1 THE EFFECTS OF SALINITY AND SALINITY UPSHIFTS ON THE IN VITRO GROWTH AND SURVIVAL OF PATHOGENIC VIBRIO SPECIES By MICHAEL AARON HUBBARD A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FU LFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Michael Aaron Hubbard
3 To my father, mother, and daughter
4 ACKNOWLEDGMENTS First and foremost I would like to thank my family and close friends for remaining by my side throughout my educational journey. Without their continued support this would not have been possible. I would like to thank my mentor and major professor, Dr. Anita Wright, for always keeping me focused and keeping h er faith in me. I would like to thank my committee members, Dr. Steve Otwell, Dr. Keith Schneider, Dr. Valarie Harwood, and Dr. Judy Johnson for providing valuable career advice and continued support in my graduate studies. Additionally, Victor and Laura G arrido helped to keep appreciated. Assistance from all of the helpful individuals from Apalachicola was invaluable, including help from Grady Levin and Tommy Ward, thoug h considerable time and energy was spent from Charlene Burke at the Oyster Industry Lab to help me graduate students who have helped me throughout the years, incl uding Brittany Martin, Xingyu Zhao, Daniel Bryan, Zhiyao Luo, Rick Swain, Dr. Melissa Jones, and Daniel Goldberg.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUC TION ................................ ................................ ................................ .... 11 Vibrio Species and the Eastern Oyster, Crassostrea virginica ................................ 11 Vibrio vulnificus ................................ ................................ ................................ 12 Vibrio parahaemolyticus ................................ ................................ ................... 13 Vibrio cholerae ................................ ................................ ................................ 15 Vibrio Species and Their Differential Response to Sal inity ................................ ..... 16 QPCR Detection and Enumeration of Vibrios ................................ ......................... 17 Oysters and Modern Post Harvest Processing ................................ ....................... 18 Environmental Conditions and Parameters for FDA Oyster Relay Studies ............. 20 Hypothesis and Specific Aims ................................ ................................ ................. 20 Vibrios ................................ .... 21 ................................ ..................... 21 Specific ................................ ......................... 21 2 MATERIALS AND METHODS ................................ ................................ ................ 22 Bacterial Strains and Culture Conditions ................................ ................................ 22 Dupont BAX Multiplex QPCR Assay ................................ ................................ ....... 22 Environmental Sample Collection and Studies ................................ ....................... 23 In Vitro Survival Studies ................................ ................................ .......................... 24 In Vitro Growth Studies ................................ ................................ ........................... 25 Statistical Analyses ................................ ................................ ................................ 26 3 VIBRIOS ................................ ................................ ................................ ................ 27 Rationale ................................ ................................ ................................ ................. 27 Comparison of MPN Enumeration Me thods ................................ ............................ 27 Relative Recovery of Vibrios in Relationship to Salinity ................................ .......... 29 4 .................... 32 Rationale ................................ ................................ ................................ ................. 32
6 Effects of Salinity and pH Upshift on Survival of Individual Vibrio Species ............. 33 Competitive Survival of Vibrios Following Salinity Upshift ................................ ....... 36 Differences in Plate Count and QPCR Data as Related to the VBNC State ........... 38 5 .......................... 40 Rationale ................................ ................................ ................................ ................. 40 Effects of Salinity o n Individual Growth of Vibrios ................................ ................... 41 Effects of Salinity on Competitive Growth of Vibrios ................................ ............... 42 6 SUMMARY AND CONCLUSIONS ................................ ................................ .......... 44 Enumeration of Vibrios in the Environment ................................ ............................. 44 Survival of Vibrio Species After Salinity Upshifts in ASW ................................ ....... 45 Effect of Salinity on Growth of Vibrio Species ................................ ......................... 4 7 APPENDIX A STANDARD CURVES GENERATED FOR ALL MEDIA AND SALINITIES ............ 49 B GROWTH OF VIBRIOS AT 30C UNDER VARIOUS SALINITIES ........................ 50 LIST OF REFERENCES ................................ ................................ ............................... 51 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 58
7 LIST OF TABLES Table page 3 1 FDA (BAM) vs. QPCR (BAX) log MPN/g of bacteria present in oyster homogenate s from Apalachicola Bay, FL. ................................ .......................... 31
8 LIST OF FIGURES Figure page 3 1 Map of sites selected for sampling in Apalachicola Bay, FL.. ............................. 28 4 1 Total reduction, or difference in initial and final bacterial concentration, due to salinity upshift of the different species in individual culture after 32 days at 23C by plate count. ................................ ................................ ........................... 35 4 2 T otal reduction, or difference in initial and final bacterial concentration, due to salinity upshift of the different species in individual culture after 32 days at 30C by plate count. ................................ ................................ ........................... 35 4 3 Comparison of concentrations (log CFU/mL) of individual species after 32 days incubation at A) 23C and B) 30C, by QPCR and plate count (PC).. ........ 37 4 4 Effects of salinity upshift on differences in mean total reduction between competitive and individual cultures after 32 days by QPCR at A) 23C and B) 30C. ................................ ................................ ................................ .................. 39 5 1 Effects of salinity on tota l growth yield of individual species in LB with modified NaCl concentration after 6 h incubation at 30C by plate count .......... 41 5 2 Comparison of individual and competitive total growth yield in L B after 6 hours incubation at 30C, by QPCR. ................................ ................................ 43 A 1 Standard curves generated for each media and the associated linear regressions relating Ct (QPCR) to log CFU/mL (plate count), for Vp (red) Vv (green), and Vc (blue). ................................ ................................ ........................ 49 B 1 Hourly growth of individual Vibrio species in LB at 30C under various salinities as determined by plate count for Vp (red), Vv (green), and Vc (blue). ................................ ................................ ................................ ................. 50
9 A bstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Require ments for the Degree of Master of Science THE EFFECTS OF SALINITY AND SALINITY UPSHIFTS ON THE IN VITRO GROWTH AND SURVIVAL OF PATHOGENIC VIBRIO SPECIES By Michael Aaron Hubbard May 2012 Chair: Anita C. Wright Major: Food Science and Human Nutrition Vi brio vulnificus (Vv), V. parahaemolyticus (Vp) and V. cholerae (Vc) cause 75% of seafood borne bacterial infections in the US, and translocation of oysters to areas with increased salinity is proposed as a strategy to reduce Vibrios contamination. This re search investigated the effects of salinity on the distribution, survival, and growth of Vibrio spp. Their relative concentration with respect to variation in salinity w as assessed from oysters collected at different sites in Apalachicola Bay, F lorida. M os t probable number (MPN) was determined using multiplex quantitative PCR (QPCR) for species confirmation Vv and Vp levels ranged from 2.7 to 4.0 log MPN/g for all sites, but similar levels of Vc (3.3 log MPN/g) were observed only at the site with 9.3 ppt, while sites with higher salinities of 20.1 and 21.9 ppt showed non detectable or much lower levels (0.4 log MPN/g) The in vitro effects of increased salinity and pH were examined for individual and co inoculated cultures These u pshifts significantly redu ced survival of Vv and Vc at 23C, but not at 30C or for Vp. QPCR analysis did not confirm reductions in plate counts, suggesting induction of the viable but nonculturable state Growth after 6 h at various salinities (5, 10, 20, 30, 35 ppt) showed highes t yields at 10 35 ppt 5 10 ppt and 10 ppt for Vp Vc, and Vv respectively. Competitive effects were minimal in co
10 inoculated cultures. These data suggest that design parameters for oyster management practices require careful evaluation in order to opti miz e the reduction of Vibrio contamination.
11 CHAPTER 1 INTRODUCTION Vibrio S pecies and the Eastern O yster, Crassostrea virginica The Eastern oyster ( Crassostrea virginica ) is an economically important commodity to the Florida seafood industry, with pro duction in the Gulf of Mexico valued at $60.2 million and representing 59% of total national production ( EPA, 2010 ) Oysters also play a vital ecological role in maintaining the water quality of Gulf Coast ecosystems. Though oysters have been harvested and consumed in Gulf waters for centuries, increased pressure from regulatory agencies to reduce the inciden ce of foodborne illness from this product has generated significant concern and cost throughout the industry ( Srivastava et al., 2009 ) Vibrio vulnificus (Vv) V. parahaemolyticus (Vp) and V. cholerae (Vc) are the three primary human pathogens responsible for the majority of disease related t o seafood borne bacterial infections ( Nishibuchi and DePaola, 2005 ) Numerous studies have evaluated the seasonal distribution of these ba cteria in estuarine waters and oyster s ( Levine and Griffin, 1993 ; Colwell, 1996 ; Hlady and Klontz, 1996 ; Wright et al., 1996 ; DePaola et al., 2000 ; DePaola et al., 2003a ; DePaola et al., 2010 ) Additionally, the effects of salinity on the growth and survival of the three species has been extensively investigated in the literature ( Singleton et al., 1982b ; Kaspar and Tamplin, 1993 ; Motes et al., 1998 ; Randa et al., 2004 ; Castaneda Chavez Mdel et al., 2005 ; Whitaker et al., 2010 ) The three sp ecies show differential preference for optimum growth and recovery from environmental samples over a range of salinities, but all grow well at moderate levels between 15 20 ppt ( Joseph et al., 1982 ; Singleton et al., 1982b ; Kaspar and Tamplin, 1993 ) The optimum for in vitro growth of Vv in nutrient media is
12 between 10 20 ppt, and recovery from environmental samples decreases with increasing salinity, with overall lower distribution of Vv at higher as compared to lower salinity sites ( Palasuntheram, 1981 ; Motes and DePaola, 1996 ) Therefore, the transport of oysters from lower to higher salinity areas has been proposed as a general management strategy for reduc tion of Vv in oysters. This process is often referred to as of Vibrio related disease. Although numerous studies have related salinity parameters to optimum growth, most have focused on a single species, and comparisons among species were based on different experimental conditions. Furthermore the effects of upshifting salinity on the survival of the individual Vibrio s have not been previously examined under controlled c onditions, nor have the effects of competition among the various species on their growth and survival. Investigations on multi species studies are hampered by the lack of practical procedures for the simultaneous detection and enumeration of the three spec ies. Therefore, we have employed a multiplex QPCR for simultaneous enumeration of the three species in order to better understand their responses to salinity changes, both independently and as a group. Vibrio vulnificus Vv is native to coastal ecosystems w orldwide and is part of the natural microflora of C. virginica in the Gulf of Mexico ( Morris and Blac k, 1985 ) and in the Chesapeake Bay ( Wright et al., 1996 ) Disease symptoms following consumption of raw or undercooked oysters can include gastroenteritis, but are generally characterized by primary septicemia. Wound infections may be caused by exposure to the bacterium from seawater or during seaf ood handling and may lead to tissue necrosis followed by
13 septicemia. Serious, life threatening infections associated with this bacterium occur primarily (perhaps exclusively) in individuals with underlying medical conditions, including hepatic or immune sy stem disorders, hemochromatosis, and/or diabetes ( Morris and Black, 1985 ) Although these at risk i ndividuals are encouraged to avoid consumption of raw oysters through government sponsored educational efforts, including mandatory warning signs posted in restaurants serving the raw product, over 95% of Vv cases between 2002 and 2007 occurred among indiv iduals who were at risk ( Oliver, 2005 ) The mortality rate from Vv infections can be over 50% in thes e affected individuals; however, an average of only 34 cases of Vv infections are reported annually ( Oliver and Kaper, 2001 ; Oliver, 2005 ) Despite this relatively low number of ca ses, the high mortality rate associated with this bacterium is largely responsible for pressure from FDA to encourage post harvest processing of the oyster to help reduce or eliminate disease incidence. Vibrio parahaemolyticus Unlike Vv, infection from Vp most often causes only mild gastroenteritis, although wound infections and fatal septicemia have been rarely reported ( Hlady and Klontz, 1996 ) In Asia, Vp has lon g been recognized as a leading cause of foodborne illness, with high annual incidence in China ( Wong et al., 2000 ; Wong et al., 2005 ) India ( Deepanjali et al., 2005 ) and Japan ( Su and Liu, 2007 ) In the US, Vp more recently became be a major concern for seafood safety, and CDC now estimates approximately 4, 500 cases occur per year domestically ( 2009 ) As a result, Vp is currently the most common Vibrio species isolated from human dise ase and is one of the leading causes of gastroenteritis due to consumption of raw or undercooked shellfish ( Daniels et al., 2000 ; Su and Liu, 2007 )
14 Unlike Vv, Vp affects most individuals exposed to a sufficiently high infectious dose, which has been suggested to be as low as 100 cells/g for pathogenic strains, or 10,000 MPN/g of total Vp ( Daniels et al., 2000 ; DePaola et al., 2000 ) Virulence of Vp is not completely understood, though several potential virulence facto rs have been investigated. Clinical isolates of Vp typically demonstrate hemolysis on Wagatsuma agar, and multiple sets of genes responsible for type III secretion system in the bacterium have been identified ( Miyamoto et al., 1969 ; Makino et al., 2003 ) However, though many clinical Vp strains contain TDH or a TDH related hemolysin (TRH), both commonly associated with virulence, some clinical isolated possess neither of these ge nes, and thus a definitive virulence marker has not been established for this species ( Miyamoto et al., 1969 ; Honda and Iida, 1993 ; Hiyoshi et al., 2010 ) Consequently, comprehensive environmental screening for pathogenic isolates of Vp is not currently practical. Since 2005, Reports of Vp outbreaks in Alask a ( McLaughlin et al., 2005 ) Oregon, and Washington ( CDC MMWR, 2006 ) have increased concern over this pathogen as the disease appears to now be endemic in the pacific northwest region of the US. Vp is also common to oysters from the Gulf Coast region and frequently recovered at levels between 1 3 log CFU/g ( Zimmerman et al., 2007 ) Outbreak disease incidence is relatively rare from Gulf Coast oysters, as only one outbreak has been reported from Galveston Bay, TX in 1998, resulting in over 200 suspected and 115 culture confirmed cases ( Daniels et al., 2000 ) However, sporadic cases are not uncommon; an estimated 4,500 cases occur annually in the US ( CDC, 2009 ) and
15 approximately 30% of confirmed cases originate from states bordering the Gulf of Mexico ( CDC, 2008 ) Vibrio cholerae Similar to Vv and Vp, Vc has also been determined to be a potential pathogen found in harvested oysters from the Gulf Coast Strains of Vc carrying specific virulence genes, including the ctx gene encoding cholera toxin, cause the disease cholera and most often associated with milder, self limiting gastroenteritis ( Hughes et al., 1978 ; Levine and Griffin, 1993 ) Although c holera is a serious and potentially fatal disease that still persists in some regions of Asia and Africa, relatively little attention has been placed on identifying and characterizing potentially epidemic Vc strains in the US during the past few decades ( Morris, 2003 ) This lack of concern was largely due to the extremely low disease incidence rate in the US Between 1995 and 2000, only six cholera cases were reported to be associated with consumption of raw or undercooked Gulf Coast seafood ( Morris, 2003 ) However, in March, 2011 an outbreak of toxigenic Vc serogroup O75 occurred in the southeastern US and was associated with the consumption of raw or lightly cooked oysters harvested in Apalachicola Bay, FL ( Onifade et al., 2011 ) Additionally, Tobin ( 2008 ) described eight cases of serogroup O75 with similar diarrheal disease in Georgia, Louisiana, Alabama and South Carolina, all occurring between 2003 and 2007. While serogroup O75 Vc isolates typically cause milder gastrointestinal disease symptoms when compared to infections from serogroups O1 and O139, these recent cases are evidence that rapid identification of toxigenic Vc in oysters should be a priority in the Florida seafood ind ustry.
16 Vibrio S pecies and T heir D ifferential R esponse to S alinity In environmental oyster samples, Vv appears to survive best under conditions of low to moderate salinity and warmer temperatures, with an optimal salinity for growth and survival at 10 20 ppt and temperatures at or above 26 C ( Kelly, 1982 ) Consequently, this bacterium is generally found in highest concentrations in coastal environments during summer months and in locations where salinities are within the optimal range. In offshore conditions where salinity may exceed 30 ppt, the levels of Vv are often below the limit of detection ( Motes and DePaola, 1996 ) Differences in coastal and offshore salinity gradients and the reduction of bacterial concentra tions at higher salinities are the rationale for using oyster relay as a management strategy to reduce the concentration of pathogenic Vv in the oyster. Vp can grow in waters with moderate salinity, though it has a much higher growth rate at elevated salin ities (20 30 ppt). Additionally, under conditions of high salinity (30 been observed ( Whitaker et al., 2010 ) Despite this increase in adaptability, in vitro studies with growth media at 3 0 ppt salinity have shown that the growth and survival of Vp is significantly inhibited when compared to salinity levels below 30 ppt ( K aspar and Tamplin, 1993 ) Thus, while elevated salinity may help eliminate Vv within the oyster and in seawater, these conditions may inhibit further growth but enhance the resilience and prevalence of Vp within the oyster. Thermally, Vp survives quite w ell in typical oceanic temperature gradients and can grow between 9 44 C, with an optimal growth range between 35 and 37 C ( Horie et al., 1966 ; Beuchat, 1973 ) Vc is extremely tole rant toward changes in salinity, and can persist in fresh water, brackish water, and offshore waters ( Colwell et al., 1981 ; Singleton et al., 1982b ;
17 Lintz, 2010 ) Thus, Vc is quite unique in regards to salinity requirements as compared the other two Vibrio species. While the optimum salinity for growth of th is bacterium has been reported to be approximately 25 ppt, the bacteria can proliferate and grow at less than 1 ppt and at salt concentrations of 45 ppt or higher under certain conditions ( Singleton et al., 1982b ) Consequently, while an increase of salinity may help to reduce or eliminate Vv within the oyster or seawater, it may actually enhance the growth and survival of Vp and have no significant reduction of Vc It has been reported that temperature has a much more significant effe ct on the survival of Vc in seawater, with the optimal temperature for recovery and survival reported to be within 20 30 C ( Huq et al., 1984 ; Venkateswaran et al., 1989 ) Vc has been shown to be incredibly resilient under the right environmental conditions, and survival of culturable cells was reported to be as long as eight months after inoculation in steri le seawater by McCarthy ( 1996 ) QPCR Detection and Enumeration of Vibrios Recent developments in quantitative polymerase chain reaction (QPCR) have allowed for the rapid detection and enumeration of many species of bacteria, yeast a nd molds, replacing many other laborious, time consuming, and expensive traditional methods. QPCR technology uses fluorescent dyes or probes that bind to specific DNA loci in the genome that are unique to the targeted species. As the PCR reaction proceeds, specialized instruments can provide real time detection of fluorescence signal, allowing for digital quantification of the number of target sequences. This process can also be used to directly enumerate multiple bacteria within the same sample. By using m ultiple probes and/or dyes in the initial design of the PCR assay, all possible ( Malorny et al., 2009 ) This technology, known as multiplex QPCR, is currently
18 in use and being further developed for many different species for application in the food science and food safety dis ciplines. While QPCR provides accurate detection and enumeration, the assay limit of detection can be reduced by the potential inhibition of the PCR reaction due to food matrices or other chemicals within the sample. Therefore, the combination of standard enrichment protocols used in most probable number (MPN) enumeration with QPCR for species detection and confirmation of positive samples has been employed to increase sensitivity ( Wright et al., 2007 ) A multiplex QPCR assay for the simultaneous detection and enumeration of pathogenic Vibrio species ( Vv, Vp, and Vc ) was rec ently developed by D uPont Qualicon and is being validated for use by the oyster industry. Th is assay is exploited in this study to enumerate the three species in vivo for evaluating competitive survival and growth. Oysters and Modern Post Harvest Processing Many different PHP methods currently used in industry have been shown to effectively reduc e or eliminate Vibrio levels, and some are in widespread use. Thermal processes including freezing ( Thomson and Thacker, 1973 ; Parker et al., 1994 ; Muntada Garriga et al., 1995 ; Seminario et al., 2011 ) pasteurization ( Cook and Ruple, 1992 ; Bryan et al., 1999 ) an temperatures of 50 60 C, ( Hesselman et al., 1999 ) have all been shown to reduce pathogenic Vibrios though correct temperature gradients are required to avoid thermal adaptation ( Bryan et al., 1999 ) High hydrostatic pressure is a process that has been shown to reduce Vibrio species to undetectable levels by applying 250 MPa to live oyster batches ( Berlin et al., 1999 ) ; irradiation has also been shown to achieve complete killing of pathogenic Vibrio s in oysters at levels less than 2 kGy exposure
19 ( Dixon and Rodrick, 1998 ; Andrews et al., 2003 ) However, all of the aforementioned processes currently used in the seafood industry are hampered by the fact that they are all lethal to the oyster, and consequently these methods will not satisfy the market Recent regulatory pressure from the FDA for post harvest processing has forced oyster processors and harves ters to consider alternative possibilities. Since many consumers prefer to purchase and consume a living, raw product, these demands have created an imminent problem for processors. One potential strategy is to focus on changes in pre harvest management, u sing a protocol frequently referred to as oyster Vv numbers in oysters. This process involves transporting batches of live oysters from coastal waters with moderate salinity and relatively high Vv levels to offshore waters with m uch higher salinity and no detectable Vv While using this process ( Motes and DePaola, 1996 ) mortality rates among the oysters have been reported to be less than 6% despite a nearly twofold increases in salinity. Although oyster re lay was shown to achieve > 4 log CFU/g reduction of Vv in this study, the authors did not evaluate the effects of oyster relay on recovery of Vp or Vc As discussed previously, increasing the salinity may actually favor an increase in the ability of Vp to adapt to pH and temperature stressors ( Whitaker et al., 2010 ) and may not significantly reduce the concentration of Vc ( Singleton et al., 1982a ) Since all three pathogenic Vibrio species have the ability to cause foodborne illness in humans after consumption of the raw product, understanding the effects of salinity on the three species simultaneously in the oyster is a critical consideration for developing oyster relay as a viable oyster management strategy.
20 Environmental C onditions and P arameters for FDA O yster R elay S tudies Studies are currently being conducted by the FDA Gulf Coast Seafood Laboratory in Dauphin Island, A L to analyze the effectiveness of oyster relay in the reduction of pathogenic Vibrios Commonly measured environmental parameters from these studies were used in the design of the in vitro experiments of the present study, and were de scribed by personal communication ( Jones, 2011 ) from the FDA laboratory as follows : Oysters were harvested in coastal waters and moved into 2 3 cages offshore, with each cage holding approximately 100 120 oysters. Sampling time points were 0, 7, 14, 21 and 32 days (variation due to inclement weather is noted when applicable). Oysters were taken from waters wit h salinities between 17 23 ppt (coastal) and relayed to a site between 29 35 ppt (offshore). Temperature ranged from 21 30 C. pH values of seawater ranged from approximately 7.4 (coastal) to 8.4 (offshore) Hypothesis and Specific Aims The focus of this st udy was to investigate the effects of salinity and temperature on the survival and growth of the three pathogenic Vibrio species using in vitro conditions that were similar to environmental parameters employed in large scale oyster relay studies currently ongoing in Mobile Bay, AL. The overarching hypothesis of these studies is that the relative growth and survival of the three different pathogenic Vibrio species will vary with salinity, and th eir response to salinity changes or upshifts can be exploited to develop effective management strategies for reduc tion of Vibrio levels in oysters.
21 The objective of this study includes the following specific aims: e numeration of Vibrios Investigate environmental distribution of pathogen ic Vibrios in Apalachicola Bay using standard MPN and multiplex QPCR MPN assa ys under harvesting conditions that vary in salinity and pH u pshift and s Examine individual and competitive survival of pathogenic Vibrios in artificial seawater after salinity and pH upshifts from 20 ppt (pH=7.4) to 30 ppt (pH=8.4), and from 20 ppt (pH=7.4) to 35 ppt (pH=8.4), at incubation temperatures of 23C and 30C. Specific aim g rowth y ield Examine individual and compet itive growth of pathogenic Vibrios in nutrient rich broth at various salinities, including 5, 10, 20, 30 and 35 ppt NaCl, at 30C.
22 CHAPTER 2 MATERIALS AND METHOD S Bacterial S trains and C ulture C onditions Vv, Vp, and Vc strains were stored frozen as sto ck cultures at 80C in Luria Bertani broth prepared with 1.0% tryptone, 0.5% yeast extract, and 1.0% NaCl (LBN) with 50% DI water and 50% glycerol. New isolates were streaked for isolation from stock freezer cultures to LBN agar (LA) for each experiment, and plates were incubated at 37C overnight. All media were purchased from Fisher Scientific (Hampton, NH), and reagents were purchased from both Fisher Scientific and Sigma Chemicals (St. Louis, MO). Dupont BAX M ultiplex QPCR A ssay Regardless of sample source (i.e., oyster, broth culture, or seawater), DNA extraction and QPCR protocols were followed directly from the user manual of the V. cholerae / parahaemolyticus / vulnificus Extraction reagents were prepared in advance by mixing 150 uL of protease reagent into 12 mL of extraction buffer, vortexing for 5 s, aliquoting 200 L of mixed extraction reagent per sample to extraction cluster tubes (Simport Scientific, Beloeil, Quebec; Part #:T11 2G) and storing at 4C. When samples were ready for extraction, 5 uL of target sample was added to the buffer, and the tubes were subjected to initial 20 min incubation at 37C followed by final 10 min incubation at 95C. DNA extracts were stor ed at 20C for the QPCR assay. Lyophilized QPCR reagent tubes included with the test kit were placed in a cooling block at 4C while performing the assay. Thirty uL of DNA extract was added to
23 the lyophilized QPCR reagent tube for each sample, and samples were loaded into the Dupont Q7 instrument (BAX Software v2.8). The limit of detection of the QPCR assay was previously described to be 4.0 log CFU/mL for each species ( Wallace, 2011 ) For each medium and salinity used in the in vitro studies, standard curves were generated for all species (Figure A 1; Appendix A) by relating Ct output from the instrument to log CFU/mL as determined by plate count. Using the appropriate equations from the l inear regressions as seen in each standard curve (Figure A 1; Appendix A), Ct output from the QPCR assay was used to estimate log CFU/mL of each species for comparison of competitive and individual cultures for each medium and salinity. Environmental S amp le C ollection and S tudies Oyster samples were collected from Apalachicola Bay, FL in June 2010 with the assistance of the Florida Department of Agriculture and Consumer Services (DACS) laboratory. Thirty six samples (3 samples of 12 oysters each) were ha rvested using oyster tongs from each of the following sites: N29.67398 W85.14764 ); Middle Site (GPS: N29.68640 W85.03324 N29.71292 W84.88168 ). Salinity, approximate total depth, pH, and water temperature were reco rded on site at each location. After harvesting, oysters were placed in separate plastic coolers for each site and immediately transported back to the laboratory in Apalachicola DACS laboratory for processing. Oyster samples were homogenized with equal we ight PBS for 90 s in blenders using sterile glass containers for each sample, as previously described by other environmental survey studies ( Wright et al., 1996 ; Motes et al., 1998 ; DePaola et al., 2003a ) After homogenization, serial dilutions in alkaline peptone water (APW) were
24 used to inoculate APW MPN enric hment tubes, which were subsequently incubated overnight without shaking at 37C. After enrichment, MPN tubes were streaked to TCBS, mCPC and ChromAGAR Vibrio (Paris, France; Part #: VB912), for presumptive positive identification of Vc, Vv and Vp, respec tively. MPN tubes were simultaneously subjected to the DNA extraction protocol for QPCR analysis described above. Presumptive positive colonies from selective media were spotted to LA and incubated overnight at 37C for subsequent freezing at 80C in LB w ith 50% glycerol. MPN calculations for traditional vs. QPCR assays were performed for Vv and Vp using MPN tables described in the FDA BAM ( FDA, 2011 ) In V itro S urvival S tudies Artificial seawater (ASW) (Instant Ocean; Part #: INS543) was mixed full strength in deionized water un til all salts were dissolved, as described by manufacturer, and appropriate salinity was adjusted (20, 30 and 35 ppt) using a portable temperature corrected glass refractometer (Fisher cat #: 13 946 27 ) with a standard error of 0.5 ppt. The pH was adjuste d using NaOH or HCl and measured by the Accumet (New Jersey, US) model AR10 pH meter with a standard error of 0.05. After preparation, ASW was filter sterilized (Millipore ExpressPLUS 0.22m filter; Part#: SCGPU 2RE) and stored at room temperature. Stoc k frozen cultures for each species were streaked to LA and incubated overnight at 37C. Individual colonies from LA were used to inoculate 250 mL flasks containing 50 mL LBN which were incubated overnight at 37C. Ten mL of LB culture from each overnight c ulture was centrifuged (Eppendorf model 8810R) in plastic 15 mL capacity Falcon tubes (Corning Part#:430791) at 10,000 x g for 15 min; supernatant was decanted, and pellets were resuspended in 10 mL of 20 ppt sterile ASW (pH=7.4)
25 for acclimation at the ap propriate temperature (23 or 30C). After acclimation, ASW samples were centrifuged at 10,000 x g for 15 min and resuspended in 10 mL of 20 ppt (pH=7.4; no shift), 30 ppt (pH=8.4), or 35 ppt (pH=8.4) ASW. The resuspended, acclimated cultures (1 ml) were us ed to inoculate 9 mL of the appropriate salinity ASW tubes for assessment of the individual species, while 3 mL total (1mL from each species) were used to inoculate 7 mL of the appropriate salinity ASW tubes for competitive cultures. At each time point (0, 14, and 32 d post inoculation), individual samples were plated to LA from serial dilutions in phosphate buffered saline (PBS); the aforementioned DNA extraction protocol performed on both individual and competitive samples. Extracted DNA samples were stor ed at 20C and thawed at room temperature for the QPCR assay. In V itro G rowth S tudies LB was mixed in deionized water, with the appropriate weight of NaCl added, i.e., 5, 10, 20, 30 and 25 g/L, for 5, 10, 20, 30 and 35 ppt NaCl, respectively The pH w as adjusted to 8.0 (standard error of 0.05) for all flasks using NaOH or HCl. After preparation, LB was sterilized under autoclave conditions for 35 min and stored at room temperature. Stock frozen cultures for each species were streaked to LA and incubat ed overnight at 37C. Individual colonies from LA were used to inoculate 250 mL flasks containing 50 mL LB at appropriate NaCl concentrations, and flasks were incubated overnight for 18 h at 37C. Serial dilutions of overnight cultures were made using PBS, and 500 uL from each ( 3) dilution tube was used to inoculate 250 mL flasks containing 45.5 mL LB of identical NaCl concentration. At each time point (0, 2, 4, 6 h), individual samples were plated to LA from serial dilutions in phosphate buffered saline ( PBS), and
26 DNA extraction for QPCR was performed on both individual and competitive samples. Extracted DNA samples were stored at 20C and thawed at room temperature for use in the QPCR assay. Statistical A nalyses All statistical calculations for these s tudies were performed in Microsoft Excel 2010 (Redmond, WA) test was used to determine statistical significance among sample points for different species and different salinities. The appropriate functions were chosen for unpaired, 2 taile d, homoscedastic comparisons. For all were presented as error bars on all relevant figures. Linear regression equations and their associated coefficients of determina tion were displayed directly on the figures.
27 CHAPTER 3 RESULTS: SPECIFIC AI ENUMERATION OF VIBRIOS Rationale Although differential salinity gradients have been reported to correspond with optimum recovery of the three pathog enic Vibrio species from the environment, their relative distribution has not been described for Apalachicola Bay, FL. This region is the primary site of oyster harvest for the state of Florida, accounting for about 95% of production. Therefore, oysters we re collected from a transect of Apalachicola Bay at three sites ( Figure 3 1 ) that differed in their salinity levels and were examined for the relative distribution of each species by the DuPont BAX multiplex QPCR MPN assay Benefits for using this method i nclude a relatively simple and rapid extraction method and straightforward output of results with a high degree of transparency for review and verification. Despite the potential of this assay to replace more traditional microbiological methods for identif ication and enumeration, validation and approval of this method by FDA and the Interstate Shellfish Sanitation Committee (ISSC) has not been achieved at this time. In this study, all three Vibrio species were assessed by both the QPCR MPN assay, and the tr aditional MPN enumeration, as described in the US FDA Bacteriological Analytical Manual (BAM ) ( FDA, 2011 ) was also used to enumerate Vv and Vp. Comparison of MPN Enumeration Methods The QPCR MPN and standard BAM MPN assay s w ere used to assess Vv and Vp in oysters (n=12) collected i n triplicate samples at the 3 sites in Apalachicola Bay FL in June 2010 (Figure 3 1) MPN enumeration of e nrichment cultures w as evaluated by the BAM method using selective agars Vibrio )
28 ( FDA, 2011 ) TCBS is a differential media commonly used in the isolation of Vc, but this medium allows for growth of many false positive typical colonies from the microflora of enriched oyster samples; thus, enrichment cultures were not evaluated by BAM MPN for Vc. Although confirmation of presumptive positive colonie s by DNA probe of individual species is described in the FDA BAM method ( FDA, 2011 ) this step was not performed for this study, as the cost was prohibitive and mCPC and media have been shown to be highly selective and differential ( Hara Kudo et al., 2001 ; Warner and Oliver, 2007 ) The same enrichment samples used for BAM MPN were also evaluated for all three species using the Dupont BAX multiplex QPCR assay. As shown in Table 3 1, recovery ranged from 2.3 to 4.6 log MPN/g by BAM and 2.8 to 4.6 log Figure 3 1. Map of sites selected for sampling in Apalachicola Bay, FL. Sites were selected based on differential salinity dist showed comparatively moderate salinity (19.9 20.1 ppt), site 2 (Middle Site) was located near the mouth of Apalachicola River and showed the lowest salinity (9.3 the
29 MPN/g by QPCR for Vp and Vv. The only significant difference in results obtained by the two assays more sensi tive and showed higher recovery than the BAM method (p<0.001). The high standard deviations among samples from each site complicated comparisons of the two methods, although no significant differences were observed between the two methods when comparing th test (p>0.50). High standard deviations of MPN/g values for Vv in oysters have been observed in previous studies; in the study by Motes et al. (1998), standard deviations for Vv MPN/g approached mean values and included sample sizes much larger than the number of samples used in this study. Relative Recovery of Vibrios in Relationship to Salinity Greatest numbers of Vp as determined by QPCR MPN w ere seen at which also showed the highest salinity (21.9 ppt), and recovery at the lower salinity site (Middle Site) was lower for Vp compared to the other 2 sites. However, enumeration by BAM MPN showed greater recovery of Vp at the Middle Site with the lowest salinity (9.3 16.8 ppt) (Table 3 1) Recovery of Vv was highest by both assays at the lower salinity Middle Site, and Vc levels also showed significantly high er levels (3.57 log MPN/g) at this site as compared to other sites where MPN/g of Vc approached the limits of detection (0.30 and 0. 48 log MPN/g) Significant differences in levels of Vv compared to Vp were not noted due to high variability of MPN values among samples from the same sites. The only significant difference between the two assays occurred at PCR assay was significantly more sensitive and showed higher recovery than the BAM method (p<0.001).
30 These results were consistent with prior data that showed the different species are differentially distributed in the environment and that their distribut ion, while highly variable among individual samples, may be related to salinity ( Kelly, 1982 ; Motes et al., 1998 ; DePaola et al., 2003a ; Zimmerman et al., 2007 ) These data do not provide comprehensive survey of the relative levels of the Vibrio species in A palachicola Bay, as they only represent a single time point. However, they provide insight into conditions that may determine the differential distribution of the three species in Apalachicola Bay and will serve as the basis for more comprehensive environm ental survey. The high variability observed among samples from the same site suggests that in future studies, more oysters should be collected and more samples from each site should be used to reduce standard deviations from each site. Despite the highly s elective and differential properties of mCPC and ChromAGAR Vibrio the potential for false positive identification exists without molecular based confirmation of presumptive positive samples. Thus, MPN evaluation based on selective agars may contribute to elevated BAM MPN values as compared to QPCR. Additionally, the presence of the oyster homogenate matrix from enriched APW tubes could potentially interfere with QPCR amplification and fluorescence for MPN confirmation. More rigorous testing of this assay s hould be completed using all applicable food products and appropriate media in this assay.
31 Table 3 1. FDA (BAM) vs. QPCR (BAX) log MPN /g of bacteria present in oyster homogenates from Apalachicola Bay FL. that went beyond the endpoint of d etection for the assay; Site name Salinity (ppt) Vv MPN/g Vp MPN/g Vc MPN/g Surface Bottom BAM QPCR BAM QPCR BAM QPCR 19.9 20.1 3.4 0.6 3.6 0.2 2.0 0. 8 3. 3 0. 6 ND 0.0 0 5 2 Middle Site 9.3 16.8 4 .4 0.6 3. 7 1.1 3. 2 1 .6 2.7 0.2 ND 3. 3 0. 6 3 21.9 21.9 3.8 0. 2 4.0 0. 0* 2 .4 0.6 4.0 0. 0* ND 0.4 0. 4
32 CHAPTER 4 RESULTS: SPECIFIC AI Rationale Understanding the conditio ns that influence the growth and survival of pathogenic Vibrio species is critical to developing and optimizing post harvest processes and pre harvest management practices in order to improve oyster safety and minimize risk to public health. Studies analyz ing oyster relay have demonstrated the effectiveness of transferring oysters to higher salinity waters in order to reduce the levels of pathogenic Vibrio species in oysters at harvest ( Motes and DePaola, 1996 ) However science base d experimental evaluation of the specific consequences of salinity and pH upshifts on the in vitro survival is lacking. Potential problems that arise during oyster relay include unpredictable weather and tidal changes that make sampling or relay difficult or impossible; inconsistent environmental or biological parameters, such as temperature, salinity, biomass, nutrient availability, etc.; and highly dynamic concentrations of initial bacteria due to biological variation or seasonal changes could adversely a ffect the reproducibility of these data. In a laboratory setting, however, in vitro survival conditions may be more closely controlled and monitored. The present study evaluated survival of the three Vibrio pathogens in artificial seawater under conditions that mimic parameters used in current oyster relay studies. Survival of the three individual species in seawater has previously been described in the literature; however, prior studies have not evaluated the effects of salinity and pH upshifts on individu al culture or the possible contribution of competitive co culture following simultaneous inoculation of all three species. Although optimal conditions for salinity showed variation among different strains, Kaspar and Tamplin
33 ( 1993 ) found that Vv exhibited optimal in vitro survival in sterile harvested seawater at moderate salinities (15 25 ppt) and at moderate temperatures (13 22C). Vc was shown to survive best at approximate ly 25 ppt and 18C in seawater ( Singleton et al., 1982b ; Huq et al., 1984 ) while DePaola et al. ( 2003b ) suggested that the optimal salinity and temperature combinations for Vp were approximately 17 ppt and 25C, based on mathematical modeling of data from environmental surveys. How ever, these prior experimental laboratory models used sterilized natural seawater, which could also influence the reproducibility of the data due to biological variability from different samples including, but not limited to, natural flora and related grow th factors present in sample, such as dissolved oxygen, chlorophyll, and pH. Additionally, many studies used natural seawater that was sterilized by autoclaving, which may significantly alter the chemical composition of this medium. Effects of Salinity and pH Upshift on Survival of Individual Vibrio Species Effects of salinity and pH upshift, from 20 ppt (pH=7.4) to 30 or 35 ppt (pH=8.4), were evaluated at two temperatures (23 or 30 C ) by incubating the individual species in ASW at the appropriate salinity and pH and subsequently measuring the relative survival of the individual Vibrio species by plate counts. Figure 4 1 shows the log reduction of bacteria relative to the inocula after 32 days incubation at 23 C for cultures that were upshifted from 20 ppt to either 30 or 35 ppt in comparison to cultures without any upshift that were maintained at 20 ppt throughout. Under these conditions, an overall decline of cultures maintained at 20 ppt was observed, but this decline was enhanced by the salinity upshift from 20 to 30 ppt, as significantly higher reductions were observed when compared to samples with no shift for both Vv and Vc at 23C (p=0.044 and 0.008, respectively). Interestingly, a salinity upshift from 20 to 35 ppt
34 produced no significant reductions compared to no upshift for Vv (p= 0.721), while Vc did show significant reduction due to the 35 ppt upshift (p=0.024). Thus, Vv showed significantly less reduction for the upshift to 35 ppt, as compared to an upshift to 30 ppt (p=0.007). No significant con sequences were observed for Vp due to salinity upshift at 23C for either 30 or 35 ppt upshift (p>0.1). Vibrio species have been shown to adapt well under conditions of high stress ( Whitaker et al., 2010 ) ; therefore, it may be possible t hat the Vp response and the higher survivability of Vv after upshift to 35 ppt shift may be due to adaptive stress responses for the higher salinity conditions. Incubation at 30C negated any effects of salinity and pH upshift as measured by plate count ( Figure 4 2). No significant differences were observed for any species when comparing the overall reduction of samples with no shift to samples subjected to the 30 ppt or 35 ppt upshift (p>0.1). However, the elevated temperature caused significantly higher reduction of Vp as compared to 23C incubation for both the 30 and 35 ppt upshifts (p=0.056 and 0.005). Significantly lower reductions were observed for Vv at the 30 ppt upshift (p=0.006) and for Vc at both the 30 and 35 ppt shifts (p<0.030) at 30C as com pared to 23C. The effects of the elevated incubation temperature suggest that Vp may be less adept at survival at higher temperatures as compared to the other two species. Overall, salinity upshift had a dramatic influence on the survival of the individual species based on plate count. Interestingly, the most effective condition was salinity upshift from 20 to 30 ppt at 23 C resulting in about a 2.0 log CFU/ml reduction for Vv and Vc after 32 days. Vp was the most resistant species to salini ty upshift as decline did
35 Figure 4 1 Total reduction, or difference in initial and final bacterial concentration, due to salinity upshift of the different species in individual culture after 32 days at 23C by plate count. Significantly higher reducti ons due to salinity shifts (as compared to samples without Figure 4 2 T otal reduction, or difference in initial and final bacterial concentration, due to salinity upshift of the different species in individual culture af ter 32 days at 3 0 C by plate count
36 not differ from cultures that were not upshifted. Increasing the differential for the upshift to 35 instead of 30 ppt also negated any significant reductions, which may be explained in part by the versatility of stress responses exhibited by Vibrio species ( Whitaker et al., 2010 ) These data showed that survival of the three species in individual culture varied with the species examined. Competitive S urvival of Vibrios F ollowing Salinity Upshift To e valuate competitive survival of the three species in co inoculated samples, multiplex QPCR was used to simultaneously and directly enumerate all three species. Standard curves for each of the three species were generated at each salinity level (20, 30 and 35 ppt) by relating Ct output from the QPCR assay to plate count log CFU/mL from individually inoculated ASW samples (Appendix A); all standard curves generated showed excellent correlation for all species at all salinities (R 2 >0.9; Appendix A, Figure A 1) Comparison by QPCR also showed no significant differences in reduction following salinity upshift for competitive co inoculated samples after 32 days incubation at either 23 or 30 C (p>0.5; Figure 4 3) with the sole exception of Vv at 23 C, where a signi ficantly higher reduction was observed in the competitive compared to the individual culture at 35 ppt upshift (p=0.034, Figure 4 3). This observation provides evidence that perhaps Vv survival may be hampered in a competitive environment at higher salinit ies typical of oyster relay conditions ( Muntada Garriga et al., 1995 ) Plate counts and QPCR results were in agreement regarding the reduction of Vp at 30 compared to 23 C, indicating increased incubation temperature causes higher reductions of the species at any salinity.
37 Standard d eviations among individual and competitive samples as measured by test comparisons showing no significant differences among nearly all samples. This may have been due to variable post acclimation levels at day 0 for bacterial concentrations among the three species. Starting inocula ranged from approximately 7 to 9 log A B Figure 4 3 Comparison of concentrations (log CFU/mL) of individual species after 32 days incubation at A ) 23C and B) 30C, by QPCR and plate count (PC). suggest VBN C state may have been triggered
38 CFU/mL based on species and replicates. While overnight culture concentrations were normali zed, variation arose after acclimation and subsequent centrifugation. F uture studies may achieve a higher degree of post acclimated normalization by generating optical density growth curves for each species at all salinities and adjusting the acclimated co ncentrations accordingly. Differences in Plate Count and QPCR Data as Related to the VBNC State In contrast to results from plate counts of individual samples, results from QPCR analysis showed no significant differences in total reduction for any speci es as a consequence of salinity upshift when compared to cultures maintained at 20 ppt. Figure 4 4 shows a comparison of CFU/mL values after day 32 by plate count and by QPCR. 4) of bacte ria were seen among the species by plate count as compared to QPCR in the majority (67%) of cases, these results strongly suggest that reductions observed after salinity upshift by plate count may have been the result of the induction of viable but noncult urable responses (VBNC) as previously described for Vibrio species. These species can become undetectable by plate count but have been shown to be viable and quantifiable via other microbial and molecular methods after exposure to temperature stress, salin ity stress, and/or starvation conditions ( Oliver et al., 1991 ) and these results are in agreement with a prior report that sho wed VBNC cells were detected via QPCR ( Campbell and Wright, 2003 ) Higher reduction as seen by plate counts lends credence to the hypothesis that the VBNC response was triggered in pat hogenic Vibrio species during long term survival in ASW suggesting that data from the QPCR enumeration may be more reliable than plate count data in evaluating survival of the species.
39 Nevertheless, data from plate counts provide insight into the patterns of survival of healthy, cultural Vibrios under these survival conditions. A B Figure 4 4 Effects of salinity upshift on differences in mean total reduction between competitive and individual cultures after 32 days by QPCR at A) 23C and B) 30 C. Significant differences among individual and competitive cultures
40 CHAPTER 5 RESULTS: SPECIFIC AI Rationale The oyster host provides nutrients that are not available in ASW; therefore, the in vitro grow th of the different Vibrio species was investigated in a nutrient rich environment under various salinities. These results may provide insight into the individual and competitive relationship of the three species within the oyster and contribute to establi shing oyster management processes that can be modeled and optimized based on initial bacterial loads and environmental conditions The salinity requirements for optimal growth of the three species in nutrient media have been extensively investigated in th e literature. V p grows the best among the three species over a broader ranges of salinities and temperature, with significant growth observed from 10 to 90 ppt ( Whitaker et al., 2010 ) at temperatures as high as 44 C. An optimal growth ra nge was reported between 35 and 37 C and 18 22 ppt ( Beuchat, 1973 ; DePaola et al., 2003b ; CDC, 2008 ) Optimal growth conditions for Vv vary among different strains for salinity, though the optimal range has been shown to be 10 to 20 ppt ( Palasuntheram, 1981 ; Kelly, 1982 ) Hsu et al. ( Hsu et al., 1998 ) also found that Vv exhibits optimal growth at pH 8.0 and at 35C. T he optimum salinity for growth of Vc was shown by Singleton et al. ( Singleton et al., 1982a ) to be approximately 25 ppt with the optimal temperature between 35 and 37 C ( Kolvin and Roberts, 1982 ; CDC, 2010 ) though the bacteria can proliferate and grow at less than 1 ppt and at 45 p pt or higher under certain conditions.
41 Effects of S alinity on I ndividual G rowth of Vibrios Total growth yield of the individual species after 6 h incubation at 30C was evaluated in this study by plate count for salinities of 5, 10, 20, 30, and 35 ppt, with starting inocula approximately 4 log CFU/mL. Figure 5 1 presents the overall growth yield of the species at all salinities after 6 h, while data for hourly growth of the individual species can be found in Appendix B (Figure B 1). Overall, Vp grew wel l at all salinities evaluated in the study and had significantly higher growth yield compared to either of the other two species after 6 h at 30 and 35 ppt (p<0.005). Vc consistently had the lowest growth yield at all salinities and was significantly lower than either Vp or Vv after 6 h incubation for 5 30 ppt (p<0.008). The total yield of Vc at 35 ppt was not significantly different from Vv (p=0.192). The greatest growth yields for Vc were declined significantly at 20 30 and 35 ppt compared to 5 and 10 pp t. No significant differences were observed between Vp and Vv at 10 and 20 ppt (p>0.1), though Vv had significantly Figure 5 1 Effects of salinity on total growth yield of individual species in LB with modified NaCl concentration after 6 h incubation at 30C by plate count
42 higher yield than Vp at 5 ppt (p=0.020). The highest total yield for Vv was obtained at 10 ppt. Overall, these data suggest that Vv and Vc grow best at a moderat e salinities, while Vp is more adapted than the other species for growth at higher salinities, specifically between 30 35 ppt. Vc consistently showed the lowest growth yield among the three species and growth inhibition for Vc at salinities as low 20 ppt did not occur for Vv until salinity reached 35 ppt. Effects of S alinity on C ompetitive G rowth of Vibrios Overall, trends for competitive growth were similar (p>0.132) to trends seen in individual growth studies based on growth yield s determined by QPCR (Figure 5 2 ). Results also were in agreement with plate count for individual samples, QPCR further demonstrated that Vp showed robust growth yield over a wide range of salinity and that Vc showed the lowest growth yield among the three species at all sali nities, with an overall decline in yield as salinity increased. However Vp seemed to exhibit a higher growth yield with competitive compared to individual culture with increasing salinity. At 35 ppt, the competitive culture was nearly 2 log CFU/ml higher g rowth yield when compared to individual culture (p=0.073). At the highest salinity of 35 ppt, competitive yield of Vp (>6.0 log CFU/mL) was significantly higher than the competitive yield for Vp (<4.5 log CFU/mL) at 10 ppt (p=0.031). Since this trend was n ot observed in individual culture, these data suggest that Vp may have a competitive advantage over Vv and Vc in a nutrient rich environment as salinity increases. No other significant differences were observed between competitive and individual cultures. All coefficients of determination in this study were considerably high in the linear regressions of the standard curves for this study, suggesting that the QPCR model was
43 appropriate for this particular application (Appendix B, Figure B 2). However, the in itial Vv inoculum of approximately 4 log CFU/mL for 30 ppt was not detected by QPCR in any of the competitive replicates; additionally, QPCR was not able to detect Vv for individual nor competitive samples at 35 ppt for all time points. Consequently, compa risons of individual and competitive growth yields were not done in this study for Vv at 30 or 35 ppt. These growth studies were repeated at 30 and 35 ppt to confirm the QPCR signal for Vv was inhibited at higher NaCl concentrations in LB. Thus, Vv was not detected despite being over the published limit of detection of the QPCR assay by plate count. In future growth studies, as suggested previously for survival studies, an additional wash step may be needed to replace experimental media during DNA extractio n in order to help eliminate the problem of high salinity inhibition. Figure 5 2 Comparison of individual and competitive total growth yield in LB after 6 hours incubation at 30C, by QPCR. ND=no data (due to absence of QPCR signal)
44 CHAPTER 6 SUM MARY AND CONCLUSIONS Enumeration of Vibrios in the Environment While this study does not provide comprehensive analysis on the distribution of Vibrios in Apalachicola Bay, these results suggested that the three species varied in their association with sp ecific environmental parameters such as salinity. These data are in agreement with previous, large scale studies on the effects of salinity on the differential distribution of the three species: Vv is normally detected at highest concentrations in lower sa linity waters ( Kelly, 1982 ; Kaspar and Tamplin, 1993 ; Randa et al., 2004 ) while total Vp counts tend to persist year round throughout the Gulf at a wide range of temperatures and salinities ( Daniels et al., 2000 ; DePaola et al., 2003a ; CDC, 2009 ) The relatively high concentration (>10 3 CFU/G) of Vc detected in oysters at Site 2 was presumably nontoxigenic Vc strains, QPCR assay used can detect both toxigenic and nontoxigenic strains, and most environmental surveys that have attempted to isolate toxigenic Vc have been unsuccessful. After the recent Vc outbreak of serogroup O75, investigations into the implicated oyster harvest areas, showed all samples tested from all areas were negative for toxigenic Vc ( DePaola et al., 2010 ; Onifade et al., 2011 ) It was noted that their results were not unexpected, as toxigenic Vc has rarely been isolated from US oysters. Since toxicity assays were not performed on the strains isolated in this study, it is not possible to determine if the Vc detected by QPCR in this study was toxigenic. Since the QPCR assay used in this study has not been extensively tested on environmental oyster samples and since the typical colonies from TCBS were not confirmed by DNA or PCR assays, the Vc detected by QP CR in this study may have been false positives.
45 The QPCR and BAM MPN methods employed in this study did not provide significantly different results based on a paired t test from pooled data, though a significantly higher concentration of Vp was detecte Higher standard deviations were seen among samples collected at the same site than samples collected at different site, suggesting that future studies using these methods of enumeration should a greater number of samples from each site to reduce variability for stronger quantitative comparisons. Survival of Vibrio S pecies A fter S alinity U pshifts in ASW Conditions specified in the experimental design for survival studies mimicked conditions of salinity, pH and tempera ture typical for oyster relay procedures currently ongoing in Mobile Bay. In agreement with the literature ( Whitaker et al., 2010 ) Vp was the most resistant toward changes in salinity since no significant reductions were observed due to salinity upshifts. The most dramatic reductions due to salinity upshift as measured by plate count were approximately 2.5 log CFU/mL for both Vv and Vc, and was only observed at the lower upshift to 30 ppt and lower temperature of 23C. These data suggest that Vv and Vc may exhibit stress responses due to changes in salinity similar to those recently described in Vp by Whitaker et al. ( 2010 ) However, no significant reductions were seen by QPCR for Vv and Vc at the 23C, suggesting that t he VBNC state may have been triggered, since QPCR can detect cells in the VBNC state that are not detectable by plate count ( Campbell and Wright, 2003 ) However, PCR may also detect de ad cells and future work should evaluate in vitro survival of the Vibrios in ASW with a side by side comparison using independent viability assays for verification of these claims. Furthermore, reductions were not observed by either plate count or QPCR at higher temperature of 30C. These data suggest that higher
46 temperatures may protect these bacteria from the effects of salinity changes and thus promote survival. Data from plate counts are in agreement with published results on oyster relay from Motes and DePaola ( 1996 ) which found 3 4 log CFU/mL reductions for Vv in all oysters after 25 days post relay to higher salinity conditions. However, the prior study did not use QPCR enumeration, and further studies should be conducted to veri fy the effects of relay on possible induction of VBNC. Also, it should be noted that any discrepancies between these studies could result from the dramatic differences between in vitro laboratory conditions and the actual environmental observations where b acteria were enumerated from oyster homogenates after harvest from ocean waters. Natural microflora in the oyster and ocean waters, climate patterns, chemical composition of the ocean waters, and many other natural variables may contribute to in situ studi es in the ocean. More studies are needed to investigate multiple variables can under conditions that can be closely controlled and modified in in vitro laboratory experiments. These experiments were the first observations on the effects of competition on survival of Vibrio species. An interesting observation from this study was that a significantly higher reduction of Vv was shown in a competitive environment as compared to individual culture at 23C following the higher 20 to 35 shift. These results sugge st that the ability of Vv to adapt to changes in high salinity upshifts may be hampered by competition with Vp, which actually showed less reduction under competitive compared to individual culture under these conditions.
47 In order to engineer optimum desi gn parameters for oyster management practices such as oyster relay, considerable effort should be made to further understand the cumulative and interactive effects of the potential stress responses of the three species due to salinity, pH, and temperature upshifts. As shown in this study for Vv and Vc and in other studies for Vp ( Whitaker et al., 2010 ) higher salinities may actually enhance the survival and adaptability of pathogenic Vibrio species under certain conditions in the environ ment, though this relationship is complex and not well understood. Effect of Salinity on Growth of Vibrio Species Results from plate counts analyzing individual growth of the three species over time showed that after 6 h incubation at 30C, Vp exhibited s ignificantly higher growth yield than Vv and Vc at 30 and 35 ppt. However, Vv had the highest yield of the three species at the lowest salinity (5 ppt). Vc exhibited the lowest growth yield among the three species at all salinities evaluated in this study. The results from plate counts generally agreed with those obtained by QPCR. However, the QPCR assay was unable to detect Vv at 35 ppt, and only detected Vv at higher concentrations for 30 ppt. Therefore, a quantitative growth comparison was not possible f or Vv at 30 and 35 ppt in this study. Data from survival and growth experiments in these studies show that an additional wash step designed to replace experimental media with DNA extraction buffer supplied in the QPCR Vibrio test kit would be advantageous to the QPCR protocol and potentially eliminate the problem of media inhibition. At all salinities investigated, no significant differences were observed among individual vs. competitive cultures by QPCR, though a competitive environment seemed to favor hi gher growth yield at increasing salinities for Vp, suggesting that Vp may have
48 a competitive advantage over Vv and Vc in an increasing salinity, nutrient rich environment. These data are in agreement with previous studies showing optimal salinity for grow th of Vp to be between 18 22 ppt ( Beuchat, 1973 ; DePaola et al., 2003b ; CDC, 2008 ) though the bacterium has been shown to grow at salinities as high as 90 ppt ( Whitaker et al., 2010 ) Conversely, Vv has been shown to grow best at low to moderate salinities ( Palasuntheram, 1981 ; Kelly, 1982 ) and Vc had the highest yield at 10 ppt, which is consistent with the minimal NaCl requirements of this sp ecies These results differ the optimal growth reported for Vc at 25 ppt in media designed to mimic aquatic microcosms ( Horie et al., 1966 ; Miller et al., 1984 ) However, studies have not evaluated optimal growth conditions for Vc for th e particular medium and associated salinities chosen for th e present study. Additionally, the incubation temperature used in thi s study wa s below the optimal range of 35 37C ( Miller et al., 1984 ; CDC, 2010 ) Thus, the discrepancy of the previously described optimal salinity in seawater and the optimal salinity found in nutrient rich media in this study is not surprising. Future studies investigating the pot ential effects of competitive on growth using different media and at different temperatures may provide further insight into the optimization of relevant design parameters for oyster management practices such as relay.
49 APPENDIX A STANDARD CURVES GENE RATED FOR ALL MEDIA AND SA LINITIES Figure A 1 Standard curves generated for each media and the associated linear regressions relating Ct (QPCR) to log CFU/mL (plate count), for Vp (red), Vv (green), and Vc (blue)
50 APPENDIX B GROWTH OF VIBRIOS AT 30C UNDER VARIOUS SALINITIES Figure B 1 Hourly growth of individual Vibrio species in LB at 30C under various salinities as determined by plate count for Vp (red), Vv (green), and Vc (blue)
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58 B IOGRAPHICAL SKETCH Michael Aaron Hubbard was born in the beautiful city of St. Augustine, FL Before starting elementary school, Michael and his parents moved to Gaines ville, where remained until finishing his graduate studies. He attended the AP/IB program at in b iological e ngineering from the University of Florida. Before pursu ing his graduate degree in the Food Science and Human Nutrition Department under the direction of Dr. Anita Wright, Michael spent several years in different labs as a laboratory technician, where he learned general laboratory technique and was e xposed to a wide range of microbiological projects. In his spare time, Michael enjoys hobbies including playing and mixing music, studying and playing strategically challenging games, building computers, and learning about everything from new electronic de vices to novel saltwater reef aquarium equipment. He is very passionate about his interests and enjoys spending time with friends and family. Michael is the proud father of Ava Hubbard.