serve as vectors for transferring serious food-borne pathogens to 
humans (35).  Millions of oysters (including raw oysters) are consumed annually in the 
United States.  Some of them carry infectious pathogens, such as Vibrio vulnificus 
(123), which can cause severe illness or even death.  Fortunately, many bacterial food-
borne pathogens present in oysters can be reduced and/or destroyed by recently 
developed post-harvest treatment technologies (6, 7, 19, 68).  These treatments try to 
reduce or eliminate consumer risks.  On the other hand, extending the shelf-life of 
oysters is an additional benefit of applying post-harvest treatments to oyster products.  
Extended shelf-life will result in increasing consumer demand for these products.  
 
Oysters industry in the Gulf of Mexico  
The eastern oyster (Crassostrea virginica Gmelin, 1791) is known as the 
American oyster, the Virginia oyster, or the Atlantic oyster.  It is one of the most 
valuable and prominent shellfish in the United States.  The eastern oyster occurs 
naturally and is widely distributed from the Gulf of St. Lawrence (Canada) to the Gulf 
of Mexico.  The eastern oyster is one of seven exotic oyster species introduced to the 
Pacific coast of North America by the late 19
th
 century in British Columbia, 
Washington, Oregon, and California.  Also, it was transported to Hawaii in early 1866 
(86).  There is no precise documentation about the importation of market oysters for the 
Gulf of Mexico, but the 60?s can be considered as the beginning of eastern oyster 
 3
industry in this area.  This corresponds to a period of decline of oyster resources in the 
Chesapeake Bay due to MSX disease (caused by the sporozoan, Haplosporidium 
nelsoni) and by increased harvesting.  Consequently, the transportation of live oysters 
between Florida, Louisiana, and Texas became profitable due to the high demand of 
oysters (86). 
Most eastern oysters are now harvested from the Gulf of Mexico, which is the 
major harvesting site for this species in the U.S.  In 2006, the amount of eastern oysters 
landed in the Gulf of Mexico accounted for roughly 89% of all the eastern oysters 
harvested from all around the U.S.  The value of landings in the Gulf has increased 
yearly from $20,138,817 in 1980 to $62,161,461 by 2006 (106).  Typically, oyster 
harvesting in the U.S. can be done throughout the year; however the quality of the meat 
varies depending on the season harvested (97).  When oysters are harvested, they are 
transported to wholesalers and/or processors for treatment and packing before being 
distributed to retailers and consumers.  Some may be directly delivered from the 
harvester to restaurants or retailers without processing.  Presentation of oysters to 
customers depends on how they were processed.  Oysters can be sold as whole oysters, 
shucked, and half-shell processed.  In addition, they can be kept fresh and alive, frozen, 
pasteurized, smoked or canned.  Oysters can be consumed raw or cooked based on 
consumer?s taste and the quality of freshness, flavor, odor, and texture.  The in-shell 
oysters seem to be in higher demand in summer than in other seasons, whereas shucked 
oyster demand tends to increase during winter season (3). 
 
 
 4
Food safety  
Although the consumption of seafood is very high, health risks associated with 
the consumption of raw or uncooked seafood are common around the world (119, 125).  
In the year 2004, seafood products accounted for 37% of food-borne illnesses in the 
U.S. (44).  Consumption of raw oysters containing food-borne pathogens can cause 
diseases in humans including primary septicemia (20, 41, 90, 92, 104, 111, 136) and 
gastroenteritis (23, 37, 38, 83, 100, 107, 110, 126, 134, 146).  Handling seafood or 
being exposed to seawater or seafood contaminated with human pathogens can cause 
severe wound infections (20, 111, 136). 
Because oysters concentrate particles from their surrounding waters, human 
pathogens present in that water are accumulated within the oysters making them a 
vector for diseases.  Typically these pathogens appear to be accumulated in oysters 
through their filtering system rather than multiply in their body (84).  Among the 
pathogens present in oysters the genus Vibrio is the main concern for the public health 
authorities (125).  Typical food-borne Vibrio pathogens include: V. vulnificus, V. 
parahaemolyticus, V. fluvialis, V. hollisae, V. mimicus, V. cholerae, V. alginolyticus, V. 
harveyi, V. pelagius, V. splendidus, and V. campbellii (125, 138).  There are some other 
typical food-borne human pathogens that can be found in oysters such as Salmonella 
spp., and Escherichia coli (122, 144).  However, these are not autochonous marine 
microbes but the result of fecal contamination. 
Vibrio vulnificus is a Gram-negative halophilic bacterium, typically found in 
marine or estuarine environments (114).  The major form of V. vulnificus infections in 
the U.S. is a primary septicemia associated with, mostly, the consumption of raw 
 5
oysters.  Vibrio vulnificus is responsible for most seafood-related death cases in the U.S. 
each year (123).  Gastroenteritis in humans can be caused by many bacterial pathogens.  
However, V. parahaemolyticus is one of the major causes of gastroenteritis in the U.S.  
Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium that occurs naturally 
in estuarine and marine environments.  It can be present in pathogenic or non-
pathogenic forms and can be isolated from fish and shellfish, including oysters.  There 
are a number of cases of infections caused by V. parahaemolyticus in the U.S. each year 
from eating raw oysters or shellfish (118). 
Because of the risks involved in consuming raw oysters, there is a growing 
concern expressed by the seafood industry as well as the consumers.  The National 
Shellfish Sanitation Program (NSSP) was launched to try to solve this problem.  The 
NSSP is a cooperative program, which was developed in 1925 for controlling diseases 
associated with the consumption of raw shellfish (55).  The program supports and 
develops the sanitation of shellfish produced for human consumption.  The members of 
NSSP include the Food and Drug Administration (FDA), the Environment Protection 
Agency (EPA), the National Ocean and Atmospheric Administration (NOAA), agencies 
from shellfish producing and non-producing states, shellfish industries, and some 
foreign governments.  In order to control the quality of the shellfish produced in the 
U.S., NSSP has issued guidelines such as the Model Ordinance and others to ensure that 
shellfish products are safe for human consumption.  The Model Ordinance lays out the 
minimum requirements necessary to regulate the interstate commerce of shellfish.  The 
Model Ordinance assures the safety of shellfish products from cultivating site, to 
 6
harvesting, transportation, shucking, packing, post-harvest processing until products are 
shipped to consumers (56). 
 
Microbial content in oysters 
Microbial contents in seafood depend on the host organism, environment where 
they live, eating habits, post-harvest handing or processing treatment and the indigenous 
microbial flora that can grow under storage condition (29, 57).  In general, the number 
of heterotrophic bacteria is greater in bivalve shellfish than in its surrounding water 
(89).  Oysters harvested under warm-water conditions are likely to have higher number 
of Vibrionaceae than the ones collected from cold water environments (25, 85, 92, 116).  
In addition, colder temperatures favor bacteria clearance from oysters better than in 
higher water temperature (67).  However, some bacteria are commonly present in 
shellfish under cold storage conditions for example members of the genera 
Pseudomonas and Moraxella/Acinetobacter (13). 
The study of microbiota in shellfish attracts interest due to public health 
concerns, since pathogenic bacteria can be concentrated in shellfish and cause severe 
illness in humans through consumption of raw or uncooked oysters.  Several studies 
have been performed in order to investigate microbial composition in shellfish.  Natural 
bacterial biota in oysters has also been investigated (32, 60).  Several genera of bacteria 
have been isolated from oysters such as Aeromonas, Acinetobacter, Alcaligenes, 
Achomobacter, Alteromonas, Campylobacter, Clostridium, Marinomonas, 
Flavobacterium/Cytophaga, Proteus, Pseudomonas, Pseudoalteromonas, Nocardia, 
Serratia, Salmonella, Escherichia, Enterococci, Enterobacter, Shewanella, 
 7
Micrococcus, Bacillus, Lactobacillus, Corynebacterium, Staphylococcus, Vibrio, and 
Corynebacterium (11, 18, 32, 61, 64, 78, 91, 93, 102, 122, 142). 
At least ten genera of bacterial pathogens have been implicated in seafood-borne 
illnesses.  Bacterial pathogens associated with fecal contamination represent only 4% of 
the shellfish-associated outbreaks, whereas naturally-occurring bacteria accounted for 
20% of shellfish-related illnesses and 99% of the deaths.  Most of these indigenous 
bacteria fall into the family Vibrionaceae which includes the genera Vibrio.  In general, 
Vibrio spp. are not associated with fecal contamination and therefore fecal indicators do 
not correlate with the presence of Vibrio (95).  The genus Vibrio consists of more than 
40 species, some of them being pathogenic to humans (26).  Among those V. vulnificus, 
V. parahaemolyticus, V. alginolyticus, V. splendidus, V. harveyi, V. phosphoreum, V. 
cholerae, V.  crassostreae, V. aestuarianus, V. natriegens, V. campbellii, V.  fluvialis, V. 
hollisae, V. mimicus, and V. pelagius are usually isolated from oysters (30, 38, 40, 42, 
43, 47, 48, 63, 120, 128, 129, 138). 
 
Post-harvest processing  
The National Shellfish Sanitation Program, FDASAN defines post-harvest 
processing of shellfish as ?processing of shellfish for the purpose of added safety or 
quality that involve hazards not addressed by controls in NSSP Model ordinance 
Chapter XI through XIV?.  The same source also defines raw shellfish as ?shellfish that 
have not been thermally processed to an internal temperature of 145
o
 or greater for 15 
seconds (or equivalent) or; 2) altering the organoleptic characteristic? (54). 
 8
Most of shellfish, including oysters, are eaten raw or lightly cooked.  These 
conditions are inadequate to eliminate the bacterial pathogens that are concentrated 
through their filtering mechanism.  There are several methods to protect consumers 
from oyster-borne illnesses including good practices in harvesting, processing, 
distribution, retailing, food-handling, preparation, and consumption behaviors (1, 145).  
Post-harvest treatment is an option that can be used to eliminate human pathogens in 
seafood including oysters.  Processors and wholesalers of oysters in the U.S. are 
required to apply post-harvest treatments to both half-shell and shucked oysters and it is 
required that all oyster traders must be qualified by NSSP in order to sell their products 
in intrastate and/or interstate markets.  The purpose of using post-harvest treatments is 
to reduce bacterial human pathogen(s) to safe levels for human consumption.  For 
example, the process that is used to reduce V. vulnificus and V. parahaemolyticus must 
be capable of reducing them to non-detectable levels (<30 MPN/gram) (53).  
Additionally, extending the shelf-life of oyster products can be brought about by post-
harvest treatments.  The available post-harvest treatment technologies for oysters 
include high-pressure processing, quick-freezing, pasteurization, and irradiation (103).  
 
High-pressure processing (HPP) 
High-pressure processing (HPP), also referred to as high hydrostatic-pressure 
processing (HHP) or ultra high-pressure processing (UHP) (51), is a novel food 
processing technology, first applied commercially to oysters in the summer of 1999 in 
Louisiana (105).  Foods processed with HPP are subjected to pressures between 100 and 
800 megapascals (MPa) at ambient temperatures.  High-pressure processing is 
 9
considered as an effective and one of the most commonly used technologies for food 
preservation since it has ability to reduce and/or to destroy the microbial community 
present in the food, lengthening shelf-life, while providing a safer and better quality 
food, and increasing market value.  Other advantages of this technology is that since it 
does not use heat, sensory and nutritional attributes of the product remain virtually 
unaffected, yielding products with better quality than those processed by traditional 
methods.  Furthermore, equipment for large-scale production of HPP processed 
products is commercially available these days.  This technology is being readily adapted 
and used by the oyster industry and has become the most promising non-thermal 
process to provide pathogen-free oysters (112, 127).  During the high-hydrostatic 
pressure process, according to Motivatit Seafoods, Inc., oysters are loaded into a water-
filled pressure chamber, which is then sealed and pressurized at 40,000 psi (pound per 
square inch).  After treated, oysters may be sold in-shell wrapped with a plastic band to 
hold the shell firmly shut and are shipped to wholesalers/retailers for distribution to 
consumers.  They can also be shucked into half-shell or frozen using liquid nitrogen in 
order to lengthen shelf-life. 
High-pressure processing induces numerous changes to the morphology, 
biochemical reactions, genetic mechanisms, cell membranes, and microorganisms 
(132).  Inhibitory effects of pressure on microorganisms perhaps are caused by the 
inactivation of essential enzymes and changes in membrane permeability (73, 128).  
Pathogenic and spoilage microorganisms in meat can be inactivated using high-pressure 
treatment but effects on the muscle ultrastructure, myofibrillar proteins, meat texture, 
myoglobin, meat color, and lipid oxidation in muscle have also been documented (28).  
 10
High-pressure processing does induce changes in color that generally imparted a 
cooked, more voluminous and juicy appearance to the oyster tissue.  The moisture 
content of oysters increased while ash and protein contents decreased (36).  It has been 
reported that sensory quality of high-pressure treated oysters is not altered from raw 
oysters after proper level of high-pressure treatment (109).  However, after high-
pressure treatment, oyster muscles became detached from their shells, resulting in 
shucking, which adds high value to the product. 
The higher the pressure, the more effective the shucking but it also has the most 
deleterious effect on oyster quality as measured by the quality index method (QIM).  
Optimum shucking pressures that cause minimum changes to oysters appearance are in 
the range of 240 to 275 MPa (68).  High pressure reduces the number of total 
microorganisms, H
2
S-producing microorganisms, lactic acid bacteria, Brochothrix 
thermosphacta, and coliforms in oysters by 10
5
 CFU/g (96).  High-pressure treatment is 
effective in reducing total bacterial loads and Vibrio spp. in raw oysters (74).  It has 
been shown that pathogenic Vibrio species are susceptible to high-pressure treatment at 
pressure levels between 200 and 300 MPa (17).  Using high-pressure treatment at the 
pressure of 345 MPa for 30 and 90 seconds numbers of V. parahaemolyticus in oysters 
can be reduced to non-detectable levels (24).  However, different serotypes of V. 
vulnificus and V. parahaemolyticus require different levels of pressure in order to treat 
them successfully (34). 
 
 
 
 11
Quick-freezing/frozen (QF) 
Freezing is a well established technology for preserving foods.  The process can 
stop the progress of the activities of spoilage microorganisms in and on foods and can 
preserve some microorganisms for long periods of time.  Frozen foods have an excellent 
overall safety record.  There are few occurrences of food-borne illness associated with 
frozen foods.  This indicates that most, although not all, human pathogens are killed by 
commercial freezing processes.  Freezing kills microorganisms by physical and 
chemical effects and even possibly through the induction of genetic changes.  Some 
studies propose that many pathogenic microorganisms may be sub-lethally injured by 
freezing (9).  However, some studies shown that frozen oysters were responsible for 
multiple outbreaks of virus infections (143). 
The cryogenic individual quick-frozen process has been in use for over a 
decade.  In this process, oysters are opened and left on the half-shell, and are then 
passed through a freezer tunnel that rapidly cooled them down using liquid CO
2
.  
Shellfish regulators have accepted the scientific data demonstrating the effectiveness of 
the cryogenic individual quick-frozen process in reducing V. vulnificus to nondetectable 
levels.  However, this process has not been adapted for shucked oysters (105, 113).  
High numbers of V. parahaemolyticus can be inactivated in freezing conditions.  The 
time of total inactivation depends on the initial number of micro-organisms and 
incubation temperature (101).  Although, Johnston and Brown (2002) showed that 
Vibrio organisms, whether in the culturable or the non-culturable form, were not 
inactivated by freezing at -20
o
C (80).  Frozen storage of edible oyster showed 
decreasing in moisture, protein, alpha amino nitrogen, and glycogen whereas free fatty 
 12
acids increased.  Viable organisms counts, staphylococci, motile aeromonads, total 
coliforms, and fecal indicator organisms decreased (14). 
 
Pasteurization 
Pasteurization is a very old food preservation method, invented by Louis Pasteur 
by the late 19
th
 century.  Pasteurization is the process of treating a food by heating it to a 
certain point for a certain time to reduce numbers of harmful organisms such as 
bacteria, viruses, and molds to safe levels for human consumption without harming the 
flavor or quality of the food.  Pasteurization has been used with milk, beer, wine, fruit 
juices, cheese, and egg products.  There are various types of pasteurization available for 
food processing.  High temperature-short time pasteurization uses temperatures from 
71.5?C (160?F) to 74?C (165 ?F) for about 15 to 30 seconds.  Ultra-high temperature 
treatment or ultra-heat treatment (UHT) uses a short time, around 1-2 seconds, at 
temperature exceeding 135
o
C (175
o
F).  UHT reduces the processing time, thereby 
minimizes the spoiling of nutrients.  Low temperature pasteurization at 50
o
C for up to 
15 minutes can reduce V. vulnificus and V. parahaemolyticus to non-detectable levels, 
thus reducing the risk of infection associated with raw oyster consumption.  Spoilage 
bacteria can also be reduced by 10
2
 to 10
3
 colony forming units (CFU)/g using 
pasteurization treatment, therefore increasing the shelf-life for up to 7 days beyond the 
life of unprocessed oysters (7).  Use of hot water pasteurization followed by cold shock 
is effective in eliminating V. vulnificus and V. parahaemolyticus from whole oysters to 
non-detectable levels (6).  A pasteurization regime of 2 minutes at 70
o
C was found to be 
effective against V. vulnificus, V. parahaemolyticus, and V. cholerae (80).  Vibrio 
 13
vulnificus and total bacterial levels in Gulf Coast oysters were significantly reduced 
from 10 to 10
4
 CFU/g in the pasteurization products.  Under the NSSP, pasteurization is 
an acceptable process for shucking shell-stock (70). 
According to Ameripure Processing Company, Inc. (Franklin, LA), after oysters 
are scrubbed, cleaned, and size graded, they are then submerge in a computer-monitored 
tank of warm water for about 24 minutes, and immediately cool down in an ice water 
tank to shock bacteria and stop their activities.  After treatment, warm-water 
pasteurization treated oysters are packed on ice with the 15 days use-by-date for in-shell 
oysters or they may be shucked into the half-shell with the 21 days use-by-date labeled. 
 
Irradiation 
Irradiation is a non-thermal technology, which is capable of preserving foods 
and eliminating bacterial pathogens in foods and was discovered in the 1920s.  This 
technology was used to preserved different types of food, such as fruits, vegetables, 
dairy products, and meat, during World War II (140).  Irradiation is considered one of 
the most efficient technological processes for the reduction of microorganisms in food.  
Since irradiation can cause damages to a cell by altering its genetic materials, it has 
been successfully used as a tool to reduce pathogenic bacteria, eliminate parasites, and 
decrease post-harvest sprouting in many products (137).  It can be used to improve the 
safety of food products, and to extend shelf-life of fresh perishable foods.  Irradiated 
foods are widely accepted in world food markets.  Irradiation processing has been 
studied extensively and is now in use worldwide for many food products (5, 77).  In the 
U.S., the Food and Drug Administration (FDA) has approved irradiation for eliminating 
 14
insects from wheat, potatoes, flour, spices, tea, fruits, and vegetables.  Approval was 
given in 1985 to use irradiation on pork to control trichinosis.  Using irradiation to 
control Salmonella and other harmful bacteria in chicken, turkey, and other fresh and 
frozen uncooked poultry was approved in May 1990 (21).  The FDA is revising the food 
additive regulations to provide the safe use of ionizing radiation for controlling of 
Vibrio species and other food-borne pathogens in fresh or frozen molluscan shellfish 
such as oysters, mussels, clams, etc.  This action is in response to a request filed by the 
National Fisheries Institute and the Louisiana Department of Agriculture and Forestry 
(59). 
Irradiation is capable of serving as a potential sanitizing treatment for improving 
the sanitary quality and increasing the safety levels of shellfish including oysters for 
human consumptions (99).  Irradiation can inactivate microbial pathogens in raw 
shellfish including Vibrio species.  Irradiation at a dose of 0.75 to 1.5 kGy (KiloGray) 
can effectively inactivate V. vulnificus, V. parahaemolyticus, and V. cholerae in raw 
oysters to the safe levels (4, 31, 39, 45, 66).  Gamma radiation (
60
Co, Cobalt-60) at a 
dose of 3.0 kGy can be considered effective in inactivating V. vulnificus in frozen 
shrimp (121) and Salmonella and V. parahaemolyticus in oysters without changing their 
odor, flavor, or appearance (77).  Although irradiation appears to be an efficient method 
for eliminating human pathogens in foods it still presents some health concerns to 
consumers based on the potential residual radiation that might persist in the treated 
foods. 
 
 
 15
Decontaminations 
Some decontamination techniques such as relaying and depuration have been 
tried in order to eliminate oyster-borne pathogens.  Depurated shellfish are often 
assumed to be a bacteriologically safe product.  Conversely, it has been reported that 
vibrios have been associated with outbreaks of gastroenteritis from consumption of 
depurated oysters.  Moreover, Vibrio does not depurate well and may proliferate in 
depurating shellfish, tank water, and plumbing systems (16).  Kelly and Dinuzo (1985) 
shown that V. vulnificus appears to be slowly depurated from oysters with complete 
elimination after 16 days (84).  However, it has been shown that relaying oysters into 
waters of higher salinity than those of harvest for 7 days can reduces number of V. 
vulnificus, although some batches required 1 month or longer to reduce to <10 cells per 
gram (81). 
 
 
 
 
 
 
 
 
 
 
 
 16
 
 
 
II. OBJECTIVES 
 
Consumption of raw or undercooked oysters can lead consumers to infections by 
bacterial pathogens that are present in oysters.  Post-harvest treatments are alternatives 
for making oysters a safer product.  High-pressure treatment is known to reduce human 
pathogens to safe levels.  However, the effect of this post-harvest treatment to the 
overall oyster flora has not been well established.  Moreover, there is evidence that 
suggests that during the storage process physiological and chemical changes in the 
oyster meat occurred altering the organoleptic properties of the product.  These changes 
might be due to bacterial flora surviving the treatment. 
The main goal of this study was to enumerate the bacteria able to survive high-
pressure and quick-frozen treatments over time.  This main goal was subdivided into the 
following objectives: 
1.   To enumerate and compare bacterial numbers in untreated (raw oysters) and 
treated oysters (high-pressure treated and quick-frozen oysters) during a 21-
day storage period under refrigeration conditions. 
2.   To identify the bacterial species composition present in untreated and treated 
oysters during the storage period. 
 
 
 17
Experimental design  
This study design included the use of two types of post-harvest treated oysters as 
well as untreated oysters.  All three samples were commercial products on their way to 
the market (from the processor to the retailer).  The samples examined were: 
1.   High-pressure treated oysters 
2.   Quick-frozen oysters 
3.   Raw oysters 
Samples were collected from three different seasons (winter, summer and fall) in 
the year 2006.  High-pressure treated and raw oysters were stored at 4
o
C and quick-
frozen oysters were maintained at -20
o
C during 21 days of study following the 
recommended storage conditions of company (Bon Secour Fisheries, Inc.).  Parameters 
examined were: 
1. Total aerobic counts were determined using a general growing medium. 
2. Presumptive Vibrio and presumptive V. vulnificus were enumerated using 
selective media 
3. Identification of randomly selected colonies by genetic methods. 
Table 1. shows details of the experimental design. 
 
 
 
 
 
TABLE 1. Experimental design for oyster sampling and bacterial enumeration. 
Season Winter Summer Fall 
Days of 
storage
a
0 7 14 21 0 7 14 21 0 7 14 21 
Number of 
oysters 
sampled
b
 10 HP 
 10 QF 
18
 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW 
 10 HP 
 10 QF 
 10 RW  
Media
c
  
 
 
 
M T C M T CMTCMTCMTCMTCMT C M TCMTCMTCMTCMTC
Number of 
plates 
replicated 
3 3 3 3 3 333333333333333 3 3 33333333333333
a, Storage temperature was 4?C for high-pressure treated and raw oysters and -20?C for quick-frozen oysters. 
b, HP, high-pressure treated oysters; QF, quick-frozen oysters; RW, raw oysters. 
c, M, Marine agar; T, Thiosulfate-Citrate-Bile-Sucrose agar; C, Cellobiose-Polymyxin B-Colistin agar. 
 
 
 19
 
 
 
III. ENUMERATION OF BACTERIA IN HIGH-PRESSURE TREATED, 
QUICK-FROZEN, AND RAW OYSTERS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 20
ABSTRACT 
 
The total number of aerobic bacteria present in oysters after post-harvest 
treatments over a 3-week storage period was investigated in this study.  Two post-
harvest treatments, high-pressure and quick-frozen oysters were compared along with 
untreated (raw) oysters.  In order to test if bacterial numbers were influenced by season, 
three sampling events were carried out independently through a year.  Results show that 
the numbers of total bacterial counts in treated oysters were significantly lower than in 
untreated oysters at day 0 by 10 to 10
5
 CFU/g in all samplings.  However, an increase in 
the total bacterial counts in high-pressure treated oysters was observed at day 7, 14, and 
21 during the storage period.  Quick-frozen oysters maintained their levels between 10
4
 
and 10
6
 CFU/g during the 21-day storage period regardless of the sampling season.  
Total bacterial numbers in high-pressure treated oysters varied between seasons; 
numbers of bacteria investigated at day 0 in fall were significantly higher (P<0.05) than 
in winter and in summer.  Total bacterial counts in quick-frozen oysters during this study 
differed in all seasons (P<0.05).  Total bacterial numbers at day 0 in raw oysters in 
summer were significantly higher than in winter and fall by 10
4
 CFU/g (P<0.05).  These 
results indicate that season have an influence on the number of total bacteria present in 
both treated and untreated oysters. 
 
 
 
 
 21
INTRODUCTION 
 
Oysters are the most numerous harvested shellfish in the world and oyster 
commerce is important industries for many countries.  Oysters are filter feeders that tend 
to concentrate pathogenic microbes that can cause severe illness in humans.  Since most 
oysters are eaten raw or poorly cooked, they can act as vectors for pathogenic microbes. 
Currently, there is high consumer demand for oysters that are safe, additive-free, retain 
original flavor, nutrients, texture, appearance, and have longer shelf-life.  Post-harvest 
treatments are used to reduce pathogenic bacteria in oysters to non-detectable levels, 
thereby extending shelf-life, maintaining freshness and quality of oysters.  Currently, 
there are several FDA approved post-harvest treatment technologies for oysters.  High-
pressure processing is a non-thermal processing technique recognized as a very effective 
process to destroy food-borne microorganisms, increase the safety and lengthen oysters 
shelf-life without causing significant changes in appearance, texture, flavor, and 
nutritional constituents (112).  High-pressure processing inactivates/destroys 
microorganisms by inducing changes to the morphology, biochemical reactions, and 
genetic system (73, 132).  Changing in enzymatic reaction restrains the accessibility of 
energy to microorganisms thereby reducing the viability of the cell (12).  Several studies 
showed that HPP has a good potential in reducing enzymatic activities and 
microbiological loads including human pathogens and spoilage bacteria in foods (17, 69, 
73, 112).  Bacterial loads in shucked oysters are effectively reduced by high-pressure 
treatment (131).  Berlin et al. (1999) showed that Vibrio spp. are susceptible to 
inactivation by high-pressure treatment and that pathogenic Vibrio spp. can be 
 22
inactivated at pressure levels between 200 and 300 MPa (17).  Counts of coliforms, 
presumptive E. coli, H
2
S producing microorganisms and total viable counts in oysters 
can be reduced to below detection levels by high-pressure at 400 MPa for 10 minutes at 
7
o
C (96).  High-pressure processing can reduce the initial total microbial load in oysters 
by 10
2
 to 10
3
 CFU/g and keep levels low during subsequent storage period of over 27 
days at 4
o
C (68).  However, Furukawa et al. (2002) showed that the initial concentration 
of bacteria has an effect on the inactivation rates of cells by high-pressure treatment (62).  
Frozen foods have an outstanding record for food safety and illnesses associated 
with frozen foods are rare.  Most frozen foods are quick-freezing to minimize crystallize 
effects.  The storage temperature of frozen foods after they have been through the 
freezing process is also important.  The storage temperature can determine the final 
quality of the product when purchased and used by the consumer (98).  Freezing is 
generally an excellent way to preserve microorganisms.  However, the effects of 
freezing on most microbial pathogens are not well documented.  Bacterial spores are 
extremely resistant to the effect of freezing.  Some outbreaks indicated that certain 
human pathogens are not killed by freezing (98).  Vibrio spp. appear be quite susceptible 
to freezing, their susceptibilities are affected by bacterial density, their physiological 
state before freezing and natural cryoprotectants associated with foods in which vibrios 
are naturally found.  Oysters subjected to freezing temperatures have shown reductions 
in viable V. vulnificus numbers by 2 to 5 orders of magnitude depending on initial 
concentration and the storage temperature (33, 113).  Initial loads of V. parahemolyticus 
in oyster meat subjected to freezing reported to affect survival numbers (101). 
 23
The microflora present in oysters depends on the environment, feeding habits and 
mode of harvesting and handling.  However, conditions during storage determine which 
microbes are responsible for spoilage.  Predominant bacterial species that can survive the 
treatment process and tolerate low temperature storage in shellfish are pseudomonads 
and members of the genera Moraxella/Acinetobacter (13).  Serratia spp., Proteus spp., 
Clostridium spp., and Bacillus spp. are also associate to seafood spoilage (79).  Factors 
that affect inactivation of bacteria in foods include bacterial strain, growth phase, growth 
temperature, and composition of food matrices (98).  Linton et al. (2003) showed that 
Gram-negative bacteria are more susceptible to the high-pressure treatment than Gram-
positive bacteria, leading to an increase in the amount of Gram-positive species during 
storage (93).  The effect of high-pressure treatment on the overall microbial composition 
in oysters has not been well established.  Additionally, there is some evidence showing 
that some bacterial flora can survive after oysters have been treated with high-pressure 
and were stored over a period of time (68).  These bacteria may cause organoleptic 
changes including texture, odor, flavor, and taste of oysters treated with high-pressure 
reported by some consumers.  On the other hand, these alterations can be due to 
chemical reactions or enzymatic activities intrinsic to the process.  The objective of this 
study was to determine if bacteria could survive the treatment and if so, if they were be 
able to multiply during the storage period. 
 
 
 
 
 24
MATERIALS AND METHODS 
 
Oyster samples and storage 
High-pressure treated and quick-frozen oysters as well as raw oysters were 
provided by Bon Secour Fisheries, Inc., Bon Secour, AL.  These oysters were 
commercial oysters on their way to the market.  Exact time/date for the post-harvest 
treatment as well as from which location they were collected was unavailable to us.  
Oysters were shipped overnight under refrigeration conditions to Auburn University.  
Post-harvest treatments were carried out according to company?s procedure: Bon Secour 
Fisheries, Inc., Bon Secour, AL (quick-frozen oysters); Motivatit Seafoods, Inc., Houma, 
LA (high-pressure treated oysters).  Oysters were sampled at three different seasons: 
winter (Feb 23 to Mar 16, 2006); summer (Jul 13 to Aug 3, 2006); and fall (Nov 6 to 
Dec 7, 2006).  High-pressure treated and raw oysters were maintained at 4?C and quick-
frozen oysters were kept at -20?C during the study. 
 
Sample preparation and bacterial isolation 
Upon arrival at the laboratory, ten oysters from each treatment were randomly 
collected and aseptically shucked.  Oyster meat from ten oysters was pooled together 
and weighed.  Sterile saline water (0.85% NaCl, w/v) was added 1:1 ratio and the 
mixture was homogenized in a sterile blender.  Ten fold serial dilutions of the 
homogenate were performed (up to 10
-9
) and 100 ?l from each dilution were spread in 
triplicate, onto three different culture media: MA (Marine Agar-Difco, Becton, 
Dickinson and Company, Sparks, MD) for total bacterial count (22); TCBS 
 25
(Thiosulphate-Citrate-Bile Salts-sucrose-Difco, Becton, Dickinson and Company, 
Sparks, MD) selective medium for presumptive Vibrio count (50); and mCPC (modified 
Cellobiose-Polymixin B-Colistin, see appendix) for presumptive V. vulnificus count 
(49).  Inoculated plates were incubated at 30?C and read daily for 7 days.  The dilutions 
yielding 30-300 CFU/plate were counted and CFU/g was calculated.  The experiments 
were repeated at 7, 14, and 21 days of storage.  The colony forming units (CFU) 
calculation was performed using the following formula: CFU/g original sample = 
CFU/plate x (1/ml aliquot plated) x dilution factor. 
 
Data analysis 
Bacterial numbers were analyzed in triplicate.  The standard error was calculated 
for all replicated treatment.  F-test, Completely Randomized Design, was performed in 
order to determine if there is difference among data set using one-way Analysis of 
Variance (ANOVA) procedure in the Statistical Analysis System, SAS 9.1.3 (SAS 
Institute, Cary, NC.). 
 
RESULTS 
Tables 2, 3, and 4 showed total bacterial counts (TBC), presumptive Vibrio spp. 
count (PV), and presumptive V. vulnificus count (PVv) from winter, summer, and fall, 
respectively.  Results showed that the TBC at day 0 in high-pressure treated oysters were 
significantly lower than in the raw oysters by 10 to 10
5
 CFU/g (P<0.05).  The TBC in 
high-pressure treated oysters at day 0 were significantly lower than in quick-frozen 
oysters in summer and in fall (P<0.05) but there was no significantly different in winter 
 26
(P>0.05).  An increase in the TBC in high-pressure treated oysters was observed at day 
7, 14, and 21 during the storage period in all three sampling seasons.  The TBC reached 
> 10
7 
CFU/g at day 7 in summer and in fall and >10
8
 CFU/g at day 14 and day 21 in all 
sampling seasons (Figures 1-4).   Numbers of presumptive Vibrio spp. and presumptive 
V. vulnificus in high-pressure treated oysters at day 0 were below detectable level (<20 
CFU/g) in all sampling seasons. 
The TBC in quick-frozen oysters were significantly lower than in raw oysters 
(P<0.05) throughout the storage period (Figures 1-3).  Levels were maintained between 
10
4
 and 10
6
 CFU/g during the study regardless of sampling season (Figure 5).  Numbers 
of presumptive Vibrio in quick-frozen oysters in winter and in summer fluctuated 
between 0 and 10
3
 CFU/g whereas no vibrios were detected in fall.  Numbers of 
presumptive V. vulnificus in quick-frozen oysters were <10
2
 CFU/g in all three seasons. 
The TBC in raw oysters were higher than in post-harvest treated oysters in all 
seasons at time 0.  The TBC in summer were 10
9
 CFU/g at day 0 and gradually 
decreased to similar levels as in winter and in fall at day 7, 14, and 21 of the storage 
period.  However, the TBC in summer were higher than in every other seasons (P<0.05) 
at all sampling times (Figure 6).  Numbers of presumptive Vibrio in raw oysters varied 
between 10
2
 and 10
6
 CFU/g, lowest in fall and maintained their concentrations of about 
10
2
 CFU/g throughout storage period.  Numbers of presumptive V. vulnificus were 
between 0 and 10
4
 CFU/g, with the highest number found in summer (between 10
3
 and 
10
4
 CFU/g). 
 
 
 27
DISCUSSION 
 
Analyses of bacterial communities in high-pressure treated oysters are relatively 
scarce.  Among the few available studies, Linton et al, (2003) analyzed the microbial 
population of pacific oysters, Crassostrea gigas treated with high-pressure over 28 days 
of refrigeration at 2
o
C.  This study showed that total aerobic counts, psychrotrophic 
counts, pseudomonads and coliform were significantly reduced after pressure treatment 
(93).  He et al. (2002) reported that high-pressure processing can reduce microbial loads 
in pacific oysters by 10
2
 to 10 
3
 CFU/g (68).  Shiu (1999) showed that high-pressure is 
effective in reducing the total bacterial loads in shucked oysters (131).  To the best of my 
knowledge, this is the first time the microflora from high-pressure treated eastern 
oysters, Crassostrea virginica has been investigated.  The results of this study agree with 
those studies mentioned above in that the numbers of bacterial flora in high-pressure 
treated oysters were reduced to lower levels in comparison to raw oysters. 
However, an increase in total number of bacterial flora after high-pressure treated 
oysters were maintained at 4
o
C for 7, 14, and 21 days was observed, showing that some 
bacterial species can not only survive the high-pressure treatment but are able to 
multiply in oysters under refrigeration.  The ability of bacteria to survive the high-
pressure treatment varies dramatically and ultimately depends on their intrinsic 
susceptibility to the pressure.  In addition, several factors that affect the effectiveness of 
high-pressure treatment in reducing bacterial numbers in oysters are: the pressure used 
during treatment; the initial concentration of bacteria; the environment surrounding 
bacteria, and the bacterial composition in the oysters (98).  Gram-positive bacteria are 
 28
typically more resistant to the high-pressure treatment than Gram-negative due to the 
differences of their membrane and cell walls (130, 135).  Linton et al. (2003) reported 
that no significant increase in total bacterial counts in high-pressure treated oysters 
during a 28 days storage period although the proportion of Gram-positive bacteria 
increased from 56% to 87% due to the inactivation of the Gram-negative species (93).  
However, Smiddy et al. (2005) reported that Gram-negative E. coli 0157:H45 was 
slightly more resistant to high-pressure than Gram-positive Listeria monocytogenes 
(133).  Calik et al. (2002) compared the efficacy of high-pressure to inactivate V. 
parahaemolyticus in buffer and in oysters.  These authors found less resistance when V. 
parahaemolyticus was embedded in oyster tissue (24).  Moreover,  sub-lethal injury of 
bacteria can also cause an over-estimation of microbial inactivation, as counts taken 
immediately after high-pressure treatment can be lower than those observed after a 
period of storage, especially for microorganisms in food or nutrient-rich media (27). 
Differences of bacterial numbers in high-pressure treated oysters collected from 
three different seasons indicated that bacterial numbers in oysters are influenced by 
season and may affect the efficiency of the treatment.  This results agrees with the study 
by Kingsley et al. (2002) in where seasonal and geographical variations in oyster 
physiology and composition were showed to have an effect on the efficacy of high-
pressure treatment (87). 
As was expected, under freezing conditions bacteria were unable to multiply, 
therefore, numbers of the TBC varied little throughout the 21-day storage at -20
o
C.  
Something to consider when isolating bacteria from frozen foods is their stressed status 
that might impede them to grown on culture media.  It has been reported that the mode 
 29
of isolation and medium used can have significant impact on the recovery of 
microorganisms after freezing and thawing (98). 
In conclusion, this study demonstrated that high-pressure treatment was effective 
in reducing microbial loads in raw oysters, which should lengthen shelf-life of products.  
However, quite large numbers of bacteria survived the treatment and were able to 
proliferate during refrigeration.  These bacteria reached high numbers, even higher than 
the TBC in raw oysters, and are likely to cause spoilage and alter the organoleptic 
properties of treated oysters.  In addition, number of microflora in oysters can differ 
from season to season and this may affects the total surviving bacteria after high-
pressure treatment. 
 
 
 
 
 
 
 
 30
TABLE 2.  Winter sampling: total bacterial counts, presumptive Vibrio spp. counts, and 
presumptive V. vulnificus counts isolated from high-pressure treated, quick-frozen, and 
raw oysters during a 21-day storage period. 
TBC
a
 PV
b
 PVv
c
 
Samples days 
(CFU/g)
d
 (CFU/g) (CFU/g) 
High-pressure treated oysters 0 1.6x10
4
 10 10 
 7 8.4x10
4
 6.1x10
4
 1.3x10
3
 
 14 2.9x10
8
 7.3x10
3
 5.3x10
3
 
 21 1.5x10
9
 2.8x10
4
 1x10
3
 
Quick-frozen oysters 0 2x10
4
 3.9x10
2
 10 
 7 1.7x10
4
 2.7x10
3
 30 
 14 1.3x10
5
 0 40 
 21 9.7x10
3
 0 0 
Raw oysters 0 3.5x10
5
 9x10
2
 5x10
2
 
 7 1.8x10
7
 8.5x10
3
 4.6x10
3
 
 14 4.9x10
6
 1.4x10
3
 5x10
2
 
 21 6.7x10
7
 2.9x10
3
 0 
a, TBC, total bacterial count 
b, PV, presumptive Vibrio count 
c, PVv, presumptive V. vulnificus count 
d, CFU/g, colony forming unit per gram 
 31
TABLE 3.  Summer sampling: total bacterial counts, presumptive Vibrio spp. counts, 
and presumptive V. vulnificus counts isolated from high-pressure treated, quick-frozen, 
and raw oysters during a 21-day storage period. 
TBC
a
 PV
b
 PVv
c
 
Samples days 
(CFU/g)
d
 (CFU/g) (CFU/g) 
High-pressure treated oysters 0 1.4x10
4
 20 10 
 7 4.8x10
7
 1.2x10
2
 2x10
4
 
 14 3.4x10
8
 4.6x10
2
 1.1x10
5
 
 21 1.2x10
8
 1.5x10
2
 0 
Quick-frozen oysters 0 9.4x10
4
 1.5x10
2
 0 
 7 4.4x10
4
 0 0 
 14 1.7x10
4
 2.4x10
2
 1x10
2
 
 21 2.2x10
4
 0 0 
Raw oysters 0 1.4x10
9
 1.1x10
5
 6.7x10
3
 
 7 3.6x10
7
 4.3x10
3
 2.4x10
3
 
 14 1.3x10
8
 1x10
3
 8x10
3
 
 21 2.9x10
8
 2.5x10
5
 4x10
3
 
a, TBC, total bacterial count 
b, PV, presumptive Vibrio count 
c, PVv, presumptive V. vulnificus count 
d, CFU/g, colony forming unit per gram 
 
 
 32
TABLE 4.  Fall sampling: total bacterial counts, presumptive Vibrio spp. counts, and 
presumptive V. vulnificus counts isolated from high-pressure treated, quick-frozen, and 
raw oysters during a 21-day storage period. 
TBC
a
 PV
b
 PVv
c
 
Samples days 
(CFU/g)
d
 (CFU/g) (CFU/g) 
High-pressure treated oysters 0 3.3x10
4
 10 0 
 7 1.5x10
8
 6.1x10
2
 0 
 14 2x10
8
 1.6x10
3
 1.3x10
4
 
 21 1.4x10
9
 1.1x10
3
 1.3x10
4
 
Quick-frozen oysters 0 1.9x10
5
 0 0 
 7 1.3x10
5
 0 10 
 14 9.2x10
4
 0 30 
 21 3.5x10
5
 0 0 
Raw oysters 0 2.8x10
5
 2x10
2
 60 
 7 1.1x10
7
 3.8x10
3
 20 
 14 1.2x10
6
 3.4x10
2
 7.7x10
3
 
 21 1.1x10
7
 2.6x10
2
 2x10
3
 
a, TBC, total bacterial count 
b, PV, presumptive Vibrio count 
c, PVv, presumptive V.  vulnificus count 
d, CFU/g, colony forming unit per gram 
 
 
 33
days
0 7 14 21 
log (
C
FU/g)
0
2
4
6
8
10
HP
QF
RW
 
Figure 1.  Winter sampling: total bacterial counts (CFU/g) from high-pressure treated 
(HP), quick-frozen (QF), and raw (RW) oysters. 
days
0 7 14 21 
l
og (CFU/
g
)
0
2
4
6
8
10
HP
QF
RW
 
Figure 2.  Summer sampling: total bacterial counts (CFU/g) from high-pressure treated 
(HP), quick-frozen (QF), and raw (RW) oysters. 
 34
days
0 7 14 21 
l
o
g (C
FU
/
g
)
0
2
4
6
8
10
HP
QF
RW
 
Figure 3.  Fall sampling: total bacterial counts (CFU/g) from high-pressure treated (HP), 
quick-frozen (QF), and raw (RW) oysters. 
days
0 7 14 21 
log (
C
FU/g)
0
2
4
6
8
10
Winter
Summer
Fall
 
Figure 4.  Total bacterial counts (CFU/g) from high-pressure treated oysters sampled in 
winter, summer, and fall 2006. 
 35
days
0 7 14 21 
l
og (CFU/
g
)
0
2
4
6
8
10
Winter
Summer
Fall
 
Figure 5.  Total bacterial counts (CFU/g) from quick-frozen oysters sampled in winter, 
summer, and fall 2006. 
days
0 7 14 21 
log (CF
U
/g)
0
2
4
6
8
10
Winter 
Summer
Fall
 
Figure 6.  Total bacterial counts (CFU/g) from raw oysters sampled in winter, summer, 
and fall 2006.
 36
 
 
 
IV. BACTERIAL COMPOSITION AND SPECIES IDENTIFICATION IN 
COMERCIAL OYSTERS  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 37
ABSTRACT 
 
More than 500 strains of heterotrophic bacteria isolated from high-pressure 
treated, quick-frozen, and raw oysters sampled in winter, summer, and fall 2006 were 
identified to the genus and/or the species level by sequencing the highly conserved 16S 
rRNA gene.  Seven classes of bacteria were found among those isolates.  The majority of 
bacterial strains belonged to the Gram-negative bacterial class Gammaproteobacteria 
(between 56% and 92% depending on the sample).  The remaining of them belonged to 
the Alphaproteobacteria, Betaproteobacteria, Flavobacteria and Sphingobacteria classes.  
A low percentage of Gram-positive bacteria were identified as members of the 
Actinobacteria and Bacilli classes (1% to 5% depending on the sample).  Four isolates 
could not be assigned to any known class and were considered unclassified.  The most 
prevalent genera were: Shewanella, Vibrio and Psychrobacter.  Only four species of 
human pathogenic bacteria were identified: V. vulnificus, V. parahaemolyticus, V. 
alginolyticus, and Aeromonas hydrophila.  Vibrio vulnificus was isolated only from 
untreated (raw) oysters.  No E. coli or other fecal coliforms were identified from any 
sample. 
 
 
 
 
 
 
 38
INTRODUCTION 
 
The safety of oysters as food is related to their potential of being contaminated 
by bacterial species that can multiply to infective levels during marketing and retailing 
operations including handling and storage.  Microbial communities in shellfish have 
been examined mainly from a public health point of view, since they tend to concentrate 
pathogenic microorganisms that can cause diseases in humans.  The recovery of human 
pathogenic bacteria from shellfish has been widely reported, with most of the studies 
focusing on fecal contamination, enteric pathogens and pathogenic species of Vibrio (46, 
60).  Although more than ten genera of bacterial pathogens have been associated to 
seafood-borne diseases, they can be categorized into three general groups: i) indigenous 
bacteria, ii) non-indigenous bacteria present in the ecosystem, and iii) non-indigenous 
bacteria added during processing.  Indigenous pathogens typically associated with the 
aquatic environment include V. cholerae, V. parahaemolyticus, V. vulnificus, 
Plesiomonas spp., Listeria monocytogenes, Clostridium botulinum and Aeromonas spp. 
(only virulent strains).  Among these bacteria Aeromonas spp. and Plesiomonas spp. 
present a minimal public health hazard, particularly in comparison with the intrinsic 
risks associated with environmental Vibrio spp.  Non-indigenous pathogens often found 
in the aquatic ecosystem are mostly associated with fecal contamination that can also 
occur during harvest and post-harvest handling.  Typical examples are Salmonella spp., 
E. coli (pathogenic strains), Shigella spp., Campylobacter spp., and Yersinia 
enterocolitica (pathogenic serotypes).  Finally, non-indigenous pathogens can be 
introduced to the final product during post-harvest handling such as Bacillus cereus 
 39
(toxigenic strains), L. monocytogenes, Staphylococcus aureus and C. perfringens (58, 
124).  Pathogenic bacteria frequently isolated from oysters include Salmonella, Shigella, 
V. cholerae, V. parahaemolyticus, C. perfringens, C. botulinum, and Y. enterocolitica, 
Campylobacter, E. coli, Aeromonas, and L. monocytogenes (8, 15, 64, 76). 
Spoilage of shellfish occurs primarily because of the metabolic activities of 
microorganisms or autolysis (13).  The spoilage microflora present in shellfish is 
determined by their environment as well as by handling and storage conditions after 
landing or harvest (13, 79).  Most microbial flora present in oysters collected from 
temperate water are psychrotrophic bacteria.  Psychrotrophic microflora are 
microorganism that can cause spoilage of foods during cold storage due to their ability to 
proliferate under low temperature condition.  Studies on microbial loads in oysters 
during storage showed that each microbial group showed a distinct response to the 
various storage conditions.  For example, Salmonellae can survive in oyster meats for up 
to 14 days at cold temperatures while levels of V. cholerae increased when oysters were 
stored at cold temperatures (72). 
Bacterial composition in oysters is dominated by Gram-negative bacteria such as 
Halomonadaceae, Pseudomonadacea, Flavobactium/Cytophaga, Photobacterium, 
Vibrio, Alteromonas, Pseudoaltermonas, and Shewanella (71, 120).  Seafood, including 
shellfish, are generally spoiled by Gram-negative bacteria, which tend to be more 
pressure sensitive than Gram-positive bacteria (65).  High-pressure treatment could 
contribute to eliminating not just the human pathogens but potential spoilage bacteria as 
well.  Pressures in the range of 300?600 MPa can inactivate many bacterial vegetative 
cells (132).  In general, Gram-positive vegetative bacteria are more resistant to 
 40
environmental stressors, including pressure, than vegetative cells of Gram-negative 
bacteria.  Among the pathogenic non-sporeforming Gram-positive bacteria, L. 
monocytogenes and S. aureus are the two most well-studied regarding the use of high-
pressure processing.  Staphylococcus aureus appears to have a high resistance to 
pressure (51).  Pathogenic Gram-negative bacteria appear to have a wide range of 
sensitivity to pressure treatment.  E. coli O157:H7 shows pressure resistance comparable 
to spores (94).  In addition, some strains of Salmonella spp. have shown relatively high 
levels of pressure resistance (115).  Because of their pressure resistance and their 
importance in food safety, E. coli O157:H7 and Salmonella spp. are the main concern in 
the development of effective high-pressure food treatments (51, 94). 
It has been shown that high-pressure treated seafood has higher amounts of 
Gram-positive bacteria, markedly lactic acid bacteria, attributable to the greater 
susceptibility of Gram-negative species to high-pressure (51, 93).  Although lactic acid 
bacteria may not be completely eliminated by high-pressure, their numbers in seafood 
can be reduced and their growth interrupted (96).  Nevertheless, off-odors associated 
with spoilage due to lactic acid bacteria are generally less objectionable than those 
produced by typical spoilage bacteria which contributes to the extended shelf-life of 
high-pressure treated seafood (139).  It has also been proposed that the inhibition of 
other spoilage bacteria by lactic acid bacteria may improve the preservation of foods 
(75).  The objective of this study was to identify the composition of bacterial species 
present in treated and untreated oyster samples during periods of storage and to 
determine bacterial species responsible for the spoilage of oysters under refrigeration. 
 
 41
MATERIALS AND METHODS 
 
Bacterial samples collection  
Single bacterial colonies isolated on solid media from previous study (see 
Chapter III) were randomly picked and re-isolated in MA (Marine Agar-Difco, Becton, 
Dickinson and Company, Sparks, MD).  More than 500 isolates were selected including 
bacterial colonies from all types of samples (high-pressure treated, quick-frozen and raw 
oysters), recovered in all three media used (MA, TCBS, and mCPC) from all three 
sampling seasons (winter, summer and fall).  After culture purity was ensured, cells were 
maintained in semi-solid marine agar (0.3% agar) in the dark at room temperature until 
identified. 
Bacterial pure cultures were recovered from semi-solid agar and were inoculated 
onto MA plates and incubated at 30?C for 18-24 hours to allow bacteria grow to mid-log 
phase.  Gram test (using 3% KOH) and oxidase test [using BD oxidase reagent droppers 
(Becton, Dickinson and company, Sparks, MD)] were performed not later than 24 hours 
of post-inoculation.  Rice grain size (3 mm. long) bacterial isolates were aseptically 
collected and placed into a 1.5 ml microcentrifuge tubes and were kept in -20
o
C for 
DNA extraction. 
 
DNA extraction 
DNA extraction was carried out using the method described by Pitcher et al. 
(1989) (117).  The Gram-positive cells were pre-incubated in 100 ?l of lysozyme at 
37
o
C for 30 minutes and then resuspended in 100 ?l of TE buffer (Tris 10 mM, EDTA 1 
 42
mM, pH 8).  The Gram-negative cells were directly resuspended in 100 ?l of TE buffer.  
Five hundred microliters of the GES reagent (Guanidine thiocyanate-EDTA-Sarkosyl, 
see appendix) was added and vortexed gently for a few minutes.  Two hundred and fifty 
microliters of 7.5 M ice-cold ammonium acetate was slowly added, mixed gently, and 
left on ice for 10 minutes to precipitate proteins.  Five hundred microliters of ice-cold 
chloroform/2-pentanol (24:1) was added to the solution and the suspension was 
vigorously mixed to form an emulsion.  The mixture was centrifuged at 13,000 rpm for 
10 minutes.  Seven hundred microliters of supernatant was transferred to a new 1.5 ml 
microcentrifuge tube and then 400 ?l of cold iso-propanol was added and mixed gently 
until DNA was precipitated.  The solution was discarded and the DNA pellet was 
washed three times with 70% cold ethanol, air dried for 30 minutes, and re-suspended in 
150 ?l of sterile milli-Q water.  DNA was quantified using a GeneQuant 
spectrophotometer (Amersham Pharmacia Biotech, Sweden), and was diluted to 50 
ng/ml, and stored at -20
o
C for amplification. 
 
PCR condition and DNA amplification 
The 16S rDNA was amplified using bacterial universal primers 63V (5?-CAG 
GCC TAA CAC ATG CAA GTC-3?) and 1387R [5?-GGG CGG (A/T)GT GTA CAA 
GGC-3?] (10) in a total volume of 50 ?l.  All PCR reagents except for primers 
(Invitrogen, Carlsbad, CA) were purchased from Promega (Promega, Madison, WI).  
One hundred and fifty nanograms of DNA was used as a template in a PCR reaction 
consisting of 1X PCR buffer, 2.5 mM MgCl
2
, 0.2 mM of dNTP mix, 0.1 mM of each 
primer, and 0.15 U of Taq polymerase.  DNA amplification was carried out in DNA 
 43
Engine (PTC0200) Peltier Thermal Cycler (Bio-Rad, Hercules, CA) using the following 
PCR cycle: hot start at 94
o
C for 5 minutes, followed by 35 cycles of denaturizing at 94
o
C 
for 30 seconds, annealing at 55
o
C for 30 seconds, and extension at 72
o
C for 1:30 
minutes; a finale step at 72
o
C for 4 was added before samples were cooled down to 4
o
C.  
In order to confirm PCR successful amplification, five microliters of each 16S rDNA 
amplified product was examined on a 1% (w/v) agarose gel in 1X TAE (0.04 M Tris, 
0.02 M acetate, 0.001M EDTA) buffer containing 0.5?g/ml of ethidium bromide.  The 
gel was run for 60 minutes at 100 V in 1X TAE buffer using electrophoresis system 
TetraSource
TM
 300 (Edvotek Inc., W. Bethesda, MD).  A 1-kb ladder (Promega, 
Madison, WI) was used as a standard marker.  The PCR products were then visualized 
under a UV Tranilluminator wave length 302 nm (DyNA Light UVP, Upland, CA). 
 
DNA sequencing and sequence analyses 
The 16S rDNA amplified products were sequenced at the Genetic Analysis 
Laboratory, Auburn University.  The DNA sequences were read and edited by Chromas 
version 1.45 (Conor McCarthy, School of Health Science, Griffith University, Gold 
Coast campus, Southport, Queensland, Australia) and were compared to the bacterial 
sequences in GenBank database using Basic Local Alignment Search Tool (BLAST) 
service (2) through the National Center for Biotechnology information (NCBI) website 
(108).  Query sequences that had between 97% to 100 % identity match to those in 
GenBank were considered identified at the species level. 
 
 
 44
RESULTS 
 
The 16S rDNA from 533 bacterial isolates was amplified and sequenced.  
Sequences were compared to the ones present in GenBank and identified on the basis 
that > 97% sequence similarity is a good match at the species level.  Most of the 
obtained sequences could be ascribed to species.  Sequences with less the 97% similarity 
were identified at the genus level.  Only four isolates lack enough similarity with known 
sequences and were considered unidentified. 
 
Bacterial composition in high-pressure treated oysters 
One hundred and seventy nine bacterial isolates from high-pressure treated 
oysters were sequenced (Table 5).  Isolates belonged to five different bacterial classes.  
The majority of them belonged to the class Gammaproteobacteria, which accounted for 
82-92% of the total sequences with a 91-99% sequence similarity.  The remaining of 
them were Alphaproteobacteria, Flavobacteria, Actinobacteria, and Bacilli (Figures 7-9).  
One bacterial isolate from the fall sample could not be ascribed to any class.  Most 
common genera in high-pressure treated oysters were Shewanella (15.7-23.9%) and 
Vibrio (21.4-22.6%).  Psychrobacter was predominant only in fall; accounting for 18.6% 
of total fall isolates (Table 5).  Some pathogenic bacteria were found in high-pressure 
treated oysters, including V. parahaemolyticus, V. alginolyticus, and A. hydrophila.  
However, no V. vulnificus was identified from any samples tested (Table 8). 
 
 
 45
Bacterial composition in quick-frozen oysters 
A total of 118 bacterial isolates from quick-frozen oysters were sequenced (Table 
6).  The results showed that bacterial composition in quick-frozen oysters fall into six 
classes.  Most of isolates belonged to the class Gammaproteobacteria, which accounted 
for 56-66% of total sequences.  The remaining were Alphaproteobacteria, 
Betaproteobacteria, Flavobacteria, Bacilli, Actinobacteria, and Sphingobacteria (Figures 
10-12).  Shewanella was the dominant genus in winter sampling.  Three genera were co-
dominant in summer: Shewanella 24%, Vibrio 22.2%, and Psychrobacter 18.5%.  In fall, 
Psychrobacter was the dominant genus, representing 30.5% followed by the genus 
Vibrio 16.6% (Table 6).  Pathogenic bacteria identified from quick-frozen oysters 
included the species V. parahaemolyticus, V. alginolyticus, and A. hydrophila.  No V. 
vulnificus was detected from any seasons during the storage period (Table 8). 
 
Bacterial composition in raw oysters 
A total of 236 bacterial isolates from raw oysters were sequenced (Table 7).  
Bacterial sequences belonged to six different bacterial classes.  The most common group 
represented was Gammaproteobacteria, which accounted for 80-89% of the total 
sequences.  The rest of sequences belonged to Alphaproteobacteria, Flavobacteria, 
Bacilli, Actinobacteria, and Sphingobacteria.  Two unclassified isolates were obtained, 
one each in summer and fall samplings (Figure 13-15).  The most predominant genera in 
all sampling seasons were Vibrio and Shewanella, which comprised 41.7 and 22.9% in 
winter, 55.7 and 11.4 % in summer and 27.8 and 21.3% in fall, respectively.  In addition, 
Pseudomonas was also predominant in fall (13.9%) (Table 7).  There were several 
 46
pathogenic bacteria isolated from raw oysters.  They belonged to the species V. 
vulnificus, V. parahaemolyticus, V. alginolyticus, and A. hydrophila (Table 8).  Most of 
them were recovered from summer and fall samplings. 
 
DISCUSSION 
 
Hazardous bacteria present in seafood can be divided in to two main groups: 
indigenous bacteria naturally present in the aquatic environments and fecal bacteria of 
human or animal origin that are introduced into the aquatic environment (124).  
Contamination of seafood with pathogenic bacteria may also occur through the 
introduction of microbial during post-harvest handling or processing (124).  Typically, 
indigenous spoilage bacteria will outgrow the pathogenic bacteria during storage, 
therefore the product will spoil before pathogens increase greatly (57). 
Bacterial composition was different between oyster samples and among them 
based on sampling season.  In general, bacterial communities investigated in this study 
were dominated by Gram-negative bacteria with Shewanella spp., Vibrio spp. and 
Psychrobacter spp. as the main genera.  These bacteria have been previously reported as 
responsible agents for causing spoilage in seafood during cold storage.  In particular, 
Shewanella putrefaciens is frequently isolated from spoiled fish (82).  My results 
contradict the report of Linton et al. (2003) that indicated that most of bacteria surviving 
high-pressure treatment are Gram-positive (93).  Gammaproteobacteria was dominant in 
all samples; although it was presented in quick-frozen oysters less than in high-pressure 
treated and raw oysters in all sampling seasons.  Moreover, quick-frozen oysters seemed 
 47
to harbor higher bacterial diversity than any other type of sample tested.  In both high-
pressure treated and raw oysters higher bacterial diversity (more bacterial classes 
identified) were found in fall and summer while winter sampling displayed the least 
bacterial diversity.  In contrast, bacterial communities in quick-frozen oysters seemed to 
be more diverse in winter than in fall or summer.  This can be related to the fact that 
winter bacterial communities might be better adapted to cold shock. 
Four species of food-borne pathogenic bacteria, among those described by the 
Center for Food Safety and Applied Nutrition (CFSAN), FDA (52), were found in this 
study: V. vulnificus, V. parahaemolyticus, V. alginolyticus, and A. hydrophila.  Only the 
last three species were recovered from high-pressure treated and quick-frozen oysters.  
However, pathogenic bacteria were detected in low numbers and were detected after 
oysters were maintained for 7 to 21 days.  This finding suggests that after high-pressure 
treatment, some of these pathogenic bacteria were killed whereas some were just 
inactivated or injured and were not able to grow immediately after treated but could be 
recovered several days post-treatment.  Vibrio parahaemolyticus is known to be a very 
heat and cold sensitive bacterium (141).  However, under the same pressurized 
condition, V. parahaemolyticus appears to be more resistant than V. vulnificus and needs 
a pressure of at least 345 Mpa at 7.7 minutes in order to reduce its number by 10
4
 CFU/g 
(88).  It has also been shown that some strains of V. parahaemolyticus and some strains 
of V. mimicus are sensitive to and can be completely inactivate by high-pressure 
treatment at 200-300 MPa for 5-15 minutes at 25
o
C (17).  My results showed that at least 
a few V. parahaemolyticus can resist both high-pressure and freezing treatments. 
 48
In conclusion, the data present in this study indicate that a broad variety of 
bacteria (mostly Gram-negative) survive the high-pressure treatment as well as freezing.  
No qualitative difference between high-pressure treated and raw oysters composition 
could be inferred from my data.  Several human pathogens were recovered and identified 
from treated and untreated (raw) oysters although only raw oysters yielded V. vulnificus 
isolates.
 49
TABLE 5.  Identification of isolates recovered from high-pressure treated oysters. 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity
Winter   
0 ?-Proteobacteria Brevundimonas sp. 1 DQ676936 99 
  Brevundimonas sp. 1 EF423374 99
 ?-Proteobacteria Citrobacter gillenii 1 AF025367 99 
  Shewanella baltica  2 CP000753 97, 98 
7 ?-Proteobacteria Aeromonas veronii 1 DQ029351 98 
  Aeromonas veronii 1 EF669480 99 
  Listonella anguillarum 1 EF579965 99 
  Oceanisphaera 1 DQ190440 98 
  Pseudoalteromonas sp. 1 EF639351 98 
  Shewanella baltica 1 CP000753 98 
  Shewanella colwelliana 1 AY653177 99 
  Vibrio aestuarianus 1 AJ845015 99 
  Vibrio ordalii 1 AY628646 99 
14 Bacilli Exiguobacterium 1 EF530574 98 
  Carnobacterium 1 AY543034 98 
 ?-Proteobacteria Psychrobacter glacincola 1 EF640972 98 
  Psychrobacter nivimaris 1 EF101544 99 
  Rahnella aquatilis 1 DQ862542 99 
  Serratia odorifera  3 AJ233432 98 
  Shewanella baltica  2 CP000753 99 
  Shewanella hafniensis 1 AB205566 99 
  Shewanella putrefaciens 1 AY321590 98 
21 ?-Proteobacteria Aeromonas salmonicida 1 CP000644 91 
  Listonella anguillarum 1 EF091704 99 
  Morganella 1 DQ358137 98 
  Morganella 1 DQ358139 99 
  Obesumbacterium proteus 1 DQ223874 98 
  Pseudomonas sp. 1 AY303291 98 
  Psychrobacter aquimaris 1 EF101547 99 
  Rahnella aquatilis  2 DQ862542 97, 99 
  Shewanella baltica  2 CP000753 98 
  Shewanella putrefaciens 1 AB208055 98 
  Vibrio aestuarianus  4 AJ845015 98, 99 
  Vibrio parahaemolyticus 4 AY245192 98, 99 
  Continued on following page
 
 
 50
TABLE 5. ?Continued 
days Class Closest species  
No.of 
seq.
GenBank 
Accession 
No. 
% 
identity
Summer      
0 Flavobacteria Flavobacterium sp.  2 AJ244702 97, 99 
  Tenacibaculum lutimaris  2 AY661693 97, 98 
 Bacilli Bacillus sp. 1 EF061440 99 
  Staphylococcus sp. 1 EF061904 98 
 ?-Proteobacteria Thalassospira sp. 1 AB265822 97 
 ?-Proteobacteria Enterobacter pulveris 1 EF614996 99 
  
Enterobacteriaceae 
1 EF151985 98 
    Pseudomonas sp. 1 AM491466 95 
  Shewanella baltica  2 CP000753 99 
  Shewanella sp. 1 AJ967028 99 
  Vibrio alginolyticus 1 EF542800 99 
  Vibrio sp. 1 DQ513192 98 
7 ?-Proteobacteria Rahnella sp.  2 AM167519 98 
  Shewanella baltica  2 CP000753 99 
  Vibrio crassostreae 1 EF094887 98 
  Vibrio pomeroyi 1 AB257329 98 
  Vibrio rumoiensis  2 DQ530289 99 
  Vibrio sp. 1 DQ068945 98 
14 Actinobacteria Kocuria sp. 1 DQ531639 99 
  Enterococcus thailandicus 1 EF197994 98 
 ?-Proteobacteria Aeromonas hydrophila 1 AY827493 99 
  Aeromonas salmonicida 1 CP000644 98 
  Pantoea sp. 1 DQ094146 95 
  Serratia grimesii  6 DQ086780 97- 99 
  Shewanella baltica  3 AB205580 98, 99 
  Shewanella baltica  3 CP000753 98, 99 
  Vibrio aestuarianus 1 AJ845014 98 
  Vibrio ordalii  2 AY628646 97 
  Vibrio rumoiensis 1 DQ530289 99 
  Vibrio sp. 1 DQ068947 98 
  Continued on following page
 
 51
TABLE 5.?Continued 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity
21 Bacilli Bacillus pumilus 1 AB301018 97 
  Carnobacterium maltaromaticum 1 AY543034 99 
 ?-Proteobacteria Enterobacter amnigenus 1 AM062693 99 
  Listonella anguillarum 1 EF091704 98 
  Pantoea sp.  2 DQ094146 98, 99 
  Pseudomonas fluorescens 1 DQ207731 99 
  Pseudomonas putida 1 AM411059 98 
  Pseudomonas sp. 1 AM491466 99 
  Psychrobacter cibarius  2 AY639872 98, 99 
  Serratia grimesii 1 AY789460 99 
  Shewanella baltica 1 CP000753 98 
  Shewanella sp. 1 CP000503 98 
  Vibrio rumoiensis 1 DQ530289 99 
  Vibrio sp. 1 DQ068945 98 
Fall      
0 Flavobacteria Gelidibacter salicanalis 1 AY694009 98 
  Tenacibaculum lutimaris  2 AY661693 98 
  Vitellibacter vladivostokensis  2 AB071382 98 
 Bacilli Bacillus pumilus 1 EF672052 98 
 ?-Proteobacteria Paracoccus sp. 1 AM231059 97 
    Pseudovibrio denitrificans  2 AY486423 97 
 ?-Proteobacteria Acinetobacter sp. 1 EU000454 99 
  Pantoea dispersa 1 DQ504305 98 
  Pantoea sp. 1 EF522820 98 
  Pseudomonas sp. 1 AY770691 99 
  Psychrobacter pulmonis  2 EF101551 98, 99 
  Shewanella baltica 1 CP000753 99 
  Vibrio alginolyticus 1 EF542800 98 
  Vibrio sp. 1 AY542526 99 
7 Bacilli Bacillus firmus 1 AB271750 98 
 ?-Proteobacteria Aeromonas salmonicida  2 CP000644 99 
  Listonella anguillarum 1 DQ247934 98 
  Psychrobacter cibarius 1 AY639872 99 
  Shewanella hafniensis 1 AB205566 98 
  Shewanella putrefaciens  2 AY321590 98, 99 
  Vibrio crassostreae 1 EF094887 98 
  Vibrio parahaemolyticus 1 AF388387 99 
  Vibrio sp. 1 DQ068945 98 
  Continued on following page
 
 52
TABLE 5.?Continued 
days Class Closest species  
No.of 
seq.
GenBank 
Accession No.
% 
identity
14 Bacteria Endophyte bacterium 1 AY842148 99 
 Bacilli Bacillus pumilus 1 EF672042 99 
 ?-Proteobacteria Aeromonas salmonicida 1 CP000644 99 
  Aeromonas sp. 1 EF550579 98 
  Listonella anguillarum 1 EF579965 99 
  Proteus hauseri  2 DQ885262 98, 99 
  Proteus vulgaris 1 DQ826507 99 
  Psychrobacter cibarius  2 AY639872 98, 99 
  Psychrobacter sp.  4 AM396916 97-99 
  Serratia grimesii 1 DQ086780 98 
  Shewanella baltica 1 AB205580 98 
  Shewanella baltica  4  CP000753 98, 99 
  Shewanella hafniensis  3 AB205566 98, 99 
  Vibrio parahaemolyticus 1 AY245179 98 
  Vibrio sp. 1 DQ068945 97 
  Vibrio sp. 1 DQ068947 98 
21 ?-Proteobacteria Acinetobacter junii 1 EF669479 98 
  Listonella anguillarum 1 EF091702 99 
  Marinomonas sp. 1 CP000749 99 
  Morganella morganii  2 DQ358145 98, 99 
  Proteus vulgaris 1 DQ885257 99 
  Psychrobacter sp.  1 AJ582399 99 
  Psychrobacter sp.  2  AM396916 99 
  Shewanella baltica  3 CP000753 98 
  Vibrio pomeroyi 1 AB257324 98 
    Vibrio rumoiensis  2 DQ530289 99 
 
 
 
 
 
 
 
 53
TABLE 6.  Identification of isolates recovered from quick-frozen oysters. 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity 
Winter   
0 Flavobacteria Formosa agariphila 1 AJ893518 98
  Gelidibacter salicanalis 1 AY694009 97
  Gelidibacter sp. 1 AF513399 98
  Salegentibacter sp. 1 DQ073102 99
 Sphingobacteria Sphingobacterium 1 AB100739 93
 ?-Proteobacteria Martelella mediterranea 1 AY649762 97
  Ochrobactrum anthropi 1 AM490611 98
 ?-Proteobacteria Bordetella sp. 1 AM402948 99
 ?-Proteobacteria Microbulbifer salipaludis 1 AF479688 98
  Pseudoalteromonas 1 DQ537520 99
  Psychrobacter glacincola 1 EF640972 98
  Shewanella sp. 1 AJ271657 98
  Shewanella sp. 1 AY566557 98
  Vibrio sp. 1 DQ146980 99
7 ?-Proteobacteria Shewanella baltica 1 AB205580 99
  Shewanella baltica 3 CP000753 97-99
  Shewanella sp. 1 AJ967028 98
  1 
14 Bacilli Bacillus megaterium 1 EF690405 98
 ?-Proteobacteria Microbulbifer celer 1 EF486352 97
  Psychrobacter glacincola 1 EF640972 99
21 ?-Proteobacteria Paracoccus marcusii 2 AY881236 99
  Sphingomonas 1 AJ871434 98
 ?-Proteobacteria Serratia marcescens 1 EF627046 99
  Shewanella baltica 1 CP000753 99
  Shewanella sp. 1 AJ967026 99
Summer   
0 Flavobacteria marine bacterium 1 AB032514 99
  Mesoflavibacter 1 AB265181 96
 Bacilli Bacillus hwajinpoensis 1 AF541966 98
  Bacillus megaterium 1 AY167862 99
  Bacillus pumilus 1 AB271753 99
 ?-Proteobacteria Phaeobacter daeponensis 3 DQ981486 98, 99
 ?-Proteobacteria Shewanella baltica 1 CP000563 98
  Shewanella colwelliana 1 AB205570 98
  Vibrio alginolyticus 2 EF542800 98, 99
  Vibrio sp. 1 DQ513193 99
  Continued on following page
 
 
 54
TABLE 6.?Continued 
days Class Closest species  
No.of 
seq.
GenBank 
Accession 
No. 
% 
identity
7 Actinobacteria Microbacterium 1 AJ277840 99 
 Flavobacteria Bizionia paragorgiae 1 AY651070 98 
 Bacilli Bacillus pumilus 1 AB271753 99 
  Halobacillus salinus 1 AF500003 99 
 ?-Proteobacteria Pseudomonas fragi 1 AM062695 99 
  Psychrobacter glacincola 1 EF640972 99 
  Psychrobacter pacificensis 1 AB016054 99 
  Psychrobacter pacificensis 1 EF179615 99 
  Shewanella baltica 1 CP000753 99 
    Shewanella baltica  3 CP000563 97-99 
14 Actinobacteria Rothia arfidiae 1 DQ673322 98 
 Bacilli Bacillus pumilus 1 EF029070 99 
  Bacillus pumilus  2 AB271753 99 
 ?-Proteobacteria Psychrobacter glacincola 1 EF640972 99 
  Shewanella algae  2 X81621 98 
  Shewanella amazonensis 1 CP000507 97 
  Shewanella loihica  2 CP000606 98, 99 
  Vibrio parahaemolyticus 1 BA000031 98 
  Vibrio parahaemolyticus 1 DQ068942 98 
  Vibrio rumoiensis 1 DQ530289 99 
  Vibrio sp.  2 AY542526 98, 99 
  Vibrio sp.  2 DQ857750 99 
21 Bacilli Bacillus pumilus 1 AB271753 99 
 ?-Proteobacteria Sphingomonas 1 AJ871434 98 
 ?-Proteobacteria Psychrobacter celer 1 EF101550 99 
  Psychrobacter celer  2 AY842259 98 
  Psychrobacter glacincola  2 EF640972 99 
  Psychrobacter pacificensis 1 AB016054 99 
  Shewanella baltica 1 AB205580 99 
  Shewanella baltica 1 CP000753 98 
  Vibrio rumoiensis  2 DQ530289 98, 99 
Fall      
0 Flavobacteria Gelidibacter salicanalis 1 AY694009 98 
 ?-Proteobacteria Pseudoalteromonas 2 AB257569 99 
  Psychrobacter glacincola 1 EF640972 98 
  Continued on following page
 
 55
TABLE 6.?Continued 
days Class Closest species  
No.of 
seq.
GenBank 
Accession 
No. 
% 
identity
7 Flavobacteria Bizionia paragorgiae  2 AY651070 97, 98 
  Formosa algae 1 AY771766 98 
 Bacilli Bacillus barbaricus 1 DQ870771 99 
  Bacillus pumilus 1 EF029070 97 
  Staphylococcus sciuri 1 AB233332 97 
 ?-Proteobacteria Pseudoalteromonas sp. 1 EF551372 98 
  Psychrobacter glacincola  4 EF640972 97-99 
  Shewanella sp. 1 AJ271657 97 
  Vibrio alginolyticus 1 EF542800 97 
  Vibrio rumoiensis  2 DQ530289 98, 99 
14 Flavobacteria Arenibacter latericius 1 AF052742 98 
  Carnobacterium 1 AY543034 99 
 ?-Proteobacteria Paracoccus marcusii 1 AY881236 99 
 ?-Proteobacteria Psychrobacter glacincola  2 EF640972 98 
  Psychrobacter marincola 1 AJ309941 97 
  Stenotrophomonas sp. 1 AJ534843 96 
  Vibrio rumoiensis  2 DQ530289 98, 99 
21 Flavobacteria Bizionia algoritergicola 1 AY694003 97 
  Gelidibacter salicanalis 1 AY694009 98 
 Bacilli Bacillus niabensis 1 DQ176423 98 
 ?-Proteobacteria Pseudoalteromonas 1 AB257569 98 
  Psychrobacter glacincola  2 EF640972 97, 98 
  Psychrobacter marincola 1 AJ309941 99 
    Vibrio rumoiensis 1 DQ530289 98 
 
 
 
 
 
 
 
 56
TABLE 7.  Identification of isolates recovered from raw oysters. 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity 
Winter      
0 Flavobacteria Olleya marilimosa 1 EF660466 98 
  Polaribacter sp. 1 DQ356493 98 
 ?-Proteobacteria 
Oceanisphaera 
1 DQ190440 98 
  Pseudoalteromonas sp.  2 EF628006 98 
  Shewanella sp.  2 AJ271657 98 
  Vibrio sp. 1 AB274765 98 
  Vibrio sp. 1 DQ068948 97 
  Vibrio sp. 1 DQ649435 100 
  Vibrio sp. 1 EF584056 96 
  Vibrio sp. 1 EF584062 88 
  Vibrio splendidus 1 AM422807 99 
7 ?-Proteobacteria Psychrobacter cibarius 1 AY639872 99 
  Shewanella baltica  4 CP000753 98 
  Vibrio nigripulchritudo 1 AB297941 98 
  Vibrio sp.  2 DQ146990 98 
  Vibrio sp. 1 EF187006 99 
  Vibrio sp. 1 EF474168 98 
  Vibrio sp. 1 EF584056 97 
  Vibrio sp. 1 EF584059 83 
14 Flavobacteria Cellulophaga sp. 1 AY035869 98 
  Olleya marilimosa  2 EF660466 99 
  Psychroserpens sp. 1 DQ073103 98 
  Salegentibacter sp. 1 DQ073102 98 
 ?-Proteobacteria Pseudoalteromonas sp. 1 AF539781 95 
  Psychrobacter nivimaris 1 EF101544 98 
  Shewanella colwelliana 1 AF530131 98 
  Shewanella colwelliana  3 AY653177 98 
  Vibrio aestuarianus 1 AJ845021 83 
  Vibrio sp. 1 DQ146990 98 
  Vibrio sp. 1 EF584059 94 
   Continued on following page
 
 
 57
TABLE 7.?Continued 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity 
21 Flavobacteria Formosa agariphila 1 AJ893518 97
 ?-Proteobacteria Pseudoalteromonas sp.  2 EF628002 98 
  Pseudoalteromonas sp. 1 EF639379 96 
  Shewanella colwelliana 1 AB205570 99 
  Vibrio aestuarianus 1 AJ845015 98 
  Vibrio sp.  2 DQ068948 97 
  Vibrio vulnificus 1 AE016795 98 
Summer      
0 Actinobacteria Curtobacterium sp. 1 EF612296 98 
  Micrococcus sp. 1 EF419329 99 
 ?-Proteobacteria Erwinia soli 1 EF540893 97 
  Pseudomonas sp. 1 DQ885459 99 
  Shewanella algae  2 X81621 98 
  Shewanella loihica 1 CP000606 98 
  Shewanella sp. 1 AF249336 98 
  Vibrio aestuarianus 1 AJ845014 98 
  Vibrio aestuarianus 1 AJ845015 98 
    Vibrio alginolyticus  2 EF542800 98 
  Vibrio parahaemolyticus 3 AF388387 98 
  Vibrio sp. 1 AY542526 97 
  Vibrio vulnificus  2 AE016795 97 
  Vibrio vulnificus 1 X76333 97 
7 Actinobacteria Curtobacterium sp. 1 EF612296 99 
 ?-Proteobacteria Pseudoalteromonas sp. 1 AB261170 98 
  Pseudoalteromonas sp. 1 EF628006 98 
  Pseudoalteromonas sp. 1 EF639350 98 
  Pseudoalteromonas sp. 1 EF639351 98 
  Pseudomonas sp. 1 DQ645482 99 
  Psychrobacter 1 EF640972 98 
  Vibrio aestuarianus 1 AJ845014 90 
  Vibrio aestuarianus 1 AJ845015 98 
  Vibrio alginolyticus 1 EF542798 98 
  Vibrio alginolyticus  4 EF542800 97, 98 
  Vibrio alginolyticus 1 X74691 99 
  Vibrio parahaemolyticus 1 BA000031 99 
  Vibrio vulnificus  4 AE016795 98 
  Vibrio vulnificus  3 X76333 98 
  Continued on following page
 
 58
TABLE 7.?Continued 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity 
14 Bacteria uncultured bacterium 1 EF379672 96 
 ?-Proteobacteria Pseudoalteromonas sp. 1 EF382708 97 
  Pseudoalteromonas sp. 1 EF628006 98 
  Pseudoalteromonas sp.  2 EF639351 99 
  Psychrobacter sp. 1 AY689064 99 
  Shewanella algae 1 X81621 99 
  Shewanella baltica 1 CP000753 99 
  Vibrio aestuarianus 1 AJ845015 98 
  Vibrio alginolyticus  5 EF542800 98, 99 
  Vibrio vulnificus 1 AE016795 99 
  Vibrio vulnificus  3 X76333 98 
21 Actinobacteria Micrococcus sp. 1 EF540464 99 
 Flavobacteria Flavobacteriaceae bacterium 1 DQ660394 99 
  Myroides odoratus  3 M58777 97 
 ?-Proteobacteria 
Enterobacteriaceae 
1 DQ436917 98 
  Listonella anguillarum 1 EF091702 97 
  Pseudomonas fluorescens 1 EF552157 99 
  Pseudomonas sp. 1 DQ645482 99 
  Shewanella baltica  2 CP000753 98 
  Shewanella putrefaciens 1 AY321590 98 
  Vibrio aestuarianus  2 AJ845014 98 
  Vibrio aestuarianus 1 AJ845015 97 
  Vibrio aestuarianus  2 AJ845016 95, 98 
  Vibrio vulnificus 1 AE016795 98 
  Vibrio vulnificus 1 X76333 98 
Fall      
0 Bacteria uncultured bacterium 1 AB175370 99 
 Sphingobacteria Sphingobacterium composta 1 AB244764 97 
 ?-Proteobacteria Roseobacter sp. 1 DQ120728 97 
  Ruegeria atlantica 1 AB255399 98 
  Silicibacter pomeroyi 1 CP000031 99 
  Stappia kahanamokuae 1 EF101503 99 
    uncultured sludge bacterium 1 AF234725 98 
  Continued on following page
 
 59
TABLE 7.?Continued 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity 
 ?-Proteobacteria Acinetobacter lwoffii 1 DQ144736 99
  Listonella anguillarum 1 AM235737 98 
  Pseudomonas oryzihabitans 1 AY850170 100 
  Pseudomonas 1 DQ837704 98 
  Pseudomonas sp. 1 AM403178 98 
  Shewanella algae 1 AF005250 97 
  Shewanella putrefaciens 1 CP000681 96 
  Stenotrophomonas 1 EF620454 99 
  Vibrio aestuarianus 1 AJ845016 97 
  Vibrio shilonii  2 AY911392 98 
  Vibrio vulnificus  7 AE016795 97-99 
7 Sphingobacteria Sphingobacterium sp. 1 EF423371 99 
 ?-Proteobacteria Phaeobacter daeponensis 1 DQ981486 99 
 ?-Proteobacteria Acinetobacter johnsonii 1 DQ257425 99 
  Aeromonas hydrophila 1 EF645798 99 
  Aeromonas salmonicida 1 CP000644 98 
  Aeromonas veronii 1 EF669480 99 
  Listonella anguillarum 1 DQ247934 99 
  Marinomonas sp. 1 EF673290 92 
  Pseudoalteromonas 1 DQ537514 99 
  Pseudoalteromonas 1 AF475096 99 
  Pseudomonas putida 1 AY277620 98 
  Pseudomonas sp. 1 AY770691 99 
  Pseudomonas sp. 1 EF190347 99 
  Rheinheimera baltica 1 AJ441082 99 
  Serratia sp. 1 EF528312 99 
  Shewanella baltica 1 CP000753 99 
  Shewanella loihica  2 CP000606 98, 99 
  Shewanella sp. 1 AJ967028 99 
  Vibrio aestuarianus 1 AJ845011 98 
  Vibrio aestuarianus 1 AJ845015 98 
  Vibrio harveyi 1 EF635306 98 
  Vibrio parahaemolyticus 1 AF388387 98 
  Vibrio rotiferianus 1 AM422800 87 
  Vibrio shilonii  2 AY911392 98, 99 
  Vibrio sp. 1 DQ068946 97 
  Vibrio vulnificus  2 AE016795 97, 99 
  Continued on following page
 
 60
TABLE 7.?Continued 
days Class Closest species  
No.of 
seq. 
GenBank 
Accession 
No. 
% 
identity
14 Flavobacteria Flavobacteriaceae bacterium 2 DQ660394 96, 99
  Flavobacterium 1 AB275999 96 
  Gelidibacter salicanalis 1 AY694009 98 
 Sphingobacteria Sphingobacterium composta 1 AB244764 97 
  Sphingobacterium faecium 1 AM411066 99 
 ?-Proteobacteria Phaeobacter daeponensis 1 DQ981486 99 
  Pseudorhodobacter 3 DQ001322 99, 100 
 ?-Proteobacteria Ferrimonas balearica 1 AB193753 98 
  Pseudoalteromonas 1 AB257569 100 
  Pseudomonas sp. 1 AY690706 98 
    Pseudomonas sp. 1 AY770691 99 
  Psychromonas sp. 1 AB238791 90 
  Shewanella arctica 1 AJ877256 99 
  Shewanella baltica  6 CP000753 97, 98 
  Shewanella hafniensis 1 AB205566 99 
  Shewanella oneidensis 1 AY881235 99 
  Shewanella sp. 1 EF639386 99 
  Vibrio aestuarianus 1 AJ845013 94 
  Vibrio aestuarianus 1 AJ845015 97 
  Vibrio aestuarianus  2 AJ845021 95, 97 
  Vibrio mimicus 1 X74713 99 
  Vibrio sp. 1 AB186497 94 
  Vibrio vulnificus 1 AE016795 99 
21 Flavobacteria Empedobacter sp. 1 AM232808 99 
 Bacilli Bacillus sp. 1 EF690430 75 
 ?-Proteobacteria Aeromonas veronii 1 AY928478 99 
  Listonella anguillarum 1 DQ247934 97 
  Pseudoalteromonas 1 AF475096 99 
  Pseudomonas anguilliseptica 1 AY771754 99 
  Pseudomonas fragi 1 AM062695 99 
  Pseudomonas sp. 1 AY770691 98 
  Pseudomonas sp. 1 EF523605 81 
  Pseudomonas stutzeri 1 AJ270454 98 
  Pseudomonas synxantha 1 AY486386 99 
  Psychrobacter glacincola 1 EF640972 99 
  Shewanella arctica 1 AJ877256 99 
  Shewanella baltica  6 CP000753 97-99 
    Vibrio aestuarianus  3 AJ845015 98 
 
 61
TABLE 8.  Pathogenic bacteria recovered from high-pressure treated, quick-frozen, and 
raw oysters versus total number of sequenced isolates. 
  sample/ Number of isolate 
  day Vv
a
 Vp
b
 Va
c
 Ah
d
 
 W
e
 S
f
 F
g
 W S F W S F W S F 
HP
h
  0 0/5 0/15 0/18 0/5 0/15 0/18 0/5 1/15 1/18 0/5 0/15 0/18
         7 0/9 0/9 0/11 0/9 0/9 1/11 0/9 0/9 0/11 0/9 0/9 0/11
       14 0/12 0/22 0/27 0/12 0/22 1/27 0/12 0/22 0/27 0/12 1/22 0/27
       21 0/20 0/16 0/15 4/20 0/16 0/15 0/20 0/16 0/15 0/20 0/16 0/15
QF
i
   0 0/14 0/13 0/4 0/14 0/13 0/4 0/14 2/13 0/4 0/14 0/13 0/4 
         7 0/5 0/12 0/15 0/5 0/12 0/15 0/5 0/12 1/15 0/5 0/12 0/15
       14 0/3 0/17 0/9 0/3 2/17 0/9 0/3 0/17 0/9 0/3 1/17 0/9 
       21 0/6 0/12 0/8 0/6 0/12 0/8 0/6 0/12 0/8 0/6 0/12 0/8 
RW
j
  0 0/13 3/19 7/25 0/13 3/19 0/25 0/13 2/19 0/25 0/13 0/19 0/25
         7 0/12 7/23 2/29 0/12 1/23 1/29 0/12 6/23 0/29 0/12 0/23 1/29
       14 0/14 4/18 1/33 0/14 0/18 0/33 0/14 5/18 0/33 0/14 0/18 0/33
       21 1/9 2/19 0/22 0/9 0/19 0/22 0/9 0/19 0/22 0/9 0/19 0/22
a, Vv, V. vulnificus 
b, Vp, V. parahaemolyticus 
c, Va, V. alginolyticus 
d, Ah, A. hydrophila 
e, W, winter 
f,  S, summer 
g, F, fall 
h, HP, high-pressure treated oysters 
I, QF, quick-frozen oysters 
J, RW, raw oysters 
 62
 
Figure 7.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from high-pressure treated oysters sampled in winter 2006. 
 
Figure 8.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from high-pressure treated oysters sampled in summer 2006. 
 
Figure 9.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from high-pressure treated oysters sampled in fall 2006. 
 63
 
Figure 10.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from quick-frozen oysters sampled in winter 2006. 
 
Figure 11.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from quick-frozen oysters sampled in summer 2006. 
 
Figure 12.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from quick-frozen oysters sampled in fall 2006. 
 64
 
Figure 13.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from raw oysters sampled in winter 2006. 
 
Figure 14.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from raw oysters sampled in summer 2006. 
 
Figure 15.  Pie diagram illustrating the class-level diversity of 16S rRNA gene bacterial 
sequences isolated from raw oysters sampled in fall 2006. 
 
 65
 
 
 
GENERAL CONCLUSIONS 
 
1) Both treated oysters (high-pressure and quick-frozen oysters) presented lower 
bacterial counts at time 0 than raw oysters. 
2) Bacterial loads in high-pressure treated oysters increased over time and were 
equal or higher than in raw oysters between 14 and 21 days post-treatment. 
3) Bacterial loads in quick-frozen oysters remained fairly constant throughout the 
study. 
4) Sampling season has an effect on bacterial loads in raw oysters. However, 
post-harvest treatments were able to reduce bacterial numbers to similar levels regardless 
of season. 
5) Bacterial communities in all samples investigated were dominated by Gram-
negative bacteria mostly by members of the Gammaproteobacteria class, regardless of 
sample type or sampling season. 
6) Most human pathogens were recovered from raw oysters.  V. vulnificus was 
detected only from raw oysters. 
7) A few pathogens were identified in both types of treated oysters investigated 
in this study.  V. parahaemolyticus was isolated from both high-pressure treated and 
quick-frozen oysters several days after treatment. 
 66
8) Even though the majority of surviving bacteria found in high-pressure treated 
oysters were not human pathogens they were in high enough numbers to potentially 
cause spoilage and organoleptic changes in oyster meat. 
 
Based on these finding, I suggest that: 
1) Shelf-life of high-pressure treated oysters (3 weeks) should be reviewed. 
2) The refrigeration conditions of oysters after high-pressure treatment should be 
re-evaluated. 
3) Combining high-pressure treatment with other techniques such as 
decontamination may increase efficacy of high-pressure in reducing/inactivating 
bacterial flora.  Also, the addition of additives may help to inhibit bacterial growth 
during storage increasing the shelf-life of treated oysters. 
4) In this study basic culture media were used to estimate Vibrio spp. and V. 
vulnificus numbers.  In order to confirm the number of human pathogens presents in the 
oysters, including V. vulnificus and V. parahaemolyticus FDA recommended methods 
should be used in the future. 
 
Propose/Future works 
My work opens new venues for the post-harvest treated oysters.  Proposed topics 
for future research are: 
1) Extensive evaluation of V. vulnificus and V. parahaemolyticus resistance to 
post-harvest treatment. 
 67
2) Repeat study using FDA recommended methods to identify human pathogens 
(V. vulnificus and V. parahaemolyticus). 
3) Compare pre- and post-treated oysters from same batch and include 
pasteurization and radiation as treatments. 
4) Quantitative identification of predominant bacteria in high-pressure treated 
oysters during storage. 
5) Study of the use of additives and investigate their effects in slowing or 
stopping bacterial growth in high-pressure treated oysters during cold storage may be 
needed, in order to expand their shelf-life. 
6) Re-evaluate the organoleptic tests in high-pressure treated oysters after 1 week 
post-treatment. 
 
 
 
 
 
 
 
 
 
 68
 
 
 
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 89
 
 
 
APPENDIX 
 
mCPC Agar (FDA,CFSAN, 2001) 
 
Solution 1 
Peptone   10 g 
Beef extract     5 g 
NaCl    20 g 
Dye stock solution    1 ml 
Agar    15 g 
Distilled water            900 ml 
Adjust to pH 7.6. Boil to dissolve agar. Cool to 48-55?C.   
 
Solution 2 
Cellobiose    10 g 
Colistin     400,000 units 
Polymixin B     100,000 units 
Distilled water            100 ml 
Dissolve cellobiose in distilled water by heating gently, cool, and add antibiotics.  
 
 90
1,000X Dye stock solution 
Bromthymol blue  4 g 
Cresol red    4 g 
Ethanol, 95%          100 ml 
For consistent medium color, use dye solution rather than repeatedly weighing 
out dry dyes. Dissolve dyes in ethanol for 4% (w/v) stock solution.  Using 1 ml of this 
solution per liter of mCPC agar gives 40 mg bromthymol blue and 40 mg cresol red per 
liter. 
 
Add Solution 2 to cooled Solution 1, mix, and dispense into petri dishes. Final 
color: dark green to green-brown.  No autoclaving required.  Store at 4
o
C. Discard after 
14 days. 
 
Tris-EDTA buffer 
 
10 mM Tris   1.21g 
1   mM EDTA   0.37g 
Adjust pH to 8.0 with HCl 
Distilled water up to  1 L 
 
 
 
 
 91
Lysozyme 
 
Lysozyme   50 mg 
Sterile water     1 ml 
Dissolve lysozyme in water and aliquot (500 ?l/tube), store at -20
o
C.  Activate at 
37
o
C for 10 minutes before use.  
 
GES solution (Guanidine thiocyanate -EDTA-Sarkosyl) 
 
Guanidium thiocyanate 60 g 
EDTA      3.7 g 
Water;  approximate  20 ml 
Heat in 65
o
C water bath with mixing until dissolved, cool. 
Add: 
N-Lauryl Sarcosine (30%)    1.7 ml 
Make volume up to 100 ml with milli-Q water and filter with 0.045? sterile filter. 
Store at room temperature. If reagent precipitates, warm up in a water bath at 60
o
C 
before use.