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Development of a sensitive and specific biosensor assay to detect Vibrio vulnificus in estuarine waters

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Development of a sensitive and specific biosensor assay to detect Vibrio vulnificus in estuarine waters
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Ulrich, Robert M
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sonication
enrichment
evanescent
fiber-optic
immunoassay
Dissertations, Academic -- Biology -- Masters -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
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ABSTRACT: Biosensor development has the potential to meet the need for rapid, sensitive, and specific detection of pathogenic bacteria from natural sources. An antibody-based fiber-optic biosensor assay to detect low levels of Vibrio vulnificus in estuarine waters following an enrichment step was developed. The principle of the sensor is based on an immuno-sandwich assay where an anti-V. vulnificus polyclonal capture antibody preparation was first immobilized on a polystyrene fiber-optic waveguide using a biotin-avidin association. The capture antibody is responsible for binding the target cells to the waveguide. Cyanine-5-conjugated anti-V. vulnificus polyclonal antibodies are subsequently allowed to bind to immobilized cells, and detection occurs when a photodetector collects emitted light (670-710 nm) from the fluorophore, which is excited with 635-nm laser light produced by the Analyte 2000 biosensor.Any detection signal greater than a pre-determined threshold signal is considered to be a positive detection event, while any signal lower than the threshold is considered no detection. This immunosensor assay proved highly specific when tested against whole cells and cell extracts from V. cholerae, V. parahaemolyticus, V. alginolyticus, and E. coli. isolates. Following a four hour enrichment in PNCC broth, and in a total of less than seven hours, the assay was able to detect cell extracts from as few as 100 V. vulnificus colony forming units suspended in sterile water. This method holds promise for detection of low numbers V. vulnificus and other autochthonous pathogens in estuarine waters.
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Thesis (M.S.)--University of South Florida, 2004.
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by Robert M. Ulrich.
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Development of a Sensitive and Specific Biosensor Assay to Detect Vibrio vulnificus in Estuarine Waters by Robert M. Ulrich A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Biology College of Arts and Sciences University of South Florida Co-Major Professor: Valerie Harwood, Ph. D. Co-Major Professor: Andrew Cannons, Ph. D. My Lien Dao, Ph.D. Date of Approval: November 12, 2004 Keywords: fiber-optic, evanescent, enrichment, sonication, immunoassay Copyright 2004, Robert M. Ulrich

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Acknowledgments I would like to extend my heartfelt th anks to the members of my graduate committee, Dr. Jody Harwood, Dr. Andrew Ca nnons, and Dr. My Lien Dao for their patience and support. I want to thank the C ooperative Institute for Coastal and Estuarine Technology for funding my research, along w ith Research International (Woodinville, WA) for their technological support pertaining to the Anal yte 2000 biosensor. I would also like to thank the members of the Ha rwood and Lim labs for their assistance and friendship throughout my stay at th e University of South Florida.

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i Table of Contents List of Tables................................................................................................................. ....iii List of Figures..................................................................................................................viii Abstract....................................................................................................................... .......ix Introduction................................................................................................................... .......1 Importance of Vibrio vulnificus .....................................................................................2 Conventional Methods of Detection..............................................................................3 Enrichment in Microbial Detection...............................................................................4 Biosensors.................................................................................................................... ..6 Piezoelectic-based Biosensors.................................................................................6 Surface Plasmon Resonance (SPR) Biosensors.......................................................7 Laser Evanescent Wave Fiber Optic Biosensors.....................................................8 Analyte 2000 Biosensor...........................................................................................9 Materials and Methods.......................................................................................................12 Bacterial Cell Preparation............................................................................................12 V. vulnificus Isolated from Environmental Waters......................................................15 Non-target Bacteria......................................................................................................16 Mixed Cultures.............................................................................................................17 Detection in Estuarine Waters.....................................................................................17 Enrichment Cultures....................................................................................................18 Antibodies.................................................................................................................... 22 Measuring Antibody Titer by ELISA..........................................................................23 Antibody Conjugation..................................................................................................25 Preparing the Polystyrene Waveguides.......................................................................26 Immunoassay Development for the Analyte 2000 Biosensor......................................27 Correlation between Mean Correct ed Detection Signals and CFU.............................30 Comparison of Mean Corrected Dete ction Signals between Clinical and Environmental Isolates.................................................................................................30 Results ................................................................................................................................31 Initial Detection of Whole Cell V. vulnificus (ATCC 27562) using the Biosensor......................................................................................................32 Initial Detection of V. vulnificus (ATCC 27562) Cell Extracts using the Biosensor......................................................................................................36 Determining the Sensitivity on Whole Cell V. vulnificus (ATCC 27562)...................40

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ii Determining the Sensitivity toward V. vulnificus (ATCC 27562) Cell Extracts.................................................................................................................43 Testing the Reproducibility of th e Biosensor for Detection of 1 4 Whole Cell V. vulnificus (ATCC 27562).....................................................................47 Testing the Reproducibility of the Biosensor for Detection of Cell Extracts from 1 3 V. vulnificus (ATCC 27562).......................................................57 Detection of a Whole Cell V. vulnificus Isolated from Environmental Waters using the Biosensor..........................................................................................68 Detecting Cell Extracts of V. vulnificus Isolated from Environmental Waters using the Biosensor..........................................................................................72 Testing the Specificity of the Biosensor Assay against Closely Related Vibrio species and E. coli ...............................................................................76 Testing the Sensitivity of the Biosensor Assay on a Mixture of Whole Cell V. vulnificus (ATCC 27562) and V. cholerae (ATCC 11623).............................93 Detection of V. vulnificus (ATCC 27562) in Estuarine Water....................................95 Detection of Cell Extracts from V. vulnificus (ATCC 27562) after Enrichment in Alkaline Peptone Water.....................................................................100 Detection of Cell Extracts of V. vulnificus (ATCC 27562) in Sterile Water after Enrichment in PNCC Enrichment Broth.....................................103 Correlation between Mean Corrected Detection Signals and CFU...........................106 Whole Cell Assays...............................................................................................106 Cell Extract Assays..............................................................................................107 Assays on Extracts from Enriched Cells..............................................................108 Comparison of Mean Corrected Detection Signals between Clinical and Environmental Isolates..........................................................................111 Discussion........................................................................................................................112 References........................................................................................................................120

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iii List of Tables Table 1 Initial detection of whole cell V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor..............................................................33 Table 2 First replicat e detecting whole cell V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor..............................................................34 Table 3 Second replicate detecting whole cell V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor..............................................................35 Table 4 Initial detection of cell extracts from V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor..............................................................37 Table 5 First replicate detecting cell extracts from V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor..............................................................38 Table 6 Second replicate detecting cell extracts from V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor.....................................39 Table 7 Determining the sensitivity of the assay on whole cell V. vulnificus (ATCC 27562)...........................................................................................40 Table 8 First replicate determining the sensitivity of the assay on whole cell V. vulnificus (ATCC 27562)......................................................................41 Table 9 Second replicate determining the sensitivity of the assay on whole cell V. vulnificus (ATCC 27562)...............................................................42 Table 10 Sensitivity of the biosensor assay on V. vulnificus (ATCC 27562) cell extracts................................................................................................44 Table 11 First replicate de termining the sensitivity of the biosensor assay on V. vulnificus (ATCC 27562) cell extracts..................................................45 Table 12 Second replicate determining th e sensitivity of the biosensor assay on V. vulnificus (ATCC 27562) cell extracts..................................................46 Table 13 Reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562).....................................................47

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iv Table 14 First replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................48 Table 15 Second replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................49 Table 16 Third replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................50 Table 17 Fourth replicate determining th e reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................51 Table 18 Fifth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................52 Table 19 Sixth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................53 Table 20 Seventh replicate determining th e reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................54 Table 21 Eighth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................55 Table 22 Ninth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562)................56 Table 23 Reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562)...................................................58 Table 24 First replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....59 Table 25 Second replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....60 Table 26 Third replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....61 Table 27 Fourth replicate determining th e reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....62 Table 28 Fifth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....63

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v Table 29 Sixth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....64 Table 30 Seventh replicate determining th e reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....65 Table 31 Eighth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....66 Table 32 Ninth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562).....67 Table 33 Detection of whole cell V. vulnificus (MC0603S) isolated from environmental waters.................................................................................69 Table 34 First replicate detecting whole cell V. vulnificus (MC0603S) isolated from environmental waters...........................................................70 Table 35 Second replicat e detecting whole cell V. vulnificus (MC0603S) isolated from environmental waters...........................................................71 Table 36 Detecting cell extracts of V. vulnificus (MC0603S) isolated from environmental waters.................................................................................73 Table 37 First replicate detecting cell extracts of V. vulnificus (MC0603S) isolated from environmental waters...........................................................74 Table 38 Second replicate de tecting cell ex tracts of V. vulnificus (MC0603S) isolated from environmental waters...........................................................75 Table 39 Testing the specificity of the biosensor assay against whole cell V. cholerae (ATCC 11623)......................................................77 Table 40 First replicate testing the speci ficity of the biosensor assay against whole cell V. cholerae (ATCC 11623)......................................................78 Table 41 Second replicate testing the speci ficity of the biosensor assay against whole cell V. cholerae (ATCC 11623)......................................................79 Table 42 Testing the specificity of the biosensor assay against V. cholerae (ATCC 11623) cell extracts....................................................80 Table 43 Testing the specificity of the biosensor assay against whole cell V. parahaemolyticus (ATCC 43938)........................................81

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vi Table 44 First replicate testing the speci ficity of the biosensor assay against whole cell V. parahaemolyticus (ATCC 43938)........................................82 Table 45 Second replicate testing the speci ficity of the biosensor assay against whole cell V. parahaemolyticus (ATCC 43938)........................................83 Table 46 Testing the specificity of the biosensor assay against V. parahaemolyticus (ATCC 43938) cell extracts.....................................84 Table 47 Testing the specificity of the biosensor assay against whole cell V. alginolyticus (ATCC 51160)................................................85 Table 48 First replicate testing the speci ficity of the biosensor assay against whole cell V. alginolyticus (ATCC 51160)................................................86 Table 49 Second replicate testing the speci ficity of the biosensor assay against whole cell V. alginolyticus (ATCC 51160)................................................87 Table 50 Testing the specificity of the biosensor assay against V. alginolyticus (ATCC 51160) cell extracts.............................................88 Table 51 Testing the specificity of the biosensor assay against whole cell E. coli (ATCC 9637)................................................................89 Table 52 First replicate testing the speci ficity of the biosensor assay against whole cell E. coli (ATCC 9637)................................................................90 Table 53 Second replicate testing the specificity of the biosensor assay against whole cell E. coli (ATCC 9637)....................................................91 Table 54 Testing the specificity of the biosensor assay against E. coli (ATCC 9637) cell extracts..............................................................92 Table 55 Testing the sensitivity of the biosensor assay on a mixture of whole cell V. vulnificus (ATCC 27562) and V. cholerae (ATCC 11623)............93 Table 56 Replicate assay testing the sensitivity of the biosensor assay on a mixture of whole cell V. vulnificus (ATCC 27562) and V. cholerae (ATCC 11623)........................................................................94 Table 57 Detection of whole cell V. vulnificus (ATCC 27562) in estuarine water.......................................................................................96 Table 58 Replicate assa y detecting whole cell V. vulnificus (ATCC 27562) in estuarine water.......................................................................................97

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vii Table 59 Detection of cell extracts from V. vulnificus (ATCC 27562) in estuarine water.......................................................................................98 Table 60 Replicate assay detecting cell extracts from V. vulnificus (ATCC 27562) in estuarine water..............................................................99 Table 61 Detection of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in APW..............................101 Table 62 Replicate detect ion of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in APW..............................102 Table 63 Detection of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in PNCC............................104 Table 64 Replicate detect ion of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in PNCC............................105 Table 65 Parametric analysis of assay se ttings that were normally distributed......110 Table 66 Non-parametric analysis of assay settings that were not normally distributed................................................................................................110 Table 67 Comparison of mean corrected detection signals between clinical V. vulnificus (ATCC 27562) and environmental V. vulnificus (MC0603S) isolates for each CFU number assayed....................................................111

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viii List of Figures Figure 1 Schematic representation s howing how CFU were estimated for assays on both whole cell, and cell extract suspensions............................14 Figure 2 Schematic representation of the enrichment assay protocol......................21 Figure 3 Measuring antibody titer after purification from antiserum using an ELISA plate system...............................................................................24 Figure 4 Schematic representation of the biosensor immunoassay..........................29 Figure 5 Corrected detection signal (detection threshold) for each whole cell assay.......................................................................................107 Figure 6 Corrected detection signal (detection threshold) for each cell extract assay......................................................................................108 Figure 7 Corrected detection signal (d etection threshold) for extracts from cells enriched in both APW and PNCC..........................................109

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ix Development of a Sensitive and Specific Biosensor Assay to Detect Vibrio vulnificus in and Estuarine Waters Robert M. Ulrich ABSTRACT Biosensor development has the potential to meet the need for rapid, sensitive, and specific detection of pathogenic bacteria from natural sources. An antibody-based fiberoptic biosensor assay to detect low levels of Vibrio vulnificus in estuarine waters following an enrichment step was developed. The principle of the sensor is based on an immuno-sandwich assay where an anti-V. vulnificus polyclonal capture antibody preparation was first immobilized on a polysty rene fiber-optic waveguide using a biotinavidin association. The capture antibody is re sponsible for binding the target cells to the waveguide. Cyanine-5-conjugated antiV. vulnificus polyclonal antibodies are subsequently allowed to bind to immobili zed cells, and detection occurs when a photodetector collects emitted light (670-710 nm ) from the fluorophore, which is excited with 635-nm laser light produced by the Anal yte 2000 biosensor. Any detection signal greater than a pre-determined threshold signa l is considered to be a positive detection event, while any signal lower than the thre shold is considered no detection. This immunosensor assay proved highly specific wh en tested against whole cells and cell extracts from V. cholerae, V. parahaemolyticus, V. alginolyticus, and E. coli. isolates. Following a four hour enrichment in PNCC broth, and in a total of less than seven hours, the assay was able to detect ce ll extracts from as few as 100 V. vulnificus colony forming units suspended in sterile water. This met hod holds promise for detection of low numbers V. vulnificus and other autochthonous pathoge ns in estuarine waters.

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1 Introduction Waterborne pathogenic microorganism s, such as those found in ground and surface waters, pose a major risk to human h ealth (4). There is a need for rapid identification of these pathogens in order to prevent disease caused by exposure to contaminated water sources or seafood (32). Conventional methods are available to detect microbial pathogens in water, but these techniques are often time consuming, require extensive training in microbiology and or molecular biology, and are not cost effective (47). Ideally, a de tection system should identify pathogenic microorganisms in real-time, or near real-time, be sensitive and specific, and utilize equipment and techniques that are eas ily operable (50). A number of autochthonous pathogeni c microorganisms are found in ground and surface waters in both freshwater and marine environments. Vibrio vulnificus is a human pathogen that is commonly found in estuar ine waters, and causes necrotizing wound infections and fulminant primary septicemia (5). Infections caused by the consumption of shellfish contaminated with V. vulnificus are the leading cause of food-borne deaths in the state of Florida (21). The mortality rate for patients having primary septicemia caused by V. vulnificus has been reported at greater than 50% (38), and death can occur within a day or two of the onset of sy mptoms (37). The potential presence of this pathogen in natural waters requires a need for quick a nd accurate detection methods. This research focuses on the use of the Analyte 2000 fiber optic evanescent-wave biosensor (Research International, Woodinv ille, WA) to detect Vibrio vulnificus in estuarine waters.

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2 Importance of Vibrio vulnificus The gram-negative bacterium, Vibrio vulnificus, inhabits estuarine waters and oysters, and poses a significant health risk to persons suffering from immune disorders, liver disease, or hemochromato sis (iron overload) (15, 68). V. vulnificus enters human hosts via wound infections or consumption of shellfish (primarily oysters which are frequently consumed raw), and infections freq uently progress to septicemia and death in susceptible individuals (5). V. vulnificus thrives in warm estuarine waters and is frequently isolated from shellfish found in co astal waters of the Gulf of Mexico (24), and both Atlantic (57) and Pacific (25) oceans, as well as Chesapeake Bay located in the northeastern U.S. (66). Incidences of in fection are most frequently reported during warm-weather months (April-November) th roughout the U.S. (21, 24). From 1988 through 1995, the Centers for Disease Control and Prevention rece ived reports of 302 V. vulnificus infections from the Gulf Coast stat es (Alabama, Florida, Louisiana, and Texas); of these 141 (47%) were due to the consumption of contaminated food, 128 (42%) were caused by wound infections, and 33 (11%) had unknown origins. Among the 242 incidences in which outcomes were known, 86 (36%) of infected individuals died from septicemia (37). Three V. vulnificus biotypes, designated 1, 2 and 3, have been established based upon characteristics such as host specificity, indole production, sero type (59), genetic sub-typing (3), and siderophore production (34). Currently, th ere are no defining characteristics that enable scientists to determine which V. vulnificus strains are more virulent than others, although po ssible virulence determinants have been suggested (33). Iron acquisition from iron-binding proteins, such as transfer rin, appears to be important

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3 in pathogenesis (34, 60, 68), as well as the pr oduction of the extra cellular lytic enzyme hemolysin (17, 46), and the formation of a polysaccharide capsule (28). The presence or absence of capsules often correlates with colony morphology, which may be used as a potential screening tool. Simps on et al. (54) described that when encapsulated (virulent) strains are grown on a number of media (thi osulfate-citrate-bile salts-sucrose, heart infusion, brain heart infusion, and lactose aga r) the colonies have an opaque appearance, while non-encapsulated (avirulent) strains appe ar translucent, although it is important to note that not all encapsulated strains are highly virulent. Conventional Methods of Detection Numerous pathogenic microorganisms can be found in raw sewage including viruses, bacteria, protozoa, and fungi (55). Se rious health risks occu r when water that is used by humans is contaminated with raw sewa ge. Fecal indicator organisms have been used for nearly a century to assess the mi crobiological status of waters affected by anthropogenic activity (31). Non-pathogenic indicator organisms are used in water quality assessment because the diversity of pathogenic species that may be present in waters is too large to allow for efficient, individual enumeration. For a species to be considered a good indicator of fecal contamin ation it must be associated with feces, should not be a naturally occurring organi sm, should outnumber pathogens, survive as well as pathogens, and should be easy and economical to detect (31). However, if the presence of a pathogen, such as V. vulnificus occurs naturally in environmental waters, and is not associated with fecal pollution, then indicator organisms cannot be expected to predict the presence of said pathogen. Thus, there are compelling reasons to develop a

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4 system for V. vulnificus detection that is rapid, sensitive, and specific to prevent infection in humans. Current methods used for detecting bacter ia from the environment rely primarily on culturing organisms on selective-differe ntial media, frequently following an enrichment step of one day or longer. Th e time needed to culture and identify various bacteria range from one day to over two w eeks. The current method specified by the U.S. Food and Drug Administration (FDA) for the isolation of V. vulnificus from oysters includes a 12-16 hour enrichment step in al kaline peptone water (APW) (5% peptone and 1.0% NaCl in sterile deionized water, pH 8.0) followed by incubating enrichments on selective media (mCPC or CC) at 39-40 C for 18-24 hours (23). Furthermore, once these organisms are cultured, it may take additional time to perform the biochemical or molecular tests necessary to determine if a sp ecific organism is present. In the time it takes to detect and confirm the presence of specific organisms using culture methods, a foodborne or waterborne outbreak may have al ready occurred, or may even have run its course before positive id entification is made. Some species of pathogenic microorganism s as well as indicator organisms can enter a viable but not culturab le state (VBNC). A review by Roszak et al. (53) describes the VBNC state as being a dormant or som nicell state wherein the cells cannot be cultured by standard microbiological met hods, but can be resuscitated upon host infection, or by a stepwise cha nge in nutrient concentration or temperature (41). There is evidence that V. vulnificus is able to enter a VBNC state when exposed to cold temperatures, making culture-based detecti on methods problematic (64). VBNC cells have been shown to cause disease in mice ( 1, 45) and Pruzzo et al. (52) have recently

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5 reported the ability of VBNC Enterococcus faecalis cells to regain viability and adhere to human tissue cells in vitro. Thus, methods for the detection of VBNC as well as culturable pathogens must be developed to ensure thorough screening of natural waters used for recreation and seafood harvesting (10). Enrichment in Microbial Detection Enrichment methods have been used for over a century to aid in the isolation of pure cultures of microorganisms (2). The goa l of enrichment techniques is to provide growth conditions that are favorable for the or ganism of interest, and are as unfavorable as possible for non-target organisms. Enrichment aids in the isolati on of target bacteria that are present in low numbers in a homogenate containing a vari ety of species that would not be possible with conventional spre ad plate methods alone (63). The method suggested by the USFDA for culturing V. vulnificus in oysters includes enriching cells for 12-16 hours at 37C in APW to increase cell num bers for spread plate isolation (23). Hsu et al. (20) recently describe d the use of an enhanced enrichment broth, designated PNCC (5.0% peptone, 1.0% NaCl 2 0.08% cellobiose, and 1 Unit of colistin per ml, pH 8.0), that out-performed standard APW in detecting low numbers of V. vulnificus (ATCC 4832) while suppressing growth of non-target Vibrio species. Enrichments have also been utilized to lower detectable lim its of microorganisms in biosensor assays. Geng et al. (14) have recently been ab le to detect low numbers of Listeria monocytogenes by incubating cells in enrichment broth for 20 hours prior to assaying with the Analyte 2000 fiber optic biosensor (Research International, Woodinville, WA). Positive de tection signals were generated by seeding enrichment broth with as little as 10 colony forming units (CFU) Listeria monocytogenes (incubated

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6 at 37C for 20 hours) prior to the immunoassay. The lowest detectable concentration of Listeria was 4.3 10 3 CFU/ml without the enrichment st ep using the same immunoassay. The ability of biosensors to meet the need for rapid and accu rate detection of microorganisms is an active res earch interest of many federal agencies and investigators. Biosensors have been develope d to detect microorganisms, toxins, and nucleic acids (12, 16, 40). These biomolecules can be detected using a number of techniques and utilize various types of biosensors. Biosensors Biosensors would ideally provide high sens itivity and selectivity combined with a significant reduction in sample preparation, a ssay time, and reagent expense compared to most conventional detection methods. Much research has been performed, and is still being performed, attempting to optimize biosen sors for practical use. Although very few devices have demonstrated commercial succes s, biosensors are finding applications in quality assurance and proce ss control in biotechnology, f ood and drink sectors, and environmental protection (11, 29). Piezoelectic-based Biosensors The presence of biomolecules can be detected using piezoelectric mass-sensing biosensors. This form of biosensor utilizes quartz crysta l microbalance (QCM) resonators that measure resonance frequenc y changes that occur when a binding event, such as an antibody to an antigen, occur (56, 62, 69). Antibodies are first absorbed to the surface of the sensor; and the sens or is then exposed to the sample to be tested. If the antigen in question is present, it binds to the antibody triggering a change in resonance frequency which is recorded using computer software. A piezoelectric mass-sensing

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7 biosensor that uses QCM resonators coupled wi th antibodies as specific capture elements has been employed to detect Vibrio cholerae 0139, and was able to detect as few as 1 5 CFU/ml (8). A flow-through QCM bios ensor was developed utilizing a broad spectrum antiE. coli antibody as the capture moiety. A lthough the sensor did capture a broad range of E. coli strains, the limit of detection was no less than 1.7 5 CFU/ml (26). Wong et al. (65) were able to differentiate between three serotypes of Salmonella enteritidis with a QCM biosensor that utilized serotype-specific murine monoclonal antibodies as capture elements. Although the test had a high degree of specificity, their lowest detectable limit was 1.0 4 CFU/ml. In addition to the relatively low sensitivity of these methods for microorganisms, drawb acks of QCM resonating biosensors include their reliance on gold plated ca thodes and precision machiner y, which are very expensive and require advanced training to use effectivel y (49). Also, the deli cate electronics used by piezoelectric mass-sensing biosensors are temperature sensitive, which make them poor candidates for use as por table field detectors (49). Surface Plasmon Resonance (SPR) Biosensors Surface plasmon resonance (SPR) biosen sors are analogous to quartz crystal microbalances in that they al so detect a change in resona nce frequency in response to binding of analyte. However, SPR systems utilize light energy to sense changes in refractive indexes of an evanescent field wh en bio-molecular hybrid ization events occur on the sensor surface (35). Like QCM sensors, these binding events include antibody/antigen intera ctions used in immunoassays and direct nucleic acid detection (51). A SPR immunosensor assay was deve loped specifically for the detection of Legionella pneumophila in which the lowest detectable limit was 1 5 CFU/ml (43).

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8 A broad spectrum antiSalmonella antibody was the basis of a SPR biosensor immunoassay developed by Bokken et al (6). The assay allowed detection of whole cell Salmonella from five different biogroups, with the lowest detectable limit of 1.7 3 CFU. Although surface plasmon resonance bi osensors are capable of detecting whole cell microorganisms and the association of specific molecules, they have drawbacks. Impurities found in natural water sources such as suspended sediment and non-target microorganisms often bind nonspecifically to the surface of the SPR sensor, which can cause a change in resonance frequency, leadi ng to false positive detection events (22). Limited success in reduction of nonspecific binding has been achieved by coating the SPR sensor surface with an albumin blocking agent, but background noise continues to be a problem (9, 22). Other drawbacks of SPR sensors include the inability to perform high-throughput assays and high cost of use (35). Laser Evanescent Wave Fiber Optic Biosensors Evanescent wave biosensors utilize a laser that travels down a fiber optic waveguide, creating a region of laser light energy (evane scent wave) extending roughly 100 nm from the surface of the waveguide. Fluorescent molecules within this region emit light energy when excited by the evanescent wave. This emitted light energy travels back through the waveguide and is detected by software (61), like the aforementioned sensors. One particular evanescent wave biosensor, the Analyte 2000 flow through injection system (Research In ternational, Woodinville, WA), has been used to detect between 3-30 CFU of Escherichia coli O157:H7 seeded in ground beef (12) and 101,000 CFU of Listeria monocytogenes seeded in hotdogs following a 20 hour enrichment step (14). An automated, portable version of the Analyte 2000, the RAPTOR fiber optic

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9 biosensor (Research Internati onal, Woodinville, WA), was recently used to detect Salmonella enteritidis Typhimurium from spent irrigation water used on alfalfa sprouts, when seeds were contaminated with 50 CFU/g pr ior to germination (27). The bacteria in both assays were detected through a sandw ich assay in which a capture antibody, bound to a polystyrene waveguide, immobilizes the target cells. The de tection antibody, which is labeled with cyanine-5, is then introduced, providing the fluorescent molecule necessary to generate a signal. Biosensors that rely on electrochemical detection (QCM) have been noted to be sensitive to extreme ends of the pH and sa linity spectrum where electric current may be affected (36). In contrast, fiber optic-based sensors can readily be us ed in field situations due to their relative imperviousness to electric and magnetic interferen ce, as well as their potential for miniaturization at relatively low cost. Furthermore, the temperaturedependence of the fiber is significantly less th an that of electrodes used in piezoelecticbased biosensors (35). Analyte 2000 Biosensor The Analyte 2000 Biosensor (Research In ternational, Woodinville, WA) emits light at 635 nm. The most common use of this biosensor system is in conjunction with a sandwich-type immunoassay that uses fluores cent dye-labeled anti bodies for generation of the detection signal. A lase r diode provides the excitation light, which is launched into the proximal end of a dual tapered fiber optic waveguide. The light energy from the laser propagates an evanescent field approximately 100 nm outside the shaft of the waveguide. Any fluorophores caught inside this evanescen t wave become excited, and light emitted is recoupled back to the sensor (39, 61). A photodiode is used to quantify the collected

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10 emission light at wavelengths of 650 nm and above. The signal, in picoamps (pA), is then displayed using computer software specific for the Analyte 2000. Non-bound fluorophores in the waveguide chamber, whic h are farther from the surface of the waveguide than analyte-bound antibody-fluorop hore complexes, experience much lower evanescent field strength and therefore are not excited by the laser. This greatly reduces the effects of background interference and the occurrence of fals e positive detection signals. All evanescent wave biosensors benefit from this strategy and thus can obtain comparatively clean signals from samples c ontaining a high degree of impurities. The absence of bulk sample fluorescence, and th e ability to examine relatively impure sample homogenates, makes the fiber optic biosensor a good candidate for the detection of waterborne pathogens. The research presented here inves tigates the possibili ty of detecting V. vulnificus in surface waters using the Analyte 2000 biosensor. Polyclonal antibodies were generated in rabbits by inoculating them with sonicated cell extracts from V. vulnificus. Aliquots of the purified IgG preparation were conjugated with eith er biotin (capture antibody) or cyanine-5 (detection antibody), forming the basis for the sandwich immunoassay. Fiber optic waveguides were prepared for antibody binding by incubating them in a streptavidin solution. The wavegui des were then incubated with the biotinconjugated capture antibodies, allowing them to adhere to the waveguide via a strong avidin/biotin association. Various concentrations of V. vulnificus, suspended in both sterile and natural water samples, were then injected into the chamber and allowed to bind to the capture antibody. Fi nally, the waveguides were in cubated with the cyanine-5conjugated detection antibody, thus comple ting the sandwich by adding the fluorophore

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11 which is excited and detected by the bios ensor. A rapid immunoassay specific for V. vulnificus was designed that is both sensitive and specific and can be performed in less than seven hours.

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12 Materials and Methods Bacterial Cell Preparation Vibrio vulnificus, ATCC 27652 (American Type Cu lture Collection, Manassas, VA) isolated from human blood (clinical) wa s used to develop the biosensor assay. V. vulnificus from frozen stocks were reanimated by inoculating cells with a sterile inoculating loop into 10.0 ml of brain heart infusion (BHI) broth (1.0% NaCl) and incubated for 24 hours at 37C with shaking in a 125-ml Erlenmeyer flask. Cells were then streaked from BHI onto thiosulfate-ci trate-bile salts-sucrose (TCBS) agar and incubated at 37C for 24 hours. Green colonies (confirmation of V. vulnificus on TCBS) were inoculated onto Marine agar 2216 (Difco, Becton Di ckinson, Sparks, MD) slants, overlayed with sterile mineral o il, and kept at room temperat ure in the dark to maintain viable cultures between biosensor assays. Growth of cultures for the biosensor a ssays was initiated by inoculating a loopfull of cells from slants into 10.0 ml of BH I broth (1.0% NaCl) and grown at 37C with shaking for 24 hours. Following incubation, the entire broth culture was centrifuged at 10,000 g for 10 minutes at room temperature to pellet cells. The supernatant was removed and the pellet was washed twice with 1.0 ml sterile artificial salt water (ASW), prepared with synthetic sea salt (Instant Ocean Aquarium Systems, Mentor, OH) (18 parts per thousand), to remove any residual broth from the cells. The pellet was resuspended in 10 ml of ASW and stored at room temperature to preserve the integrity of the cells while inhibiting replication by de nying nutrients, as described by Campbell and

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13 Wright (7) for enumerating V. vulnificus via culturable plate counts. Serial dilutions of the suspension were spread onto Marine agar 2216 and allowed to incubate at 37C overnight. Culturable plate c ounts were performed on serial dilutions to determine the concentration, in colony forming units per m illiliter (CFU/ml), of the cell suspension. Accurate CFU counts to be assayed with the biosensor were acquired by pipeting the appropriate volume of suspension, which would gi ve the target CFU, into a sterile 1.5 ml microcentrifuge tube. The cells were pe lleted by centrifugati on at 10,000 g for 10 minutes at room temperature and the supernatant was removed. The cells were then resuspended in 200 l (capacity of the waveguide chamber) sterile deionized water. The entire volume of sterile deionized water cont aining target CFU was injected into the waveguide chamber during biosensor assays (figure 1a). For assays performed on V. vulnificus cell extracts, target CFU suspended in 200 l sterile deionized water were subjecte d to probe sonication (Sonic Dismembrator, model 100, Fisher Scientific, Pi ttsburg, PA) for 5 minutes at 14 Watts on ice. The entire 200 l volume of cell extract suspension was exposed to the waveguide during the biosensor assays (figure 1b). To confir m cell lysis by sonication, the waveguide along with the entire volume of target effluent (post assay), was placed into 10.0 ml of BHI broth (1.0% NaCl) and was allowed to incuba te overnight at 37C for 24 hours with shaking in a 125-ml Erlenmeyer flask. Th e entire volume of broth, along with the waveguide, was placed in a 10-ml test tube and centrifuged at 10,000 g for 10 minutes at room temperature to improve recovery of cells. The supernatant was removed and the pellet was resuspended in 1.0 ml of sterile ASW. The entire volume of cell extract suspension was spread onto Marine agar 2216 and allowed to incubate at 37C for 24

PAGE 25

hours. No colonies formed from suspensions of cell extracts, confirming loss of viability by sonication. Cells grown 37C for 24h Entire broth centrifuged Pellet washed twice and resuspended in ASW Plate count on serial dilutions 37C grown overnight -1-2-3Appropriate volume required for desired CFU count Cells pelleted(a) Cells resuspended in 200 l sterile deionized water or estuarine water and assayed (b) Cells resuspended in 200 l sterile water or estuarine water, sonicated, and then assayed -1-2-3 Figure 1. Schematic representation showing how CFU were estimated for assays on both whole cell (a), and cell extract suspensions (b). 14

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15 V. vulnificus Isolated from Environmental Waters V. vulnificus (designated MC0603S) was isolated in June 2003 from sediment (cultured in the Harwood lab at the University of South Florida, Tampa, FL) at a site on Marshall Creek in the Guana-Tolamato-Matan zas National Estuarine Research Reserve (St. Augustine, FL). The isolate was identified as V. vulnificus using the API-20E Enteric Identification System (B ioMerieux, Inc. Hazelwood, MO). Species identification was confirmed by PCR using sp ecies-specific primers (forward: 5-GAC TAT CGC ATC AAC AAC CG-3, reverse: 5-AGG TAG CGA GTA TTA CTG CC-3) targeting a portion of the cytolysin ( vvhA ) gene (GenBank accession number M34670 ) (18). Cells were reanimated from cryogeni c stocks by incubating in BHI broth (1.0% NaCl) at 37C for 24 hours with shaking. Cells were streaked from BHI broth onto TCBS agar and incubated at 37C for 24 hours. Green colonies we re inoculated onto Marine agar 2216 slants, overlayed with sterile mineral oil, an d kept at room temperature in the dark to maintain cultures between biosensor assays. For biosensor assays targeting V. vulnificus MC0603S, cells from slants were inoculated into 10.0 ml of BHI broth (1.0% NaCl) and grown at 37C with shaking for 24 hours. Following incubation, the entire broth culture was centrifuged at 10,000 g for 10 minutes at room temperature to pellet cel ls. The supernatant was removed and the pellet was washed twice with 1.0 ml of ASW to remove any residual broth. The pellet was re-suspended in 10.0 ml of ASW and stor ed at room temperature while plate count media was incubated. Serial dilutions of th e suspension were spread onto Marine agar 2216 and allowed to incubate at 37C overnight Direct plate count s were performed on serial dilutions to determine the concentration of the cell suspension. Target CFU to be

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16 assayed with the biosensor were acquire d by pipeting the appropriate volume of suspension, containing the target CFU, into a 1.5 ml microcentrif uge tube. The cells were pelleted by centrifuging at 10,000 g fo r 10 minutes at room temperature and the supernatant was removed. The cells were then resuspended in 200 l of sterile deionized water. The entire volume of sterile deioni zed water containing target CFU from the environmental isolate was injected into the waveguide chamber during biosensor assays (figure 1a). Cell extract pr eparation for the environmen tal isolate was performed by sonication for 5 minutes on ice according to the aforementioned sonication protocol used for the clinical (ATCC 27652) isolate of V. vulnificus (figure 1b). Non-target Bacteria The specificity of the biosensor immunoassay was tested by assaying several nontarget bacteria. Closely related Vibrio species ( Vibrio cholerae ATCC 11623, Vibrio parahaemolyticus ATCC 49398, and Vibrio alginolyticus ATCC 51160) and Escherichia coli ATCC 96370 were grown in BHI broth (1.0% NaCl for non-target Vibrios; 0.5% NaCl for E. coli ) at 37C with shaking for 24 hours. Cells were centrifuged at 10,000 g for 10 minutes at room temperature, the supernatant was removed, and cells were resuspended in 10.0 ml of sterile ASW for non-target Vibrios, and 10.0 ml of sterile phosphate buffered saline (PBS) (137 mM NaCl 2.7 mM KCl, 41.4 mM KH 2 PO 4 10.1 mM Na 2 HPO 4 pH 7.0) for the non-target E. coli Enumeration of culturable cells was accomplished by plate counts performed on serial dilutions of cells grown on Marine agar 2216 (non-target Vibrios ) or tryptic soy agar (TSA) ( E. coli ) at 37C for 24 hours. Target concentrations of cells were diluted into sterile deionized water at a total volume of 200

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17 l. The entire volume of whole cell dilution was exposed to the waveguide during the immunoassays (figure 1a). Mixed Cultures V. vulnificus ATCC 27652 and V. cholerae ATCC 11623 cells were grown in BHI broth (1.0% NaCl) at 37C w ith shaking for 24 hours. Following incubation, the entire broth culture from each cell line was centr ifuged at 10,000 g for 10 minutes at room temperature to pellet cells. The supernat ant was removed and the pellet was washed twice with 1.0 ml of ASW to remove any resi dual broth. The pellet was re-suspended in 10.0 ml of ASW and stored at room temperat ure while plate count media was incubated. Serial dilutions of the suspension were sp read onto Marine agar 2216 and allowed to incubate at 37C overnight. Direct plate c ounts were performed on serial dilutions to determine the concentration of the cell suspensi ons. Target CFU to be assayed with the biosensor were acquired by pipeting volumes of suspensions from both cell lines (giving a 1:1 ratio of V. vulnificus to V. cholerae) into the same microcen trifuge tube. The mixed suspension was centrifuged at 10,000 g for 10 minutes at room temperature and the supernatant was removed. The cells were then resuspended in 200 l of sterile deionized water. The entire volume of sterile deioni zed water containing mixed cell species was injected into the waveguide chamber dur ing biosensor assays (figure 1a). Detection in Estuarine Waters Estuarine water (salinity at 24.1 parts per thousand) was acquired from Northern Tampa Bay (Tampa, FL) in July 2004 at th e same location where other environmental V. vulnificus were isolated from oyster tissue. The estuarine water was filter-sterilized by passing it through a sterile 0.2 m syringe filter. V. vulnificus 27562 was grown in BHI

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18 broth (1.0% NaCl) at 37C w ith shaking for 24 hours. Following incubation, the entire broth culture was centrifuged at 10,000 g for 10 minutes at room temperature to pellet cells. The supernatant was removed and th e pellet was washed twice with 1.0 ml of ASW to remove any residual broth. The pelle t was re-suspended in 10.0 ml of ASW and stored at room temperature while plate count media was incubated. Serial dilutions of the suspension were spread onto Marine agar 2216 and allowed to incubate at 37C overnight. Culturable plate c ounts were performed on serial dilutions to determine the concentration of the cell suspen sion. Target CFU to be assayed with the biosensor were acquired by pipeting the appropr iate volume of suspension, c ontaining the target CFU, into a microcentrifuge tube. The cells we re pelleted by centrifuging at 10,000 g for 10 minutes at room temperature and the supernatant was removed. The cells were then resuspended in 200 l of estuarine water and assayed (figure 1a). For the cell extract assays, cell dilutions in estuarine water were sonicated for 5 minutes on ice prior to the assay as previously described (figure 1b). Assays were performed on both whole cells and cell extracts from V. vulnificus 27562 diluted in estuarine water. Enrichment Cultures V. vulnificus 27562 was grown at 37C for 24 hours in 10.0 ml of BHI broth (1.0% NaCl) with shaking. The entire br oth culture was centrifuged at 10,000 g for 10 minutes at room temperature to pellet cells. The pellet was re-suspended in 10.0 ml of ASW and stored at room temperature while plate count media was incubated overnight. Serial dilutions of the ASW suspension were spread onto Marine agar 2216 and allowed to incubate at 37C overnight. Culturable plat e counts were performed on serial dilutions to determine the concentration of the cell suspension. Two enri chment cultures inoculated

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19 with the same CFU (prepared above) were prep ared for each assay as detailed below: one was eventually injected into the waveguide chamber and assayed (figure 2a), and one was used for enumeration after enrichment to enumerate the culturable cells that were exposed to the waveguide at the time of th e assay (post-enrichment) (figure 2b). The appropriate ASW dilutions were centrifuged at 10,000 g for 10 minutes at room temperature and the pellet was resuspended in alkaline peptone water (APW) (5% peptone and 1.0% NaCl in sterile deionized water, pH 8.0) in a 15-ml conical test tube. The entire volume of APW seeded with known CFU was poured into sterile 125-ml Erlenmeyer flasks (allowing room for pr oper agitation) and inc ubated at 37C with shaking for 4 hours. The entire volume of APW enrichment was poured into 15-ml sterile conical test tubes and centrifuge d at 10,000 g for 10 minutes at room temperature to pellet cells. The pellet to be assayed with the biosensor was resuspended in 200 l sterile water, sonicated for 5 minut es on ice, and assayed with the biosensor (figure 2a). The pelle t to be enumerated was resuspe nded in ASW and plate counted on Marine agar 2216 incubated at 37 C overnight (figure 2b). Enrichment cultures were also established in PNCC broth (20). V. vulnificus 27562 was grown at 37C for 24 hours in 10.0 ml of BHI broth (1.0% NaCl) with shaking. The entire broth culture was cen trifuged at 10,000 g for 10 minutes at room temperature to pellet cells. The pellet was re-suspended in 10.0 ml of ASW and stored at room temperature while plate count media was incubated overnight. Serial dilutions of the ASW suspension were spread onto Marine agar 2216 and allowed to incubate at 37C overnight. Culturable plate c ounts were performed on serial dilutions to determine the concentration of the cell susp ension. Two enrichment cultures inoculated with the same

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20 CFU (prepared above) were prepared for each assay as detailed below: one was eventually injected into the waveguide ch amber and assayed (figure 2a), and one was used for enumeration after enrichment to enumerate the culturable cells that were exposed to the waveguide at the time of th e assay (post-enrichment) (figure 2b). The appropriate ASW dilutions were centrifuged at 10,000 g for 10 minutes at room temperature and resuspended in PNCC en richment broth in 15-ml sterile conical test tubes. Both pellets we re resuspended in 10.0 ml of PNCC enrichment broth. The entire volume of PNCC seeded with k nown CFU was poured into sterile 125-ml Erlenmeyer flasks (allowing room for pr oper agitation) and inc ubated at 37C with shaking for 4 hours. The entire volume of PNCC enrichment was poured into 15-ml sterile conical test tubes and centrifuge d at 10,000 g for 10 minutes at room temperature to pellet cells. The pellet to be assayed with the biosensor was resuspended in 200 l sterile water, sonicated for 5 minutes on ice, and assayed (figure 2a). The pellet to be enumerated was resuspended in ASW and plate counted on Marine agar 2216 incubated at 37C overni ght (figure 2b).

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Cells grown 37C for 24h Entire brothcentrifuged Plate count on serial dilutions 37C grown overnight -1 -2 -3 -1 -2 -3 Appropriate volume required for desired CFU count Cells pelleted Resuspended in enrichment broth (APW or PNCC) Incubated at 37C for 4h with shaking Enrichment transferred, pelleted, and resuspended in 200 l sterile water Enrichment transferred, pelleted, and resuspendedin 200 l ASW (b) Plate count for enumeration 37C overnight Sonication (a) Assayed with biosensor (a) (b)Pellet washed twice and resuspended in ASW Figure 2. Schematic representation of the enrichment assay protocol. Preparation (a) represents cell extract suspensions that were assayed with the biosensor, and (b) represents the enumeration of CFU exposed to the waveguide after enrichment. 21

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22 Antibodies Polyclonal antiV. vulnificus antibodies targeting intern al epitopes were made at Strategic Biosolutions (Newark, DE) by i noculating two New Zealand White rabbits (Specific Pathogen Free, designated A 7310 and A7311) with cell extracts from V. vulnificus 27652. Antiserum was bled from both ra bbits on five different dates in 2003 (January 1, January 17, January 23, Ma y 8, May 12, August 26, and August 22). Antibodies were purified from th e rabbit antiserum using a HiTrap rProtein A affinity purification kit (Amersham Biosciences Arlington Heights, IL) according to manufacturer instructions. Antiserum (0.8 ml) was first filtered using a 0.45 m syringefilter. A volume of 20 mM sodium phosphate buffer (2.0 M monobasic sodium phosphate monohydrate and 2.0 M dibasic so dium phosphate, pH 7.0) was pushed through the filter until the tota l volume of filtrate reached 1. 0 ml. The affinity column was washed with 10.0 ml of 20 mM sodium phosphate buffer (pH 7.0) at a rate of 1.0 ml/min. The antiserum filtrate (1.0 ml) was then applied through the affinity column twice, allowing the majority of antibody to bind to the column. The column was again washed with 10.0 ml of 20 mM sodium phosphate buffer (pH 7.0) at a rate of 1.0 ml/min. Antibodies were then eluted from the column in 0.5 ml fractions into 200 l of 0.1 M carbonate buffer (0.2 M sodium carbonate Na 2 CO 3 and 0.2 M sodium bicarbonate NaHCO 3 pH 9.3). Protein in twenty fractions of purified antibody was estimated using ultraviolet spectrophotometry (Beckman Coulter, model DU 640 spectrophotometer, Fullerton, CA) at 280 nm. Two fractions with the highest A 280 were pooled and titered using an enzyme-linked immunosorbent assa y (ELISA). Conjugates of this antibody

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23 preparation were used as both capture and detection moieties in all subsequent immunoassays. Measuring Antibody Titer by ELISA V. vulnificus 27562 was grown in 10.0 ml of BH I broth (1.0% NaCl) at 37 C with shaking for 24 hours. The entire broth culture was centrifuge d at 10,000 g for 10 minutes at room temperature to pellet cells The supernatant was removed and the pellet was washed twice with 1.0 ml of sterile 0.025 M carbonate buffer (pH 9.3). Cells were resuspended in 10.0 ml of sterile 0.025 M carbonate buffer (pH 9.3). The entire cell suspension was sonicated for 5 minutes on i ce as previously described. The protein concentration of the cell extract susp ension was measured using ultraviolet spectrophotometry at 280 nm (0.025 M carbonate buffer, pH 9.3 as blank). The cell extract suspension was diluted to a final con centration of 10.0 g/ml of total protein in 0.025 M carbonate buffer (pH 9.3). One hundred micr oliters of this diluent was used as a coating solution for wells in a 96-well mi crotiter plate (pol ystyrene 96-well round bottom, Corning Inc., Corning, NY) The plate was incubated at 4 C for 24 hours and washed 3 times with sterile deionized water. One hundred microliter s of PBT (PBS with 2% bovine serum albumin and 0.1% Tween 20) was added to each well. After incubation at room temperature for 1 hour, the plate wa s washed 3 times with sterile deionized water. Antibody suspensions from each bleed date were diluted 1:50, serial dilutions were made in PBT, and 100 l of each diluen t was added to the antigen-coated wells. The plate was again washed with sterile de ionized water 3 times after incubating for 1 hour at room temperature. A 100 l volume of peroxidase-conjugated goat anti-rabbit IgG (1.0 mg/ml, Sigma-Aldrich, Saint Louis, MO) diluted in PBT (1:8000) was added to

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each well and incubated at room temperature for 1 hour. Plates were washed with sterile deionized water and 100l of substrate (1.5 mg/ml of phenylenediamine dihydrochloride in 0.2 M sodium phosphate buffer, pH 5.0) was added. The substrate was allowed to incubate in the wells for 10 minutes, and sulphuric acid (50 l of 4 M H 2 SO 4 ) was added to stop the reaction. The plates were read at 490 nm using a fluorescent plate reader (CytoFluor 4000 Series Fluorescence Multi-well Plate Reader, Applied Biosystems, Foster City, CA). Antiserum from both A7310 and A7311 rabbits for five different bleed dates were measured for antibody titer (data not shown). Two bleed dates (January 23, 2003 and August 22, 2003) from the A7311 rabbit had the highest antibody titer (figure 3). Antibodies purified from antiserum obtained on these two dates were used in all subsequent assays. 010000200003000040000500006000070000800009000010000050100200400800160032006400128002560051200102400Antibody Dilutionrelative fluorescence units 1/23/03 bleed date 8/22/03 bleed date Negative (no antigen) Figure 3. Measuring antibody titer after purification from antiserum using an ELISA plate system. Antibodies purified from antiserum, bled on 1/23/03 and 8/22/03 from rabbit A7311, were used in forming both capture and detection moieties for all immuno-assays. 24

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25 Antibody Conjugation AntiV. vulnificus polyclonal antibodies purifie d from the aforementioned antiserum were used as the basis for deve loping the immuno-sandwic h biosensor assay. The capture antibody was formed by conjugating purified polyclonal antibodies with biotin using the E-Z Link NHS-LC-LC Bi otin (Pierce Biotechnology, Rockford, IL) buffer exchange system, according to manufactur er specifications. Final concentrations of purified antibody from the pooled elutions with the highest A 280 were estimated using ultraviolet spectrophotometry (using 0.025 carbo nate buffer, pH 9.3 as a blank) at 280 nm. Antibodies were diluted to 2.0 mg/ml in 0.025 carbonate buffer (pH 9.3). After 0.5 mg of EZ link NHS-LC-LC Biotin was dissolved in 1.0 ml N N -dimethylformamide (DMF), 75 l of this solution was added to 475 l of purified antibody solution. The sample was then inverted several times to mix and allowed to incubate on ice for 2 hours at room temperature. Unincorporated biotin was removed by gel filtration on a Bio-Gel P10 column (Bio-Rad, Hercules, CA) equili brated with PBS and 0.02% sodium azide. Stock solutions of the biotin labeled antiV. vulnificus polyclonal antibodies were estimated for total protein concentration us ing ultraviolet spectrophotometry at 280 nm and stored in the dark at 4C until needed. For each channe l assayed with the biosensor, 200 l of the biotinylated capture an tibody (diluted to 100 mg/ml total protein concentration in PBS) was incubated on the waveguide, as described by Tims and Lim (58), for detecting Bacillus anthracis spores using the Analyte 2000 biosensor. The capture antibody adheres to the streptavidin coated waveguide utilizing the strong, noncovalent, biotin/avidin association. The capture antibody is responsible for adhering the target antigen to the surface of the waveguide awaiting detection.

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26 The detection antibody was formed by c onjugating purified polyclonal antibodies with cyanine-5 using the FluoroLink Cy5 Reactive Dye pack (Amersham Life Sciences, Arlington Heights, IL) according to manufacturer specifications. Purified antibody concentrations from the pool ed elutions with the highest A 280 were estimated using ultraviolet spectrophotometry (using 0.1 M carbonatebicarbonate buffer, pH 9.3 as a blank) at 280 nm. Antibodies were diluted to 2.0 mg/ml in 0.1 M carbonate bicarbonate buffer (pH 9.3) for a total volume of 500 l. The entire 500 l was added to the dye vial, capped, mixed thoroughly, and inc ubated in the dark at room temperature for 30 minutes. Labeled antibody was purified from free dye by gel filtration on a BioGel P10 column equilibrated with PBS and 0.02% sodium azide. Efficient labeling was confirmed by estimating the protein to dye ra tio using ultraviolet spectrophotometry at 280 nm and 650 nm respectively. No aliquot of labeled antibody with less than a 2:1 dye to antibody ratio was used in any immunoassay. Stock solutions of cyanine-5 labeled antiV. vulnificus antibodies were estimated for total protein concentration using ultraviolet spectrophotometry at 280 nm and stor ed in the dark at 4C until needed. For each channel assayed with the biosensor, 200 l of the biotinylated capture antibody (diluted to 10.0 g/ml total protein concen tration in blocking buffer) was incubated on the waveguide as described by Tims and Lim (58). The detection an tibody is responsible for finishing the detection sandwich in the immunoassay by incorpor ating the fluorescent moiety (cyanine-5) which is excite d and measured by the biosensor. Preparing the Polystyrene Waveguides Optics grade polystyrene fibers (wav eguides) (Research International, Woodinville, WA) were washed by sonication in an isopropanol bath (FS30 Ultrasonic

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27 Bath Cleaner, Fisher Scientific, Pittsbur g, PA) for 30 seconds at 130 Watts. The waveguides were rinsed with st erile deionized water and allowe d to dry by inverting in a waveguide holder (Research Internationa l, Woodinville, WA). The tips of the waveguides (approximately 1.0 mm of the distal end) were di pped into flat black enamel paint (Testors, Rockford, IL) and allowed to dry for 30 minutes. The waveguides were then placed into sealed glass capillary t ubes and incubated in a streptavidin (SigmaAldrich, Saint Louis, MO) solution at100 g/m l in PBS overnight at 4C, allowing the streptavidin to adsorb to the surface of the waveguide. Immunoassay Development for the Analyte 2000 Biosensor Streptavidin-coated waveguides were placed into four chambers attached to each of the four channels of the biosensor. On e milliliter of sterile PBST (PBS with 0.1% Tween 20, pH 7.0) was injected with a syri nge through the chamber to wash away any non-adsorbed streptavidin from the wavegui de. Two hundred microliters of biotinconjugated antV. vulnificus capture antibody (100 g/ml) was injected into the waveguide chamber and allowed to incubate at room temperature for 30 minutes. The waveguide was then rinsed with 1.0 ml PBST to wash away any unbound capture antibody. The waveguides were then inc ubated with 200 l blocking buffer (PBS containing 2.0 mg/ml bovine serum albumin, 2.0 mg/ml casein) for 30 minutes at room temperature to inhibit capture antibo dy from adsorbing to the waveguide nonspecifically. The threshold signal level that must be exceeded to constitute a positive detection event was determined separately fo r each for each channel of the Analyte 2000, which includes four channels. Two hundred mi croliters of detection antibody (10.0 g/ml cyanine-5 conjugated antiV. vulnificus antibody diluted in blocking buffer) was injected

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28 into each chamber and allowed to incubate at room temperature for 5 minutes. The chambers were then rinsed twice with 1.0 ml PBST, the laser was activated, and fluorescence measurements (pA) were taken. The incubation with detection antibody, rinses, and readings were repeated five more times to acquire six total background readings for each channel. The threshol d signal for each channel was determined by calculating three times the standard deviati on of the signal changes between each consecutive background signal, added to the mean signal change (threshold signal = [(standard deviation of signal between each consecutive background signal 3) + (mean background signals)]. After the last background signal was taken, 200 l of the diluted V. vulnificus sample was injected into the chamber and in cubated at room temperature for 10 minutes. The waveguide was again rinsed with 1.0 ml PBST. Detection antibody (same composition and volume used in background readings) was injected into the chamber and incubated at room temperature for 5 minutes, allowing the cyanine-5-conjugated antibodies to bind to the captured V. vulnificus antigen. A final rinse of 1.0 ml PBST was injected to wash away any unbound detec tion antibody from the surface of the waveguide. Fluorescence measurements were taken following the last wash. Any detection signal greater than the threshold signal determined for a given wave guide is considered a positive detection event, while a signal less than the threshold, is considered no detection. A schematic representa tion of the components comprising the immunoassay is given in figure 4.

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Step 1 Step 2 Step 3 Step 4 streptavidin target antigen biotinylated capture antibody cyanine-5-conjugated detection antibody Figure 4. Schematic representation of the biosensor immunoassay. The polystyrene waveguide is first coated with streptavidin (step 1). The biotin-conjugated capture antibody binds to the streptavidin (step 2). The target antigen is specifically bound by the capture antibody (step 3). The cyanine-5-conjugated detection antibody binds to the captured antigen, completing the immuno-sandwich assay (step 4). 29

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30 Correlation between Mean Corrected Detection Signals and CFU Corrected detection signals for all assay settings were acquired by subtracting the threshold signal from the detection signal for each detection event. Signals acquired from whole cell and cell extract assays for V. vulnificus 27562 assayed in sterile water, V. vulnificus (MC0603S) assayed in sterile water, and V. vulnificus 27562 assayed in estuarine water were compared. Also, signa ls acquired from both enrichment assays (APW and PNCC) were compared. The mean corrected detection signals were compared with CFU in each of the assay settings. Correlation coefficients were calculated for both normally distributed assay settings (Pearson s r value), and settings that were not normally distributed (Spearma ns r). Also, p values ( = 0.05) and 95% confidence intervals were calculated for each assay setting. Comparison of Mean Corrected Detect ion Signals between Clinical and Environmental Isolates Corrected detection signals were compar ed between whole cell and cell extract assays on both clinical (ATCC 27562) and environmental (MC0603S) V. vulnificus isolates suspended in sterile water. The sample sizes for some assay settings were too small to determine accurate distribution. In these instances, normal distribution was assumed, and unpaired t tests with Welch corre ctions were performed on mean corrected detection signals. Two-tailed p values ( = 0.05) were calculated for each comparison to determine if the differences between detection signals for both isolates were statistically significant.

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31 Results All subsequent biosensor assays were pe rformed using the same general protocol for the detection of V. vulnificus and non-target antigen. St reptavidin-coated waveguides were placed into four chambers attached to each of the four channels of the biosensor. PBST was injected through the chamber to wa sh away any non-adsorbed streptavidin. Biotin-conjugated capture antibody was inje cted into the waveguide chamber and allowed to incubate at room temperature fo r 30 minutes. The waveguide was then rinsed with PBST and incubated in blocking buffer for 30 minutes at room temperature. The threshold signal for each channel was dete rmined by incubating cyanine-5 detection antibody on the waveguide for 5 minutes, rinsed with PBST, and fluores cent signals (pA) were recorded by the biosensor. This was repeated 5 more times to acquire a signal for each consecutive background reading. The threshold signal for each channel was determined by calculating three times the st andard deviation of the signal changes between each consecutive background signal, ad ded to the mean signal change (threshold signal = [(standard deviation of signal between each consecutive background signal 3) + (mean background signals)]. After the last background signal was taken, diluted V. vulnificus or non-target antigen (equal volumes of PBS for negative channels) was injected into the chamber and incubated at room temperature for 10 minutes. The waveguide was rinsed with PBST and detection antibody was incubated on the waveguide at room temperature for 5 minut es. Waveguides were rinsed PBST, and fluorescence measurements were taken. Any detection signal over th e threshold signal

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32 determined for a given chamber is considered a positive detection event, while a signal under the threshold, is cons idered no detection. Initial Detection of Whole Cell V. vulnificus 27562 using the Biosensor An initial assay was performed with a high number of whole cell CFU to confirm the usefulness of the antiV. vulnificus polyclonal antibodies in the immunoassays. Cells were grown in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU were enumerated and suspended in 200 l of st erile water. Three whole cell suspensions (1 4 1 5 and 1 6 ) were assayed, all having positive detection events with no positive signal in the negative (no target antigen) channel (table 1). It is important to note that the numbers given in all subsequent tables are not conc entrations (i.e. CFU/ml), but are actual cell numbers (CFU) exposed to th e waveguides. The assay was replicated twice (tables 2 and 3), each time with similar results.

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33 CFU number 1 4 1 5 1 6 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 3.2 9.5 2.6 1.6 2. signal over previous background 4.6 11.2 12.6 15.6 3. signal over previous background -9.2 -6.3 1.6 -4.6 4. signal over previous background 1.6 1.0 4.6 4.6 5. signal over previous background 6.2 7.3 -7.6 1.8 6. Standard deviation of signals 6.1 7.2 7.2 7.4 7. Standard deviation 3 18.3 21.6 21.6 22.2 8. Average of signals 1.3 4.5 2.8 3.8 9. Threshold signal (standard deviation 3) + (mean signal) 19.6 26.1 24.4 26.0 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 70.6 249.2 529.6 4.5 12. Detection results Positive Positive Positive Negative Table 1. Initial dete ction of whole cell V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor. Values represented are the ch ange in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target tr eatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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34 CFU number 1 4 1 5 1 6 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 2.6 9.6 -7.6 16.2 2. signal over previous background -6.5 -8.3 4.6 -2.6 3. signal over previous background 11.2 4.2 4.2 6.9 4. signal over previous background 5.2 2.7 8.4 1.6 5. signal over previous background 1.9 1.3 10.5 7.5 6. Standard deviation of signals 6.4 6.5 7.0 7.1 7. Standard deviation 3 19.2 19.5 21.0 21.3 8. Average of signals 2.9 1.9 4.0 5.9 9. Threshold signal (standard deviation 3) + (mean signal) 22.1 21.4 25.0 27.2 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 75.2 229.4 625.8 9.5 12. Detection results Positive Positive Positive Negative Table 2. First replicate detecting whole cell V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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35 CFU number 1 4 1 5 1 6 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background -4.6 3.2 5.6 11.3 2. signal over previous background 5.3 7.0 1.6 1.2 3. signal over previous background 11.2 -5.6 8.3 9.3 4. signal over previous background -9.2 9.3 4.3 -1.6 5. signal over previous background 5.6 6.7 -2.3 4.2 6. Standard deviation of signals 8.3 5.9 4.0 5.4 7. Standard deviation 3 24.9 17.7 12.0 16.2 8. Average of signals 1.7 4.1 3.5 4.9 9. Threshold signal (standard deviation 3) + (mean signal) 26.6 21.8 15.5 21.1 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 45.2 310.6 469.2 11.3 12. Detection results Positive Positive Positive Negative Table 3. Second replicat e detecting whole cell V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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36 Initial Detection of V. vulnificus (ATCC 27562) Cell Extracts using the Biosensor Cell extracts were assayed with the bi osensor in attempt to lower the limit of detection (minimum CFU required to produ ce a positive detection event) by sonicating cells prior to the assay. Cells were grow n in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU were enumerated and suspended in 200 l of sterile water. Suspensions were then sonicated for 5 minutes on ice. Three cell extract suspensions from high CFU (1 3 1 4 and 1 5 ) were assayed, all having positive detection events with no positive signal in the negative ch annel (table 4). The assay was replicated twice (tables 5 and 6), each time with similar results.

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37 CFU number 1 3 1 4 1 5 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 8.2 10.3 8.2 11.2 2. signal over previous background -4.2 4.9 -4.2 -1.2 3. signal over previous background 1.2 5.0 4.6 7.5 4. signal over previous background 5.6 -1.4 1.8 4.6 5. signal over previous background -4.6 -1.9 -1.7 -2.3 6. Standard deviation of signals 5.7 5.1 4.9 5.7 7. Standard deviation 3 17.1 15.3 14.7 17.1 8. Average of signals 1.2 3.4 1.7 4.0 9. Threshold signal (standard deviation 3) + (mean signal) 18.3 18.7 16.4 21.1 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 32.5 98.6 394.1 8.2 12. Detection results Positive Positive Positive Negative Table 4. Initial detectio n of cell extracts from V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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38 CFU number 1 3 1 4 1 5 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 5.3 10.5 4.6 9.6 2. signal over previous background 2.6 8.6 1.6 -4.3 3. signal over previous background 1.3 -7.6 2.0 1.6 4. signal over previous background -7.2 1.3 3.6 4.6 5. signal over previous background 1.5 -8.3 -1.5 3.2 6. Standard deviation of signals 4.7 8.8 2.3 5.0 7. Standard deviation 3 14.1 26.4 7.9 15.0 8. Average of signals 0.7 0.9 2.1 2.9 9. Threshold signal (standard deviation 3) + (mean signal) 14.8 27.3 9.0 17.9 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 28.6 111.2 414.2 6.8 12. Detection results Positive Positive Positive Negative Table 5. First replicate detecting cell extracts from V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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39 CFU number 1 3 1 4 1 5 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 11.2 10.2 11.3 12.3 2. signal over previous background 4.6 -3.2 4.6 -1.2 3. signal over previous background 7.3 4.0 2.3 2.3 4. signal over previous background -4.3 5.3 5.3 2.6 5. signal over previous background 1.8 7.6 2.3 4.9 6. Standard deviation of signals 5.8 5.0 3.7 5.0 7. Standard deviation 3 17.4 15.0 11.1 15.0 8. Average of signals 4.1 4.8 5.2 4.2 9. Threshold signal (standard deviation 3) + (mean signal) 21.5 19.8 16.3 19.2 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 35.9 116.3 416.2 7.7 12. Detection results Positive Positive Positive Negative Table 6. Second replicate de tecting cell extracts from V. vulnificus (ATCC 27562) using the Analyte 2000 biosensor. Values represented are the change in signals between each of the six consecutive background r eadings (rows 1-6), calculati ons used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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40 Determining the Sensitivity on Whole Cell V. vulnificus (ATCC 27562) The initial biosensor assay was repeated with lower CFU in order to determine the sensitivity of the assay in detecting whole cells. Cells were grown in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU we re enumerated and suspended in 200 l of sterile water. Suspensions of 1 2 1 3 and 1 4 whole cells were assayed (table 7) and replicated twice (tables 8 and 9). None of the assays were able to detect 1 2 or 1 3 dilutions, yet all detected 10 4 There were no positive signals for any of the negative channels. CFU number 1 2 1 3 1 4 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 1.6 7.8 8.4 5.2 2. signal over previous background 4.2 11.5 -2.5 4.1 3. signal over previous background -8.2 -8.2 9.2 6.2 4. signal over previous background 4.1 2.5 4.0 -5.2 5. signal over previous background 7.5 3.2 1.2 1.2 6. Standard deviation of signals 6.0 7.4 4.9 4.6 7. Standard deviation 3 18.0 22.2 14.7 13.8 8. Average of signals 1.8 3.4 4.1 2.3 9. Threshold signal (standard deviation 3) + (mean signal) 19.8 25.6 18.8 16.1 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 4.2 20.2 52.3 1.3 12. Detection results Negative Negative Positive Negative Table 7. Determining the sensi tivity of the assay on whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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41 CFU number 1 2 1 3 1 4 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 6.2 11.2 6.1 8.4 2. signal over previous background 8.2 6.2 7.5 -4.3 3. signal over previous background -4.3 1.3 6.2 7.8 4. signal over previous background 9.0 7.5 -1.2 4.1 5. signal over previous background 4.2 -4.2 11.3 1.6 6. Standard deviation of signals 5.3 6.0 4.5 5.2 7. Standard deviation 3 15.9 18.0 13.5 15.6 8. Average of signals 4.7 4.4 6.0 3.5 9. Threshold signal (standard deviation 3) + (mean signal) 20.6 22.4 19.5 19.1 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background -1.6 14.5 62.8 3.8 12. Detection results Negative Negative Positive Negative Table 8. First replicate determining th e sensitivity of th e assay on whole cell V. vulnificus (ATCC 27562). Values repr esented are the change in signals between each of the six consecutive background r eadings (rows 1-6), calculati ons used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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42 CFU number 1 2 1 3 1 4 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 6.2 -2.3 11.6 3.5 2. signal over previous background 7.2 7.1 3.2 -9.3 3. signal over previous background -3.6 2.3 -1.6 7.2 4. signal over previous background 10.2 -8.3 4.8 1.5 5. signal over previous background 4.9 3.2 8.6 6.2 6. Standard deviation of signals 5.2 5.9 5.1 6.6 7. Standard deviation 3 15.6 17.7 15.3 19.8 8. Average of signals 5.0 0.4 5.3 1.8 9. Threshold signal (standard deviation 3) + (mean signal) 20.6 18.1 20.6 21.6 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 4.1 6.4 49.8 2.0 12. Detection results Negative Negative Positive Negative Table 9. Second replicate determining the sensitivity of th e assay on whole cell V. vulnificus (ATCC 27562). Values repr esented are the change in signals between each of the six consecutive background r eadings (rows 1-6), calculati ons used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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43 Determining the Sensitivity toward V. vulnificus (ATCC 27562) Cell Extracts The initial biosensor assay was repeated on cell extracts from a lower CFU in order to determine the sensitivity of the assay in detecting cell extracts. Cells were grown in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU were enumerated, suspended in 200 l of sterile water, and soni cated for 5 minutes on ice. Suspensions of extracts from 1 2 1 3 and 1 4 cells were assayed (table 10) and replicated twice (tables 11 and 12). None of the thr ee assays were able to detect 1 2 suspensions, yet all detected the 1 3 and 1 4 suspensions. There were no positive signals for any of the negative channels.

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44 CFU number 1 2 1 3 1 4 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 5.3 2.6 11.3 1.3 2. signal over previous background -1.2 6.3 4.2 1.5 3. signal over previous background 6.2 6.0 7.9 -8.3 4. signal over previous background 8.3 -4.2 8.3 4.6 5. signal over previous background 2.3 4.6 9.2 4.9 6. Standard deviation of signals 3.7 4.3 2.5 5.4 7. Standard deviation 3 11.1 12.9 7.8 16.2 8. Average of signals 4.2 3.1 8.2 0.8 9. Threshold signal (standard deviation 3) + (mean signal) 15.3 16.0 16.0 17.0 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 10.2 36.3 94.5 6.3 12. Detection results Negative Positive Positive Negative Table 10. Sensitivity of the biosensor assay on V. vulnificus (ATCC 27562) cell extracts. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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45 CFU number 1 2 1 3 1 4 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 11.3 6.3 14.3 7.3 2. signal over previous background 3.6 4.5 6.3 8.0 3. signal over previous background -2.6 3.6 1.6 -4.6 4. signal over previous background 3.6 -4.6 4.3 6.3 5. signal over previous background 4.2 1.6 6.3 4.6 6. Standard deviation of signals 4.9 4.2 4.7 5.1 7. Standard deviation 3 14.7 12.6 14.1 15.3 8. Average of signals 4.0 2.3 6.6 4.3 9. Threshold signal (standard deviation 3) + (mean signal) 18.8 14.9 20.7 19.6 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 9.6 30.8 110.6 4.6 12. Detection results Negative Positive Positive Negative Table 11. First replicate determining th e sensitivity of th e biosensor assay on V. vulnificus (ATCC 27562) cell extracts. Values repr esented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

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46 CFU number 1 2 1 3 1 4 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 9.6 11.6 10.3 7.6 2. signal over previous background -2.3 1.6 1.9 7.0 3. signal over previous background 6.5 -1.6 -4.6 5.6 4. signal over previous background 3.6 7.8 7.7 -4.5 5. signal over previous background 5.5 9.0 7.6 4.6 6. Standard deviation of signals 4.4 5.5 6.0 5.0 7. Standard deviation 3 13.2 16.5 18.0 15.0 8. Average of signals 4.6 5.7 4.6 4.0 9. Threshold signal (standard deviation 3) + (mean signal) 17.8 22.2 22.6 19.0 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 8.5 38.9 123.2 10.6 12. Detection results Negative Positive Positive Negative Table 12. Second replicate determining th e sensitivity of the biosensor assay on V. vulnificus (ATCC 27562) cell extracts. Values repr esented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

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47 Testing the Reproducibility of th e Biosensor for Detection of 1 4 Whole Cell V. vulnificus (ATCC 27562) The reproducibility of biosensor detection of whole cell CFU was tested by assaying suspensions containing 1 3 and 1 4 cells (table 13) and was replicated nine times (tables 14-22). Cells were grown in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU were enumerated, suspended in 200 l of sterile water, and assayed. To more rigorously control the accuracy of the a ssays, an extra negative (total of 2) was added to each assay. In all ten assays the biosensor was ab le to detect 1 4 cells with no positive signals in either the 1 3 or the negative channels. CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 14.2 12.5 2.3 6.4 2. signal over previous background 5.3 11.2 4.2 7.5 3. signal over previous background -2.3 3.5 12.5 2.3 4. signal over previous background 12.6 -4.0 -4.2 7.1 5. signal over previous background 7.5 6.2 3.6 -4.9 6. Standard deviation of signals 6.6 6.6 6.0 5.2 7. Standard deviation 3 19.7 19.9 17.9 15.7 8. Average of signals 7.5 5.9 3.7 3.7 9. Threshold signal (standard deviation 3) + (mean signal) 27.1 25.8 21.6 19.4 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background -1.2 50.6 2.5 -1.1 12. Detection results Negative Positive Negative Negative Table 13. Reproducibility of the bios ensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values repr esented are the change in signals between each of the six consecutive background r eadings (rows 1-6), calculati ons used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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48 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 6.2 10.2 14.2 -2.3 2. signal over previous background 4.5 2.3 -2.1 9.2 3. signal over previous background 1.2 -4.6 2.5 2.7 4. signal over previous background 11 -2.3 9.2 6.9 5. signal over previous background -3.6 7.8 4.2 -4.5 6. Standard deviation of signals 5.5 6.3 6.3 5.8 7. Standard deviation 3 16.5 18.9 18.9 17.4 8. Average of signals 3.9 2.7 5.6 2.4 9. Threshold signal (standard deviation 3) + (mean signal) 20.4 21.6 24.5 19.8 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background -11.2 75.6 6.5 -1.9 12. Detection results Negative Positive Negative Negative Table 14. First replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection result s (row 12) for each chamber. All values are in pico amps (pA).

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49 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 11.2 3.2 -5.2 -1.5 2. signal over previous background 2.5 11.3 2.3 8.4 3. signal over previous background 8.2 4.3 9.2 2.5 4. signal over previous background -4.2 6.9 10.2 3.0 5. signal over previous background 4.9 1.3 7.6 10.2 6. Standard deviation of signals 5.9 3.9 6.4 4.7 7. Standard deviation 3 17.7 11.7 19.2 14.1 8. Average of signals 4.5 5.4 4.8 4.5 9. Threshold signal (standard deviation 3) + (mean signal) 22.2 17.1 24.0 18.6 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 4.6 45.5 2.4 -1.2 12. Detection results Negative Positive Negative Negative Table 15. Second replicate determining the re producibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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50 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 9.6 5.5 8.4 11.3 2. signal over previous background -2.3 8.6 4.6 7.6 3. signal over previous background 6.9 4.6 -2.3 5.6 4. signal over previous background 1.2 -1.6 5.6 4 5. signal over previous background 4.6 5.3 6.2 -2.5 6. Standard deviation of signals 4.7 3.7 4.0 5.1 7. Standard deviation 3 14.1 11.1 12.0 15.3 8. Average of signals 4.0 4.5 4.5 5.2 9. Threshold signal (standard deviation 3) + (mean signal) 18.1 15.6 16.5 20.5 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 5.9 66.5 6.9 10.5 12. Detection results Negative Positive Negative Negative Table 16. Third replicate determining the re producibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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51 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 6.9 11.2 10.2 13.6 2. signal over previous background 4.2 6.1 1.3 -1.6 3. signal over previous background -1.8 7.0 2.6 4.6 4. signal over previous background 7.6 4.6 1.9 4.9 5. signal over previous background 7.6 1.6 -1.6 4.0 6. Standard deviation of signals 4.0 3.5 4.4 5.4 7. Standard deviation 3 12.0 10.5 13.2 16.2 8. Average of signals 4.9 6.1 2.9 5.1 9. Threshold signal (standard deviation 3) + (mean signal) 16.9 16.6 16.1 21.3 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 9.5 50.9 4.6 8.0 12. Detection results Negative Positive Negative Negative Table 17. Fourth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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52 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 5.6 12.3 11.3 4.9 2. signal over previous background 7.9 2.6 -1.6 8.9 3. signal over previous background 5.6 6.9 5.6 8.0 4. signal over previous background 8.6 4.6 6.2 -3.6 5. signal over previous background -1.6 1.2 5.6 4.1 6. Standard deviation of signals 4.0 4.4 4.6 4.9 7. Standard deviation 3 12.0 13.2 13.8 14.7 8. Average of signals 5.2 5.5 5.4 4.5 9. Threshold signal (standard deviation 3) + (mean signal) 17.2 18.7 19.2 19.2 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 10.3 75.2 9.8 11.3 12. Detection results Negative Positive Negative Negative Table 18. Fifth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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53 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 10.2 6.5 10.3 4.6 2. signal over previous background -2.6 6.8 4.3 7.0 3. signal over previous background 6.6 4.5 8.6 -5.3 4. signal over previous background 7.6 -2.6 -4.6 4.8 5. signal over previous background 3.3 4.0 7.6 3.5 6. Standard deviation of signals 4.9 3.8 5.9 4.8 7. Standard deviation 3 14.7 11.4 17.7 14.4 8. Average of signals 5.0 3.8 5.2 2.9 9. Threshold signal (standard deviation 3) + (mean signal) 19.7 15.2 22.9 17.3 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 4.6 45.3 8.1 6.4 12. Detection results Negative Positive Negative Negative Table 19. Sixth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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54 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 10.6 10.7 6.5 7.7 2. signal over previous background -4.2 4.5 1.3 7.0 3. signal over previous background 8.5 4.6 -1.6 2.3 4. signal over previous background 7.9 2.0 6.8 8.4 5. signal over previous background 3.0 6.3 2.3 1.6 6. Standard deviation of signals 5.9 3.2 3.6 3.2 7. Standard deviation 3 17.7 9.6 10.8 9.6 8. Average of signals 5.2 5.6 3.1 5.4 9. Threshold signal (standard deviation 3) + (mean signal) 22.9 15.2 13.9 15.0 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 11.2 50.2 10.8 4.2 12. Detection results Negative Positive Negative Negative Table 20. Seventh replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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55 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 10.5 17.3 5.2 6.2 2. signal over previous background -1.2 5.6 9.4 4.1 3. signal over previous background 5.0 7.6 4.8 7.2 4. signal over previous background 6.2 3.2 -1.2 -2.4 5. signal over previous background 7.5 1.9 5.0 6.4 6. Standard deviation of signals 4.3 6.1 3.8 3.9 7. Standard deviation 3 12.9 18.3 11.4 11.7 8. Average of signals 5.6 7.1 4.6 4.3 9. Threshold signal (standard deviation 3) + (mean signal) 18.5 25.4 16.0 16.0 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 6.6 46.8 2.3 3.7 12. Detection results Negative Positive Negative Negative Table 21. Eighth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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56 CFU number 1 3 1 4 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 10.6 11.3 9.8 7.9 2. signal over previous background 7.2 6.5 4.3 3.8 3. signal over previous background -1.6 7.6 1.6 4.6 4. signal over previous background 3.6 6.0 1.5 7.5 5. signal over previous background 4.9 4.6 3.6 -2.6 6. Standard deviation of signals 4.5 2.5 3.4 4.2 7. Standard deviation 3 13.5 7.5 10.2 12.6 8. Average of signals 4.9 7.2 4.2 4.2 9. Threshold signal (standard deviation 3) + (mean signal) 18.4 14.7 14.4 16.8 10. Target treatment add cells add cells add PBS add PBS 11. Detection signal signal over the last background 5.5 59.3 4.2 7.8 12. Detection results Negative Positive Negative Negative Table 22. Ninth replicate determining the reproducibility of the biosensor assay for detection of 1 4 whole cell V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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57 Testing the Reproducibility of the Biosen sor for Detection of Cell Extracts from 1 3 V. vulnificus (ATCC 27562) The reproducibility of the biosensor for detection of cell extracts was tested by performing an assay on extracts from 1 2 and 1 3 CFU (table 23), and replicated nine times (tables 2432). Cells were gr own in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU were enumerated, su spended in 200 l of sterile water, and sonicated for 5 minutes on ice prior to the a ssay. Again, to more rigorously test the accuracy of positive detection events, an extra negative was added to each assay. In nine of the ten assays, the biosensor was able to detect extracts from 10 3 CFU, with no positive signals in either the 1 2 suspensions or the negative channels. One of the replicate assays was not able to detect extract from 1 3 CFU (table 26).

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58 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 12.3 10.3 11.3 8.3 2. signal over previous background 4.2 2.5 6.5 7.8 3. signal over previous background 1.6 1.8 2.5 -1.6 4. signal over previous background -1.5 -2.0 1.5 5.6 5. signal over previous background 9.5 6.5 8.5 6.2 6. Standard deviation of signals 5.7 4.7 4.1 4.0 7. Standard deviation 3 17.1 14.1 12.3 12.0 8. Average of signals 5.2 3.8 6.1 5.3 9. Threshold signal (standard deviation 3) + (mean signal) 22.3 17.9 18.4 17.3 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 2.5 20.6 6.0 4.8 12. Detection results Negative Positive Negative Negative Table 23. Reproducibility of the biosensor assa y for detection of cell extracts from 110 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (row s 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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59 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 11.5 16.4 9.9 10 2. signal over previous background 6.5 7.2 8.5 4.8 3. signal over previous background -2.3 4.5 6.1 -2.3 4. signal over previous background 6.5 4.6 -2.6 3.3 5. signal over previous background 5.6 5.9 3.4 5.6 6. Standard deviation of signals 5.0 5.0 5.0 4.4 7. Standard deviation 3 15.0 15.0 15.0 13.3 8. Average of signals 5.6 7.7 5.1 4.3 9. Threshold signal (standard deviation 3) + (mean signal) 20.6 22.7 20.1 17.6 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 11.6 31.5 12.6 8.2 12. Detection results Negative Positive Negative Negative Table 24. First replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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60 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 15.3 11.5 11.2 9.5 2. signal over previous background 6.2 4.8 7.3 6.5 3. signal over previous background -2.3 3.3 4.2 8.6 4. signal over previous background 3.6 2.3 1.6 6.5 5. signal over previous background 1.6 -2.3 -5.3 -3.5 6. Standard deviation of signals 6.6 5.0 6.2 5.2 7. Standard deviation 3 19.8 15.0 18.6 15.6 8. Average of signals 4.9 3.9 3.8 5.5 9. Threshold signal (standard deviation 3) + (mean signal) 24.7 18.9 22.4 21.1 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 12.3 22.6 11.2 10.8 12. Detection results Negative Positive Negative Negative Table 25. Second replicate determining the re producibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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61 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 14.2 10.2 11.6 9.9 2. signal over previous background 6.0 7.6 6.5 4.3 3. signal over previous background -1.5 2.6 5.3 3.4 4. signal over previous background 6.3 -2.3 4.6 -2.2 5. signal over previous background 5.3 2.5 -1.2 1.6 6. Standard deviation of signals 5.6 4.9 4.6 4.4 7. Standard deviation 3 16.8 14.7 13.8 13.2 8. Average of signals 6.1 4.1 5.4 3.4 9. Threshold signal (standard deviation 3) + (mean signal) 22.9 18.8 19.2 16.6 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 14.5 18.4 12.2 10.2 12. Detection results Negative Negative Negative Negative Table 26. Third replicate determining the re producibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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62 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 9.8 12.2 6.8 7.9 2. signal over previous background 6.7 4.8 5.0 4.2 3. signal over previous background -2.3 3.5 2.2 1.5 4. signal over previous background 3.5 6.5 -2.3 -4.2 5. signal over previous background 8.3 -2.3 9.6 6.5 6. Standard deviation of signals 4.8 5.2 4.5 4.8 7. Standard deviation 3 14.4 15.6 13.5 14.4 8. Average of signals 5.2 4.9 4.3 3.2 9. Threshold signal (standard deviation 3) + (mean signal) 19.6 20.5 17.8 17.6 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 6.5 30.5 6.5 7.9 12. Detection results Negative Positive Negative Negative Table 27. Fourth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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63 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 11.6 9.8 4.6 12.3 2. signal over previous background 6.7 8.2 5.6 1.3 3. signal over previous background -2.3 7.0 6.2 1.9 4. signal over previous background 3.3 -1.2 -2.3 6.4 5. signal over previous background 5.6 6.1 8.6 3.4 6. Standard deviation of signals 5.1 4.2 4.1 4.5 7. Standard deviation 3 15.3 12.6 12.3 13.5 8. Average of signals 5.0 6.0 4.5 5.1 9. Threshold signal (standard deviation 3) + (mean signal) 20.3 18.6 16.8 18.6 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 12.3 29.5 3.2 10.5 12. Detection results Negative Positive Negative Negative Table 28. Fifth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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64 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 13.5 10.3 6.5 12.3 2. signal over previous background 7.6 5.3 6.0 4.9 3. signal over previous background -2.4 6.2 4.6 -2.2 4. signal over previous background 6.2 -3.0 -1.4 3.2 5. signal over previous background 3.1 4.2 6.1 3.4 6. Standard deviation of signals 5.9 4.8 3.3 5.2 7. Standard deviation 3 17.7 14.4 9.9 15.6 8. Average of signals 5.6 4.6 4.4 4.3 9. Threshold signal (standard deviation 3) + (mean signal) 23.3 19.0 14.3 19.9 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 12.2 27.3 4.6 10.5 12. Detection results Negative Positive Negative Negative Table 29. Sixth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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65 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 6.9 10.0 5.2 4.3 2. signal over previous background 7.6 6.1 3.3 2.0 3. signal over previous background -2.2 9.7 -3.2 9.6 4. signal over previous background 6.5 -2.2 6.5 6.5 5. signal over previous background 8.7 6.5 9.0 -3.5 6. Standard deviation of signals 4.4 4.9 4.6 4.9 7. Standard deviation 3 13.2 14.7 13.8 14.7 8. Average of signals 5.5 6.0 4.2 3.8 9. Threshold signal (standard deviation 3) + (mean signal) 18.7 20.7 18.0 18.5 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 13.5 36.2 9.0 8.4 12. Detection results Negative Positive Negative Negative Table 30. Seventh replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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66 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 12.5 11.2 6.4 5.6 2. signal over previous background 6.5 4.6 7.0 3.1 3. signal over previous background -2.3 -5.9 7.9 6.4 4. signal over previous background 6.5 3.1 -1.6 -4.6 5. signal over previous background 3.3 1.6 3.4 7.2 6. Standard deviation of signals 5.4 6.1 3.9 4.8 7. Standard deviation 3 16.2 18.3 11.7 14.4 8. Average of signals 5.3 2.9 4.6 3.5 9. Threshold signal (standard deviation 3) + (mean signal) 21.5 21.2 16.3 17.9 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 15.2 35.1 9.0 7.9 12. Detection results Negative Positive Negative Negative Table 31. Eighth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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67 CFU number 1 2 1 3 Negative Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 6.4 7.6 10.4 12.5 2. signal over previous background -4.2 6.4 -1.3 5.4 3. signal over previous background 3.4 4.2 7.6 4.6 4. signal over previous background 7.7 -4.3 4.0 3.4 5. signal over previous background 5.6 6.4 3.7 6.0 6. Standard deviation of signals 4.7 4.8 4.4 3.6 7. Standard deviation 3 14.1 14.4 13.2 10.8 8. Average of signals 3.8 4.1 4.9 6.4 9. Threshold signal (standard deviation 3) + (mean signal) 17.9 18.5 18.1 17.2 10. Target treatment add extract add extract add PBS add PBS 11. Detection signal signal over the last background 11.0 30.5 6.5 14.1 12. Detection results Negative Positive Negative Negative Table 32. Ninth replicate determining the reproducibility of the biosensor assay for detection of cell extracts from 1 3 V. vulnificus (ATCC 27562). Values represented are the change in signals between each of the six consecutive background readings (rows 16), calculations used to determine the thresh old signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detec tion results (row 12) for each chamber. All values are in pico amps (pA).

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68 Detection of a Whole Cell V. vulnificus Isolated from Enviro nmental Waters using the Biosensor V. vulnificus (MC0603S) isolated in June 2003 fr om sediment (cultured in the Harwood lab) at a site on Marshall Creek in the Guana-Tolamato-Matanzas National Estuarine Research Reserv e were identified by API-20E Enteric Identification System (B ioMerieux, Inc. Hazelwood, MO) and confirmed by PCR using species-specific primers (18). This isolate was assayed to bett er represent the final goal of the biosensor, which is to be able to detect autochthonous V. vulnificus in marine and estuarine waters. Cells were grown in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU were enumerated and suspended in 200 l of steril e water. Three whole cell CFU suspensions (1 4 1 5 and 1 6 ) were assayed (table 33) and replicated twice (tables 34 and 35), all having positive detection events with no positive signal in any of the negative channels.

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69 CFU number 1 4 1 5 1 6 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 12.3 10.6 6.5 6.1 2. signal over previous background -2.2 5.0 4.1 3.7 3. signal over previous background 6.1 3.4 -2.2 7.6 4. signal over previous background 3.4 2.2 6.0 -3.3 5. signal over previous background 1.6 -1.6 8.6 9.4 6. Standard deviation of signals 5.4 4.5 4.1 4.9 7. Standard deviation 3 16.2 13.5 12.3 14.7 8. Average of signals 4.2 3.9 4.6 4.7 9. Threshold signal (standard deviation 3) + (mean signal) 20.4 17.4 16.9 19.4 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 25.6 154.9 298.2 6.7 12. Detection results Positive Positive Positive Negative Table 33. Detection of whole cell V. vulnificus (MC0603S) isolated from environmental waters. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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70 CFU number 1 4 1 5 1 6 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 8.6 3.7 9.0 4.6 2. signal over previous background 3.5 7.6 10.3 8.6 3. signal over previous background -5.6 4.7 2.5 5.5 4. signal over previous background 10.2 -2.2 6.7 -8.0 5. signal over previous background 1.6 9.5 4.3 3.3 6. Standard deviation of signals 6.3 4.5 3.2 6.3 7. Standard deviation 3 18.9 13.5 9.6 18.9 8. Average of signals 3.7 4.7 6.6 2.8 9. Threshold signal (standard deviation 3) + (mean signal) 22.6 18.2 16.2 21.7 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 23.7 144.0 301.2 11.3 12. Detection results Positive Positive Positive Negative Table 34. First replicat e detecting whole cell V. vulnificus (MC0603S) isolated from environmental waters. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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71 CFU number 1 4 1 5 1 6 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 13.6 10.3 7.7 9.3 2. signal over previous background 6.6 5.1 3.8 7.3 3. signal over previous background 2.1 -1.6 6.4 6.4 4. signal over previous background 3.5 4.5 -5.6 -4.6 5. signal over previous background 4.5 3.7 9.4 6.5 6. Standard deviation of signals 4.5 4.2 5.9 5.5 7. Standard deviation 3 13.5 12.6 17.7 16.5 8. Average of signals 6.1 4.4 4.3 5.0 9. Threshold signal (standard deviation 3) + (mean signal) 19.6 17.0 22.0 21.5 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 23.6 153.4 286.3 3.7 12. Detection results Positive Positive Positive Negative Table 35. Second replicate detecting whole cell V. vulnificus (MC0603S) isolated from environmental waters. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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72 Detecting Cell Extracts of V. vulnificus Isolated from Environmental Waters using the Biosensor Cell extracts from cultures of the environmental V. vulnificus (MC0603S) isolate were assayed to determine if the sensitivity could be improved, as it was with extracts from clinical (ATCC 27652) isolate. Cells were grown in BHI broth (1.0% NaCl) at 37C with shaking for 24 h. CFU were enumerat ed, suspended in 200 l of sterile water, and sonicated for 5 minutes on ice prior to the assay. Extracts from three cell suspensions (1 3 1 4 and 1 5 ), were assayed (table 36) and replicated twice (tables 37 and 38). Two of the three assays were positive for extracts from 1 3 CFU (tables 36 and 38) and one was negative (table 37). No positive signals occurred in any of the negative channels.

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73 CFU number 1 3 1 4 1 5 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 12.5 8.5 6.4 17.2 2. signal over previous background 6.1 5.3 7.0 3.2 3. signal over previous background 2.6 -4.5 9.6 10.2 4. signal over previous background 4.2 3.7 -7.3 4.4 5. signal over previous background 3.6 4.3 2.2 6.2 6. Standard deviation of signals 4.0 4.8 6.6 5.7 7. Standard deviation 3 12.0 14.4 19.8 17.1 8. Average of signals 5.8 3.5 3.6 8.2 9. Threshold signal (standard deviation 3) + (mean signal) 17.8 17.9 23.4 25.3 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 18.9 65.2 298.3 16.5 12. Detection results Positive Positive Positive Negative Table 36. Detecting cell extracts of V. vulnificus (MC0603S) isolated from environmental waters. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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74 CFU number 1 3 1 4 1 5 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 3.6 10.2 14.6 8.4 2. signal over previous background 8.8 6.2 4.0 6.4 3. signal over previous background -6.2 3.4 5.7 6.1 4. signal over previous background 5.4 -2.2 3.4 -6.5 5. signal over previous background 3.3 9.6 8.6 1.6 6. Standard deviation of signals 5.6 5.1 4.6 6.0 7. Standard deviation 3 16.8 15.3 13.8 18.0 8. Average of signals 3.0 5.4 7.3 3.2 9. Threshold signal (standard deviation 3) + (mean signal) 19.8 20.7 21.1 21.2 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 17.6 75.6 255.3 13.6 12. Detection results Negative Positive Positive Negative Table 37. First replicate de tecting cell extracts of V. vulnificus (MC0603S) isolated from environmental waters. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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75 CFU number 1 3 1 4 1 5 Negative Chamber 1 Chamber 2 Chamber 3 Chamber 4 1. signal over previous background 12.6 11.3 4.6 6.6 2. signal over previous background -3.6 4.7 1.9 8.5 3. signal over previous background 6.5 3.2 6.8 -1.6 4. signal over previous background 4.4 -2.5 -4.6 6.4 5. signal over previous background 3.2 3.7 9.6 -3.5 6. Standard deviation of signals 5.8 4.9 5.4 5.4 7. Standard deviation 3 17.4 14.7 16.2 16.2 8. Average of signals 4.6 4.1 3.7 3.3 9. Threshold signal (standard deviation 3) + (mean signal) 22.0 18.8 19.9 19.5 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 24.6 82.3 263.4 6.5 12. Detection results Positive Positive Positive Negative Table 38. Second replicate detecting cell extracts of V. vulnificus (MC0603S) isolated from environmental waters. Values represen ted are the change in signals between each of the six consecutive background readings (row s 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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76 Testing the Specificity of the Bios ensor Assay against Closely Related Vibrio species and E. coli To study the possible cross-reactivity of the V. vulnificus -specific polyclonal antibodies with other ATCC Vibrio spp. strains, whole cells and cell extracts from V. cholerae (ATCC 11623) (tables 39-42), V. parahaemolyticus (ATCC 49398) (tables 4346), and V. alginolyticus (ATCC 51160) (tables 47-50) were assayed with the biosensor. Whole cells and cell extr acts from a non-target E. coli (ATCC 9637) were also assayed for cross-reactivity (tables 51-54). Cells were grown in BHI broth (1.0% NaCl for Vibrio spp. and 0.5% NaCl for E. coli) at 37C with shaking for 24 h. CFU were enumerated, suspended in 200 l of sterile water (sonicated for extract assays), and then assayed. No positive detection events occurred with any non-target Vibrio spp. or E. coli (whole cell or extracts), while the positive control V. vulnificus (ATCC 27562) was detected in all assays. There were no positive detection events for any of the negatives used with each assay.

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77 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 11.6 14.3 9.2 8.6 2. signal over previous background 6.6 3.5 4.6 7.6 3. signal over previous background -1.3 2.1 2.0 -1.9 4. signal over previous background 3.9 4.6 -3.4 4.3 5. signal over previous background 4.3 1.6 7.4 -1.6 6. Standard deviation of signals 4.7 5.2 4.9 5.0 7. Standard deviation 3 14.1 15.6 14.7 15.0 8. Average of signals 5.0 5.2 4.0 3.4 9. Threshold signal (standard deviation 3) + (mean signal) 19.1 20.8 18.7 18.4 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 76.5 17.9 12.2 3.3 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. cholerae (ATCC 11623) Chamber 3 1 4 V. cholerae (ATCC 11623) Chamber 4 Negative Table 39. Testing the specificity of th e biosensor assay against whole cell V. cholerae (ATCC 11623). Values represented are the ch ange in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target tr eatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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78 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 11.0 12.6 10.6 11.2 2. signal over previous background 6.2 4.7 6.3 -1.2 3. signal over previous background -5.6 1.6 7.6 3.5 4. signal over previous background 3.0 -1.2 -3.5 4.9 5. signal over previous background 2.6 3.8 2.0 -3.6 6. Standard deviation of signals 6.1 5.2 5.5 5.8 7. Standard deviation 3 18.3 15.6 16.5 17.4 8. Average of signals 3.4 4.3 4.6 3.0 9. Threshold signal (standard deviation 3) + (mean signal) 21.7 19.9 21.1 20.4 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 85.6 16.2 6.9 4.9 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. cholerae (ATCC 11623) Chamber 3 1 4 V. cholerae (ATCC 11623) Chamber 4 Negative Table 40. First replicate testing the specificity of the bios ensor assay agai nst whole cell V. cholerae (ATCC 11623). Values re presented are the change in signals between each of the six consecutive background readings (row s 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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79 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 5.6 7.6 11.5 6.2 2. signal over previous background 10.1 -3.2 4.3 1.9 3. signal over previous background -2.2 8.1 -2.8 3.7 4. signal over previous background 3.7 4.0 1.9 -2.8 5. signal over previous background 9.8 1.9 8.6 9.5 6. Standard deviation of signals 5.1 4.6 5.6 4.6 7. Standard deviation 3 15.3 13.8 16.8 13.9 8. Average of signals 5.4 3.7 4.7 3.7 9. Threshold signal (standard deviation 3) + (mean signal) 20.7 17.5 21.5 17.5 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 56.2 14.8 10.0 7.7 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. cholerae (ATCC 11623) Chamber 3 1 4 V. cholerae (ATCC 11623) Chamber 4 Negative Table 41. Second replicate testing the specifi city of the biosensor assay against whole cell V. cholerae (ATCC 11623). Values represented ar e the change in signals between each of the six consecutive background read ings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

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80 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 10.4 9.7 11.2 8.9 2. signal over previous background 4.6 4.5 6.7 4.4 3. signal over previous background 3.7 2.9 1.3 6.5 4. signal over previous background 2.8 4.7 4.6 2.7 5. signal over previous background 1.1 -2.2 5.3 -2.4 6. Standard deviation of signals 3.5 4.3 3.6 4.3 7. Standard deviation 3 10.5 12.9 10.8 12.9 8. Average of signals 4.5 3.9 5.8 4.0 9. Threshold signal (standard deviation 3) + (mean signal) 15.0 16.8 16.6 16.9 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 114.2 14.0 9.1 3.8 12. Detection results Positive Negative Negative Negative Chamber 1 extracts from 1 4 V. vulnificus (ATCC 27562) Chamber 2 extracts from 1 5 V. cholerae (ATCC 11623) Chamber 3 extracts from 1 4 V. cholerae (ATCC 11623) Chamber 4 Negative Table 42. Testing the specificity of the biosensor assay against V. cholerae (ATCC 11623) cell extracts. Values represented are the change in si gnals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target tr eatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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81 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 4.6 8.1 11.0 7.5 2. signal over previous background 10.8 1.8 6.7 9.4 3. signal over previous background -1.5 3.7 4.9 -3.9 4. signal over previous background 5.8 -6.5 -4.3 2.4 5. signal over previous background 6.0 5.5 3.1 7.0 6. Standard deviation of signals 4.4 5.6 5.6 5.3 7. Standard deviation 3 13.2 16.8 16.8 15.9 8. Average of signals 5.1 2.5 4.3 4.5 9. Threshold signal (standard deviation 3) + (mean signal) 18.3 19.3 21.1 20.4 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 66.2 18.1 16.8 10.5 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. parahaemolyticus (ATCC 43938) Chamber 3 1 4 V. parahaemolyticus (ATCC 43938) Chamber 4 Negative Table 43. Testing the specificity of th e biosensor assay against whole cell V. parahaemolyticus (ATCC 43938). Values represented ar e the change in signals between each of the six consecutive background read ings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

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82 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 15.6 11.8 16.2 7.9 2. signal over previous background 9.5 7.0 6.7 3.7 3. signal over previous background 7.2 3.3 4.5 -2.6 4. signal over previous background 1.2 1.6 8.8 4.6 5. signal over previous background 7.5 -4.3 5.2 6.3 6. Standard deviation of signals 5.2 6.0 4.7 4.0 7. Standard deviation 3 15.6 18.0 14.1 12.0 8. Average of signals 8.2 3.9 8.3 4.0 9. Threshold signal (standard deviation 3) + (mean signal) 23.8 21.9 22.4 16.0 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 86.5 17.5 12.6 7.7 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. parahaemolyticus (ATCC 43938) Chamber 3 1 4 V. parahaemolyticus (ATCC 43938) Chamber 4 Negative Table 44. First replicate testing the specificity of the bios ensor assay agai nst whole cell V. parahaemolyticus (ATCC 43938). Values represente d are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

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83 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 6.5 8.0 10.6 7.8 2. signal over previous background 7.2 11.6 -2.6 11.3 3. signal over previous background -1.6 4.6 10.5 6.5 4. signal over previous background 3.5 -2.2 1.6 3.2 5. signal over previous background 8.6 6.5 3.3 -3.3 6. Standard deviation of signals 4.1 5.1 5.8 5.5 7. Standard deviation 3 12.3 15.3 17.4 16.5 8. Average of signals 4.8 5.7 4.7 5.1 9. Threshold signal (standard deviation 3) + (mean signal) 17.1 21.0 22.1 21.6 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 69.1 18.6 7.5 6.6 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. parahaemolyticus (ATCC 43938) Chamber 3 1 4 V. parahaemolyticus (ATCC 43938) Chamber 4 Negative Table 45. Second replicate testing the specifi city of the biosensor assay against whole cell V. parahaemolyticus (ATCC 43938). Values represente d are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

PAGE 95

84 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 12.4 9.7 10.9 10.7 2. signal over previous background 6.4 6.1 8.1 9.1 3. signal over previous background 4.7 7.2 6.3 6.4 4. signal over previous background 4.8 3.7 2.7 3.9 5. signal over previous background 7.9 1.7 3.8 4.2 6. Standard deviation of signals 3.2 3.1 3.3 3.0 7. Standard deviation 3 9.6 9.3 9.9 9.0 8. Average of signals 7.2 5.7 6.4 6.9 9. Threshold signal (standard deviation 3) + (mean signal) 16.8 15.0 16.3 15.9 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 95.4 12.3 7.7 4.8 12. Detection results Positive Negative Negative Negative Chamber 1 extracts from 1 4 V. vulnificus (ATCC 27562) Chamber 2 extracts from 1 5 V. parahaemolyticus (ATCC 43938) Chamber 3 extracts from 1 4 V. parahaemolyticus (ATCC 43938) Chamber 4 Negative Table 46. Testing the specificity of the biosensor assay against V. parahaemolyticus (ATCC 43938) cell extracts. Values represente d are the change in signals between each of the six consecutive background readings (row s 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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85 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 15.2 10.5 14.3 9.8 2. signal over previous background 7.5 5.0 9.4 6.1 3. signal over previous background -2.2 1.6 3.0 4.1 4. signal over previous background 1.6 6.2 -3.6 6.6 5. signal over previous background 3.8 -3.3 5.2 -4.6 6. Standard deviation of signals 6.6 5.2 6.7 5.4 7. Standard deviation 3 19.8 15.6 20.1 16.2 8. Average of signals 5.2 4.0 5.7 4.4 9. Threshold signal (standard deviation 3) + (mean signal) 25.0 19.6 25.8 20.6 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 49.2 18.9 15.5 10.6 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. alginolyticus (ATCC 51160) Chamber 3 1 4 V. alginolyticus (ATCC 51160) Chamber 4 Negative Table 47. Testing the specificity of th e biosensor assay against whole cell V. alginolyticus (ATCC 51160). Values represented are the change in sign als between each of the six consecutive background readings (row s 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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86 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 14.0 12.6 13.6 4.5 2. signal over previous background 6.2 -1.6 4.6 8.1 3. signal over previous background 3.3 7.5 2.1 5.8 4. signal over previous background 9.4 7.5 3.8 3.0 5. signal over previous background 2.0 7.9 8.8 -5.6 6. Standard deviation of signals 4.8 5.2 4.6 5.2 7. Standard deviation 3 14.4 15.6 13.8 15.6 8. Average of signals 7.0 6.8 6.6 3.1 9. Threshold signal (standard deviation 3) + (mean signal) 21.4 22.4 20.4 18.7 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 67.8 20.3 14.4 13.6 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. alginolyticus (ATCC 51160) Chamber 3 1 4 V. alginolyticus (ATCC 51160) Chamber 4 Negative Table 48. First replicate testing the specificity of the bios ensor assay agai nst whole cell V. alginolyticus (ATCC 51160). Values represented ar e the change in signals between each of the six consecutive background read ings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

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87 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 8.2 5.6 7.5 2.9 2. signal over previous background 1.9 6.9 8.8 7.0 3. signal over previous background 2.7 2.2 -6.5 -1.9 4. signal over previous background -5.7 -5.8 1.8 7.6 5. signal over previous background 5.9 8.2 3.0 4.2 6. Standard deviation of signals 5.3 5.6 6.0 3.8 7. Standard deviation 3 15.9 16.8 18.0 11.4 8. Average of signals 2.6 3.4 2.9 4.0 9. Threshold signal (standard deviation 3) + (mean signal) 18.5 20.2 20.9 15.4 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 55.1 16.2 5.8 7.8 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 V. alginolyticus (ATCC 51160) Chamber 3 1 4 V. alginolyticus (ATCC 51160) Chamber 4 Negative Table 49. Second replicate testing the specifi city of the biosensor assay against whole cell V. alginolyticus (ATCC 51160). Values represente d are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each cham ber. All values are in pico amps (pA).

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88 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 8.3 12.4 10.9 7.7 2. signal over previous background 7.1 7.6 7.2 6.1 3. signal over previous background 3.4 3.5 3.7 10.5 4. signal over previous background 2.4 5.4 4.8 4.7 5. signal over previous background -2.4 4.9 2.6 1.3 6. Standard deviation of signals 4.2 3.5 3.3 3.4 7. Standard deviation 3 12.6 10.5 9.9 10.2 8. Average of signals 3.8 6.8 5.8 6.3 9. Threshold signal (standard deviation 3) + (mean signal) 16.4 17.3 15.7 16.3 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 124.2 13.8 8.7 7.6 12. Detection results Positive Negative Negative Negative Chamber 1 extracts from 1 4 V. vulnificus (ATCC 27562) Chamber 2 extracts from 1 5 V. alginolyticus (ATCC 51160) Chamber 3 extracts from 1 4 V. alginolyticus (ATCC 51160) Chamber 4 Negative Table 50. Testing the specificity of the biosensor assay against V. alginolyticus (ATCC 51160) cell extracts. Values represented are the change in si gnals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target tr eatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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89 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 10.6 9.6 6.1 11.2 2. signal over previous background 5.3 6.0 7.8 6.4 3. signal over previous background -1.3 2.5 3.8 3.1 4. signal over previous background 3.3 0.6 -3.9 5.5 5. signal over previous background 2.4 5.9 4.2 -2.0 6. Standard deviation of signals 4.4 3.5 4.5 4.8 7. Standard deviation 3 13.2 10.5 13.5 14.4 8. Average of signals 4.1 4.9 3.6 4.8 9. Threshold signal (standard deviation 3) + (mean signal) 17.3 15.4 17.1 19.2 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 23.1 11.2 6.4 3.7 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 E. coli (ATCC 9637) Chamber 3 1 4 E. coli (ATCC 9637) Chamber 4 Negative Table 51. Testing the specificity of th e biosensor assay against whole cell E. coli (ATCC 9637). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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90 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 13.6 14.5 11.1 6.9 2. signal over previous background 5.5 10.2 -3.6 5.2 3. signal over previous background 6.2 2.5 5.7 2.4 4. signal over previous background -3.9 3.5 6.6 -3.9 5. signal over previous background 2.4 4.2 6.0 7.8 6. Standard deviation of signals 6.4 5.2 5.4 4.7 7. Standard deviation 3 19.2 15.6 16.2 14.1 8. Average of signals 4.8 7.0 5.2 3.7 9. Threshold signal (standard deviation 3) + (mean signal) 24.0 22.6 21.4 17.8 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 27.0 9.9 6.2 5.1 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 E. coli (ATCC 9637) Chamber 3 1 4 E. coli (ATCC 9637) Chamber 4 Negative Table 52. First replicate testing the specificity of the bios ensor assay agai nst whole cell E. coli (ATCC 9637). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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91 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 16.3 14.0 9.4 9.7 2. signal over previous background 7.9 8.6 -2.6 9.5 3. signal over previous background 3.2 -2.3 3.1 -2.1 4. signal over previous background 6.5 5.5 4.1 5.8 5. signal over previous background 6.6 4.6 1.9 6.4 6. Standard deviation of signals 4.9 6.0 4.3 4.8 7. Standard deviation 3 14.7 18.0 12.9 14.4 8. Average of signals 8.1 6.1 3.2 5.9 9. Threshold signal (standard deviation 3) + (mean signal) 22.8 24.1 16.1 20.3 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 46.8 19.4 4.8 -5.6 12. Detection results Positive Negative Negative Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 5 E. coli (ATCC 9637) Chamber 3 1 4 E. coli (ATCC 9637) Chamber 4 Negative Table 53. Second replicate testing the specifi city of the biosensor assay against whole cell E. coli (ATCC 9637). Values represented are th e change in signals between each of the six consecutive background r eadings (rows 1-6), calculati ons used to determine the threshold signal (rows 6-9), target treatment (row 10), detection si gnal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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92 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 14.7 10.4 8.9 11.1 2. signal over previous background 9.3 8.2 6.4 6.1 3. signal over previous background 5.0 6.2 2.7 4.8 4. signal over previous background 6.1 3.7 8.6 3.6 5. signal over previous background 5.2 1.7 1.9 3.1 6. Standard deviation of signals 4.1 3.5 3.3 3.2 7. Standard deviation 3 12.3 10.5 9.9 9.6 8. Average of signals 8.1 6.0 5.7 5.7 9. Threshold signal (standard deviation 3) + (mean signal) 20.4 16.5 15.6 15.3 10. Target treatment add extract add extract add extract add PBS 11. Detection signal signal over the last background 117.8 14.7 6.7 2.9 12. Detection results Positive Negative Negative Negative Chamber 1 extracts from 1 4 V. vulnificus (ATCC 27562) Chamber 2 extracts from 1 5 E. coli (ATCC 9637) Chamber 3 extracts from 1 4 E. coli (ATCC 9637) Chamber 4 Negative Table 54. Testing the specificity of the biosensor assay against E. coli (ATCC 9637) cell extracts. Values represented are the ch ange in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target tr eatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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93 Testing the Sensitivity of the Biosensor Assay on a Mixture of Whole Cell V. vulnificus (ATCC 27562) and V. cholerae (ATCC 11623) Cells were grown in BHI broth (1.0% NaCl ) at 37C with shaking for 24 h. CFU were enumerated and suspended in 200 l st erile water. Mixed suspensions containing 1 4 and 1 5 V. vulnificus (ATCC 27562) and V. cholerae (ATCC 1162) (1:1), were assayed and replicated once with the biosenso r. The assay was able to detect mixed suspensions containing 1 4 and 1 5 CFU in both assays, with no positive detection signal in either negative channels (tables 55 and 56). Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 12.2 10.8 6.7 15.7 2. signal over previous background 8.1 9.4 8.6 7.5 3. signal over previous background -2.6 4.6 4.4 4.0 4. signal over previous background 3.9 2.2 -3.7 5.6 5. signal over previous background 4.2 -5.6 2.7 6.4 6. Standard deviation of signals 5.5 6.5 4.7 4.6 7. Standard deviation 3 16.5 19.5 14.1 13.8 8. Average of signals 5.2 4.3 3.7 7.8 9. Threshold signal (standard deviation 3) + (mean signal) 21.7 23.8 17.8 21.6 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 46.7 62.3 227.3 9.2 12. Detection results Positive Positive Positive Negative Chamber 1 1 4 V. vulnificus (ATCC 27562) Chamber 2 1 4 V. vulnificus (ATCC 27562) + 1X10 4 V. cholerae (ATCC 11623) Chamber 3 1 5 V. vulnificus (ATCC 27562) + 1X10 5 V. cholerae (ATCC 11623) Chamber 4 Negative Table 55. Testing the sensitivity of the bi osensor assay on a mixture of whole cell V. vulnificus (ATCC 27562) and V. cholerae (ATCC 11623). Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold si gnal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection result s (row 12) for each chamber. All values are in pico amps (pA).

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94 Chamber 1 Chamber 2 Chamber 3 Chamber 4 (Negative) 1. signal over previous background 9.9 9.2 10.8 15.2 2. signal over previous background 11.6 3.9 2.4 6.6 3. signal over previous background2.3 3.8 5.4 4.0 4. signal over previous background-2.8 -6.7 -3.3 6.2 5. signal over previous background5.1 5.9 5.3 5.4 6. Standard deviation of signals 5.8 6.0 5.1 4.4 7. Standard deviation 3 17.4 18.0 15.3 13.2 8. Average of signals 5.2 3.2 4.1 7.5 9. Threshold signal (standard deviation 3) + (mean signal) 22.6 21.2 19.4 20.7 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 27.0 49.0 186.3 6.2 12. Detection results Positive Positive Positive Negative Chamber 1 14 V. vulnificus (ATCC 27562) Chamber 2 14 V. vulnificus (ATCC 27562) + 1X104 V. cholerae (ATCC 11623) Chamber 3 15 V. vulnificus (ATCC 27562) + 1X105 V. cholerae (ATCC 11623) Chamber 4 Negative Table 56. Replicate assay testing the sensitiv ity of the biosensor assay on a mixture of whole cell V. vulnificus (ATCC 27562) and V. cholerae (ATCC 11623). Values represented are the ch ange in signals between each of the six consecutive background readings (rows 1-6), calculations used to dete rmine the threshold signa l (rows 6-9), target treatment (row 10), detection signal (row 11) and detection results (row 12) for each chamber. All values are in pico amps (pA).

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95 Detection of V. vulnificus (ATCC 27562) in Estuarine Water Estuarine water (salinity at 24.1 parts per thousand) was acquired from Northern Tampa Bay (Tampa, FL) at the same location where environmental V. vulnificus were isolated. The estuarine water was filter sterilized using a 0.2 m syringe filter. V. vulnificus (ATCC 27562) was grown in BHI (1.0% NaCl) overnight at 37C with shaking. Cells were enumerated, suspended in 200l estuarine water, and assayed using the biosensor. For the cell extract assays, cells were suspended in estuarine water and sonicated for 5 minutes prior to detection. Assays were performed, with one replicate each, on both whole cell (tables 57 and 58) and cell extract (tables 59 and 60) suspensions. In both whole cell assays in es tuarine water (tables 57 and 8), there was no detection for 14 suspensions, which was a detectable CFU number, when assayed in sterile water. In both cell extract assays in estuarine water (tables 59 and 60), there was no detection for 13 suspensions, which was a detectable limit when assayed in sterile water. No positive detection events occurred in any of the negative channels.

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96 CFU number 141516Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background4.6 10.5 7.4 4.5 2. signal over previous background-2.8 5.8 4.1 6.7 3. signal over previous background6.7 -1.6 3.3 10.7 4. signal over previous background1.1 3.8 -1.8 1.5 5. signal over previous background9.6 2.7 4.6 1.6 6. Standard deviation of signals 4.8 4.4 3.4 3.9 7. Standard deviation 3 14.4 13.2 10.2 11.7 8. Average of signals 3.8 4.2 3.5 5.0 9. Threshold signal (standard deviation 3) + (mean signal) 18.2 17.4 13.7 16.7 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 10.4 28.9 77.7 6.7 12. Detection results Negative Positive Positive Negative Table 57. Detection of whole cell V. vulnificus (ATCC 27562) in estuarine water. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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97 CFU number 141516Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background3.9 8.6 2.5 9.8 2. signal over previous background-1.3 4.7 3.0 4.1 3. signal over previous background6.8 -2.2 6.2 3.3 4. signal over previous background9.8 10.1 9.4 -2.1 5. signal over previous background4.9 2.4 -1.6 5.5 6. Standard deviation of signals 4.1 4.9 4.1 4.3 7. Standard deviation 3 12.3 14.7 12.3 12.9 8. Average of signals 4.8 4.7 3.9 4.1 9. Threshold signal (standard deviation 3) + (mean signal) 17.1 19.4 16.2 17.0 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 11.9 26.4 62.0 8.2 12. Detection results Negative Positive Positive Negative Table 58. Replicate assa y detecting whole cell V. vulnificus (ATCC 27562) in estuarine water. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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98 CFU number 131415Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background9.9 10.8 4.8 11.5 2. signal over previous background4.0 2.1 7.7 2.3 3. signal over previous background4.6 4.7 -1.2 6.4 4. signal over previous background-3.2 5.1 3.0 2.4 5. signal over previous background5.7 2.5 2.9 1.9 6. Standard deviation of signals 4.7 3.5 3.2 4.1 7. Standard deviation 3 14.1 10.5 9.6 12.3 8. Average of signals 4.2 5.0 3.4 4.9 9. Threshold signal (standard deviation 3) + (mean signal) 18.3 15.5 13.0 17.2 10. Target treatment add extract add extractadd extract add PBS 11. Detection signal signal over the last background 10.0 18.4 48.9 1.8 12. Detection results Negative Positive Positive Negative Table 59. Detection of cell extracts from V. vulnificus (ATCC 27562) in estuarine water. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detec tion signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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99 CFU number 131415Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background11.2 8.7 8.9 7.7 2. signal over previous background4.6 9.9 3.7 5.4 3. signal over previous background3.1 4.1 -3.5 3.4 4. signal over previous background-1.2 2.2 2.1 -2.1 5. signal over previous background6.7 3.1 6.0 6.7 6. Standard deviation of signals 4.6 3.5 4.6 3.9 7. Standard deviation 3 13.7 10.4 13.9 11.6 8. Average of signals 4.9 5.6 3.4 4.2 9. Threshold signal (standard deviation 3) + (mean signal) 18.6 16.0 17.3 15.8 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 11.8 22.1 89.0 4.1 12. Detection results Negative Positive Positive Negative Table 60. Replicate assay de tecting cell extracts from V. vulnificus (ATCC 27562) in estuarine water. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target tr eatment (row 10), detection signal (row 11), and detection results (row 12) for each chamber. All values are in pico amps (pA).

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100 Detection of Cell Extracts from V. vulnificus (ATCC 27562) after Enrichment in Alkaline Peptone Water V. vulnificus (ATCC 27562) was grown at 37 C for 24 hours in 10.0 ml of BHI broth (1.0% NaCl) with shaking. The entire broth culture was centrif uged to pellet cells. The pellet was re-suspended in ASW, and e numerated by culturable counts of serial dilutions. Two enrichment cult ures inoculated with the sa me CFU (prepared above) were prepared for each assay: one was eventually injected into the waveguide chamber and assayed (figure 2a), and one was used for en umeration after enrichment to enumerate the culturable cells that were e xposed to the waveguide at the time of the assay (postenrichment) (figure 2b). The appropriate ASW dilutions were centrifuged, and resuspended in alkaline peptone water (APW) enrichment broth. The entire volume of APW enrichment broth seeded with known CFU was incubated at 37C with shaking for 4 hours. The entire volume of APW enrichme nt broth was centrif uged to pellet cells. The pellet to be assayed with the biosensor was resuspended in 200 l sterile water, sonicated for 5 minutes on ice, and assayed with the biosensor (figure 2a). The pellet to be enumerated was resuspended in ASW and plate counted on Marine agar 2216 incubated at 37C overni ght (figure 2b). Cell extracts from enrichments seeded with 12, 52, and 13 CFU were assayed twice with the biosensor (tables 61 a nd 62). Positive dete ction events occurred for extracts from both 52 and 13 CFU in both assays, while no detection occurred for extracts from 12 CFU in either assay. Cells from the enumeration suspension in ASW were enumerated by plate counts to approximate the CFU exposed to the waveguide at the time of the pa rallel assay (t ables 61 and 62).

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101 CFU numbers seeded in APW 100 500 1000 Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background10.5 3.8 6.1 10.2 2. signal over previous background4.1 9.4 4.0 6.7 3. signal over previous background6.7 -3.4 10.4 -2.9 4. signal over previous background1.9 1.5 3.6 4.8 5. signal over previous background-0.5 2.6 -1.9 1.7 6. Standard deviation of signals 4.3 4.6 4.5 5.0 7. Standard deviation 3 12.9 13.8 13.5 15.0 8. Average of signals 4.5 2.8 4.4 4.1 9. Threshold signal (standard deviation 3) + (mean signal) 17.4 16.6 17.9 19.1 10. Target treatment add extract add extractadd extract add PBS 11. Detection signal signal over the last background 13.2 32.6 86.1 8.7 12. Detection results Negative Positive Positive Negative 13. Approximate CFU exposed to waveguide after enrichment 760 1360 7040 0 Table 61. Detection of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in APW. Values represented are the change in signals between each of the six consecutive background read ings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each chamber (pA) Row 13 represents the approximate CFU exposed to the waveguide at the time of assay determined by a parallel plate count.

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102 CFU numbers seeded in APW 100 500 1000 Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background11.2 8.8 7.9 10.5 2. signal over previous background5.6 4.4 3.7 8.7 3. signal over previous background5.0 -2.4 4.8 3.3 4. signal over previous background4.3 6.1 8.4 3.7 5. signal over previous background1.8 5.7 -3.4 4.9 6. Standard deviation of signals 3.5 4.2 4.7 3.2 7. Standard deviation 3 10.5 12.6 14.1 9.6 8. Average of signals 5.6 4.5 4.3 6.2 9. Threshold signal (standard deviation 3) + (mean signal) 16.1 17.1 18.4 15.8 10. Target treatment add extract add extractadd extract add PBS 11. Detection signal signal over the last background 10.6 22.4 66.7 4.1 12. Detection results Negative Positive Positive Negative 13. Approximate CFU exposed to waveguide after enrichment 680 1160 6680 0 Table 62. Replicate detec tion of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in APW. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each chamber (pA) Row 13 represents the approximate CFU exposed to the waveguide at the time of assay determined by a parallel plate count.

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103 Detection of Cell Extracts of V. vulnificus (ATCC 27562) in Sterile Water after Enrichment in PNCC Enrichment Broth V. vulnificus (ATCC 27562) CFU were enriched for a short time in PNCC enrichment broth (5.0% peptone, 1.0% NaCl2, 0.08% cellobiose, and 1 U of colistin per ml, pH 8.0) (20) in attempt to further lower the limit of detection of the biosensor assay. V. vulnificus (ATCC 27562) was grown at 37C fo r 24 hours in 10.0 ml of BHI broth (1.0% NaCl) with shaking. The entire broth culture was centrifuged to pellet cells. The pellet was re-suspended in ASW, and enumerated by culturable counts of serial dilutions. Two enrichment cultures inocul ated with the same CFU (prepared above) were prepared for each assay: one was eventually injected into the waveguide chamber and assayed (figure 2a), and one was used for enumer ation after enrichment to enumerate the culturable cells that were e xposed to the waveguide at the time of the assay (postenrichment) (figure 2b). The appropriate ASW dilutions were centrifuged and resuspended PNCC. The entire volume of PNCC seeded with known CFU was incubated at 37C with shaking for 4 hours then centrifuged to pellet cells. The pellet to be assayed with the biosensor was resuspended in 200 l sterile water, sonicated for 5 minutes on ice, and assayed (figure 2a). The pellet to be enumerated was resuspended in ASW and plate counted on Marine agar 2216 incubate d at 37C overnight (figure 2b). Extracts from enrichments seeded w ith dilutions of 50, 100, and 500 CFU were assayed twice on the biosensor. Neither of the two assays (Tables 63 and 64) were able to detect extracts from 50 CFU seeded in PNCC, while extracts from 100 CFU were detected in both assays. CFU from the enumeration suspension in PNCC enrichment

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104 broth were enumerated by direct plate counts to approximate the CFU exposed to the waveguide at the time of the pa rallel assay (t ables 63 and 64). CFU numbers seeded in PNCC 50 100 500 Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background11.2 5.7 5.5 11.8 2. signal over previous background8.4 7.3 10.4 9.3 3. signal over previous background4.6 9.8 6.7 3.7 4. signal over previous background3.9 -2.1 2.9 6.6 5. signal over previous background8.4 5.3 4.7 4.1 6. Standard deviation of signals 3.0 4.4 2.8 3.5 7. Standard deviation 3 9.0 13.2 8.4 10.5 8. Average of signals 7.3 5.2 6.0 7.1 9. Threshold signal (standard deviation 3) + (mean signal) 16.3 18.4 14.4 17.6 10. Target treatment add extract add extractadd extract add PBS 11. Detection signal signal over the last background 14.2 20.1 29.6 4.7 12. Detection results Negative Positive Positive Negative 13. Approximate CFU exposed to waveguide after enrichment 880 1640 7840 0 Table 63. Detection of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in PNCC. Values repr esented are the change in signals between each of the six consecutive background read ings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), targ et treatment (row 10), de tection signal (row 11), and detection results (row 12) for each chamber (pA) Row 13 represents the approximate CFU exposed to the waveguide at the time of assay determined by a parallel plate count.

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105 CFU numbers seeded in PNCC 50 100 500 Negative Chamber 1Chamber 2Chamber 3 Chamber 4 1. signal over previous background9.1 6.0 12.3 10.4 2. signal over previous background3.9 12.8 6.1 7.3 3. signal over previous background4.7 6.7 6.2 8.4 4. signal over previous background-2.4 3.7 4.1 5.2 5. signal over previous background5.6 5.9 7.7 5.1 6. Standard deviation of signals 4.2 3.4 3.1 2.2 7. Standard deviation 3 12.5 10.3 9.3 6.7 8. Average of signals 4.2 7.0 7.3 7.3 9. Threshold signal (standard deviation 3) + (mean signal) 16.7 17.3 16.5 14.0 10. Target treatment add cells add cells add cells add PBS 11. Detection signal signal over the last background 9.3 19.7 27.4 6.4 12. Detection results Negative Positive Positive Negative 13. Approximate CFU exposed to waveguide after enrichment 800 1560 7680 0 Table 64. Replicate detec tion of cell extracts of V. vulnificus (ATCC 27562) in sterile water after a four hour enrichment in PNCC. Values represented are the change in signals between each of the six consecutive background readings (rows 1-6), calculations used to determine the threshold signal (rows 6-9), target treatment (row 10), detection signal (row 11), and detection results (ro w 12) for each chamber (pA) Row 13 represents the approximate CFU exposed to the waveguide at the time of assay determined by a parallel plate count.

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106 Correlation between Mean Corrected Detection Signals and CFU Whole Cell Assays: Corrected detection signals for all w hole cell assay settings were acquired by subtracting the threshold signal from the de tection signal for each detection event. Signals acquired from whole cell V. vulnificus (ATCC 27562) in ster ile water, whole cell V. vulnificus (MC0603S) in sterile water, and whole cell V. vulnificus (ATCC 27562) in estuarine water were compared. The mean corrected detection signal was compared with CFU in each of the assay settings (figure 5). The correlation between CFU and mean corrected detection si gnals for whole cell V. vulnificus ATCC 27562 (table 66) and whole cell environmental (table 65), as well as whole cell ATCC assayed in estuarine water (table 65), was statistically significant.

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Corrected Detection Signals for Whole Cell Assays-100.00.0100.0200.0300.0400.0500.0600.0700.01.0E+061.0E+051.0E+041.0E+031.0E+02cell number (CFU)mean corrected detection signal (pA) ATCC assayed in sterile water Environmental (MC0603S)assayed in sterile water ATCC assayed in estuarinewater Figure 5. Corrected detection signal (detection threshold) for each whole cell assay. There were no 12 or 13 CFU suspensions assayed for the environmental (MC0603S) or V. vulnificus ATCC 27562 isolates in estuarine water. Cell Extract Assays: Corrected detection signals for all cell extract assay settings were acquired by subtracting the threshold signal from the detection signal for each detection event. Signals acquired from cell extracts from V. vulnificus 27562 in sterile water, cell extracts from V. vulnificus (MC0603S) in sterile water, and cell extracts from V. vulnificus 27562 in estuarine water were compared. The mean corrected detection signal was compared with CFU in each of the assay settings (figure 6). The correlation between CFU and mean corrected detection signals for extracts from V. vulnificus 27652 (table 66) and the 107

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108environmental isolate (table 65) assayed in sterile water, as well as V. vulnificus ATCC 27562 extracts assayed in estuarine water (table 65) was statistically significant. Corrected Detection Signals for Cell Extract Assays-500501001502002503003504004501.0E+051.0E+041.0E+031.0E+02cell number (CFU)mean corrected detection signal (pA) ATCC extracts assayed in sterilewater Environmental (MC0603S) extractsassayed in sterile water ATCC extracts assayed in estuarinewater Figure 6. Corrected detection signal (detection threshold) for each cell extract assay. There were no 12 CFU extract suspensions assayed for the environmental (MC0603S) or V. vulnificus ATCC 27562 isolates in estuarine water. Assays on Extracts from Enriched Cells: Corrected detection signals for the cell extract enrichment assays were acquired by subtracting the threshold signal from the detection signal for each detection event. Signals acquired from V. vulnificus 27652 extracts enriched in APW and PNCC (assayed in sterile water) were compared. The mean corrected detection signal was compared with CFU in each of the assay settings (figure 7). The correlation between CFU and mean

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109corrected detection signals for extracts from cells enriched in APW and PNCC (table 65) was statistically significant. Corrected Detection Signals for Cell Extracts from Enriched Cells-20-1001020304050607080100050010050cell number (CFU)mean corrected detection signal (pA) Cell extracts from APWenrichment Cell extracts from PNCCenrichment Figure 7. Corrected detection signal (detection threshold) for extracts from V. vulnificus ATCC 27562 enriched in both APW and PNCC. There were no 50 CFU extract suspensions from cells enriched in APW assayed, and no 13 CFU extract suspensions from cells enriched in PNCC assayed.

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110 Assay setting Correlation coefficient (Pearson's r) P value ( = 0.05) 95% Confidence intervals Whole cell environmental (MC0603S) in sterile water 0.9149 0.0005 0.6390-0.9822 Cell extract environmental (MC0603S) in sterile water 0.9869 <0.0001 0.9369-0.9974 Whole cell clinical (ATCC 27562) in estuarine water 0.9650 0.0018 0.7077-0.9063 Cell extract clinical (ATCC 27562) in estuarine water 0.9262 0.0080 0.4614-0.9921 Clinical (ATCC 27562) whole cell APW enrichment in sterile water 0.9479 0.0040 0.5906-0.9945 Clinical (ATCC 27562) whole cell PNCC enrichment in sterile water 0.9260 0.0080 0.4064-0.9920 Table 65. Parametric statistical analysis of assay readings th at were normally distributed. Correlations were calculated comparing the m ean corrected detecti on signal (detection threshold) vs. CFU for each setting. Assay setting Correlation coefficient (Spearman's r) P value ( = 0.05) 95% Confidence intervals Whole cell clinical (ATCC 27562) in sterile water 0.8839 <0.0001 0.8022-0.9331 Cell extract clinical (ATCC 27562) in sterile water 0.9389 <0.0001 0.8826-0.9686 Table 66. Non-parametric analysis of assay se ttings that were not normally distributed. Correlations were calculated comparing the m ean corrected detecti on signal (detection threshold) vs. CFU for each setting.

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111 Comparison of Mean Corrected Detect ion Signals between Clinical and Environmental Isolates Corrected detection signals were compar ed between whole cell and cell extract assays on both clinical (ATCC 27562) and environmental (MC0603S) V. vulnificus isolates suspended in sterile water. Two-tailed t tests ( = 0.05) were calculated performed for each comparison to determine if the differences between the detection signals for both isolates were statistically si gnificant. It was determined that the mean signals for 14 whole cell CFU (limit of detection for the whole cell V. vulnificus 27562 isolate), between the clin ical and environmental isol ates, were significantly different (table 67). It was also determined that the mean signals for cell extracts from 13 CFU in sterile water (limit of detection for cell extracts from the clinical V. vulnificus 27562 isolate), between the clinical and environmental isolates, were statistically different (table 67). Whole cell Cell extracts CFU t value p value t value p value 13NA NA 5.429 0.0016 1410.011 < 0.0001 5.539 0.0052 154.255 0.0510 9.612 0.0024 165.579 0.0307 NA NA Table 67. Comparison of mean corrected detection signals between clinical V. vulnificus (ATCC 27562) and environmental V. vulnificus (MC0603S) isolates for each CFU number assayed. Two-tailed t and p values ( = 0.05) were calculated using parametric analysis. All differences in the mean co rrected detection sign als between the two isolates, other than 15 whole cell (p = 0.0510), were statis tically significant.

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112 Discussion V. vulnificus strains ATCC 27652 (clinical) and MC0603S (environmental) were assayed to determine the usefulness of the antiV. vulnificus polyclonal antibodies for the detection of whole cells, as th ey would be found naturally in estuarine waters. Although the polyclonal antibodies were generated from an immune response from rabbits toward cell extracts of V. vulnificus 27652, nothing is known about th e epitope(s) recognized by the antibodies. Whole cell assays for both isolat es were first carried out in sterile water, in an attempt to exclude any biological or chemical impurities which may interfere with antibody-mediated binding events. The biosenso r assay was able to detect as few as 14 CFU whole cell V. vulnificus (ATCC 27652) suspended in sterile water in 30 out of 30 assays. The assay was also ab le to detect as few as 14 CFU of whole cell V. vulnificus isolated from an environmental source (MC0603S) in sterile water (3 out of 3 assays), but with a significantly lower (p< 0.0001) mean corrected detect ion signal than the clinical (ATCC 27652) whole cell assa ys. This data implies that the antiV. vulnificus polyclonal antibodies used in the biosensor assay had a lower affinity for the environmental isolate than the clinical isolate. In a recent study performed by Nilsson et al. (42), two distinct genotypes of V. vulnificus were revealed using terminal restri ction fragment length polymorphism (TRFLP) analysis of the 16S rRNA gene. The sequence groups were termed type A and type B. Clinical (highly vi rulent) and environmental (gener ally less virulent) isolates were tested using universal prokaryotic 16S rRNA primers to produce a 492-bp amplicon

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113 (corresponding to amplification of nucleotid es 46 to 537 of the 16S rRNA gene). The purified PCR products were digested with Alu I and Hae III, yielding distinctly different patterns for the two types. The majority of the environmental isolat es (33 tested) grouped together as type A (31/33 or 93.9%) and the clinical isolates (34 tested) grouped together as type B (26/34 or 76.5%). It was suggested that this genotypic difference could be associated with virulence (42). Both clinical (ATCC 27562) and environmental (MC0603) isolates used in the developmen t of the biosensor assay were typed by comparing RLFP patterns to the patterns establ ished in the aforementioned Nilsson et al. study. The clinical (ATCC 27562) isolate was classified as type B, and the environmental (MC0603) isolate was classified as type A. The comparatively low affinity of the antibodies for the environmenta l isolate compared to the clinical isolate further support the theory that there are at least two distinct genotypes of V. vulnificus. Cell extracts containing pre-determined CFU were assayed to determine if the addition of a short sonication step prior to the assay would increase the sensitivity of the biosensor assay. It was theorized that cell lysis by sonication would expose epitopes that might not be present on the surface of intact ce lls. As previously stated the quantity and location(s) of the target antigens are not know n. The biosensor assay was able to detect sonicated cell extracts in sterile water from as few as 13 V. vulnificus (ATCC 27652) CFU in 15 out of 16 assays. Also, the assay wa s able to detect cell extracts from as few as 13 V. vulnificus (MC0603S) CFU in sterile water (2 out of 3 assays), but with a significantly lower (p = 0.0016) me an corrected detection signal than the clinical (ATCC 27652) cell extract assays. Soni cating cells prior to the assa y increased the sensitivity

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114 (for both clinical and envir onmental isolates) by nearly te nfold, suggesting that lysing cells exposed additional target antigens for both capture and detection events. The specificity of the antibody used in the biosensor assay was tested against closely related Vibrio spp., not only because of physiological relatedness, but because they are often present in the same natural waters as V. vulnificus (23). Whole cells and cell extracts from three non-target Vibrio spp. ( V. cholerae, V. parahaemolyticus, and V. alginolyticus ) and one E. coli strain were assayed with the biosensor. No positive detection occurred for either 14 or 15 CFU suspensions assayed (whole cell or extracts), for any of the non-target spp., while the positive control V. vulnificus was detected in all assays. This evidence suggest s that the polyclonal antibodies generated in this study are highly specific. It was theorized that assaying mixe d cultures (cultures containing positive V. vulnificus (ATCC 27652), mixed with an equal number of a non-target Vibrio cells) would lead to decreased assay sensi tivity caused by binding competition between V. vulnificus and non-target Vibrios. Mixed, whole cell suspensions containing V. vulnificus (ATCC 27562) and V. cholerae (ATCC 1162) (1:1), were assa yed (one replicate) with the biosensor. The assay was still able to detect as few as 14 CFU of V. vulnificus in mixed suspensions, as was the case with pure suspensions of whole cell V. vulnificus. The final goal in designing the biosen sor assay was to detect autochthonous V. vulnificus in estuarine water without the need for any purification or filtering steps. As a step toward that goal, assays were performed on V. vulnificus 27652 seeded into estuarine water. In both whole cell assa ys in estuarine water, 14 CFU were not detected, although this quantity was reliably detected when assayed in sterile water. In both cell

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115 extract assays in estuarine water, there was no detection for 13 CFU suspensions, which was detectable when assayed in sterile water. These experiments demonstrated that the assay conducted in es tuarine water was less sensi tive by approximately one log CFU. It is possible that high salt concentrations or dissolved ions in estuarine water may have lessened the antibodies affinity for the V. vulnificus antigen, thus reducing the sensitivity of the biosensor assay. In an attempt to increase assay sensitivity in estuarine water, a short enrichment step was added prior to the biosensor assa y. Two enrichment broths (APW and PNCC) were evaluated for their abil ity to support rapid cell grow th over a 4-hour incubation period. Cell extracts from enrichment broths seeded with known CF U were assayed with the biosensor (one replicate for each broth). Both assays on extracts from cells enriched in APW were unable to detect fewer than 500 CFU seeded into enrichment broth, while assays on PNCC extracts were able to detect as few as 100 CFU seeded in enrichment broth. Cell suspensions we re enumerated after enrich ment in both APW and PNCC enrichment broths, to estimate CFU exposed to the waveguides at the time of assays. For initial seeds of 500 CFU in APW, post-enrichment extract s contained approximately 1,260 CFU (average of two assays) exposed to the waveguide at the time of assay, and for seeds of 100 CFU in PNCC, post-enrichme nt extracts contained approximately 1,600 CFU (average of two assays) exposed to the waveguide at the time of assay. As previously stated, the limit of detecti on for cell extracts from the control V. vulnificus 27562 isolate assayed in sterile water was a pproximately 1,000 CFU, implying that seeds of 500 CFU (APW) and 100 CFU (PNCC) are near, yet greater than, the limit of detection for the enrichment assays performed on cell extracts. When cells were enriched

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116 in PNCC, as apposed to APW, there were an average of 880 more CFU in enrichments seeded with 100 CFU, and an average of 6,500 greater CFU in enrichments seeded with 500 CFU. Thus, PNCC was more efficient in enhancing CF U numbers over the 4-hour incubation period than APW. PNCC also contai ns a selective agent (colistin) that is designed to inhibit replica tion of non-target species pr ior to the bi osensor assay. Rapid detection of V. vulnificus would help reduce the incidence of illness and fatality that result from ingestion of contaminated shellfish. The current method specified by the U.S. FDA (23) for the isolation of V. vulnificus from seafood samples using most probable number (MPN) analysis includes a 12-16 hour enrichment step followed by incubating enrichments on selectiv e media for 18-24 hours. This is then coupled with identification of suspect isolates using biochemical (API 20E) profiles (~24 hours), DNA probe colony hybridization targeting the species specific ( vvhA ) cytolysin gene (~48-72 hours) (13, 67), or PCR (~ 8 hours with DNA extraction) (19). A gas chromatographic assay develope d at he U.S. FDA (30) has also been successful for identifying V. vulnificus by comparing cellular fatty acid profiles searched against a computer-generated library, although it is n ecessary to incubate cultures for 24 hours prior to the assay. Some of the most prom ising research for the rapid detection and quantification of V. vulnificus has been in the development of real-time PCR assays. A V. vulnificus -specific SYBR Green I-based real-time PCR assay was recently developed that was able to detect DNA at the equivalent of 1 cell seeded in en richments in less than 8 hours (48). Another realtime PCR assay specific for V. vulnificus was developed utilizing the TaqMan quantitative detection system. This assay was able to detect as few as 100 CFU g-1 and 17 g-1 VBNC cells from oyster hom ogenates, using purified DNA

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117 templates, in approximately 6 hours (7). The V. vulnificus -specific biosensor assay developed in this study was ab le to detect cell extracts from 100 CFU seeded in PNCC enrichment broth. The entire assay includi ng a 4-hour enrichment step, centrifugation, and sonication can be performed in less than 7 hours. Also, ther e was a statistically significant linear correlation between CFU a nd detection signal noticed for all assay settings (whole cell, cell extr acts, and cell extracts from enri chments). This suggests that V. vulnificus quantification from suspect samples with unknown cell co ncentrations may be possible using linear regres sion analysis based on a standard curve generated from detection signals establishe d from known cell numbers. Guidelines established by the Intersta te Shellfish Sanitation Conference (ISSC) (2000) suggest a target limit of 30 V. vulnificus per gram oyster meat. The Analyte 2000 biosensor has been successfully used to detect 3-30 CFU E. coli O157:H7 seeded in ground beef (12) and 10-1,000 CFU Listeria monocytogenes seeded in hotdogs (14). These studies support the ability of the Analyte 2000 biosensor to be useful in detecting V. vulnificus present in meat matrices such as oyster tissue. Although the limit of detection for the V. vulnificus biosensor assay developed in this study was no less than 100 CFU seeded in PNCC enrichment broth, ther e may be ways to further increase the sensitivity without significantly increasing the assay time. The possibility of increasing the sensitivity of the biosensor assay by enriching CFU for a longer period has been suggested. When 100 CFU were enriched for 4 hours in PNCC enrichment broth, numbers were in creased to approximately 1,600 CFU. The optimum doubling time for V. vulnificus is approximately 20 minutes once logarithmic (log) growth phase is reached (44). This suggests that enri ching the ISSC target of 30

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118 CFU (assuming in log growth phase) for 2 hours (~6 generations) would increase numbers to approximately 1,920 CFU which is a detectable limit of the biosensor assay. However, this assumption does not cons ider the length of lag phase that V. vulnificus cells may experience when transferred from su spect samples to enrichment broth, or that the optimum generation rate may not be achieved in PNCC enrichment broth. Optimization of enrichment incubation time n eeds to be performed in attempt to reach target sensitivity, without signifi cantly increasing the assay time. It was suggested that the sensitivity of the enrichment assays may have been underestimated due to a significant die-off of V. vulnificus suspended in ASW, while plate count enumeration was carried out overn ight. Enumeration of CFU seeded into enrichment broth was estimated by direct pl ate counting cells that were suspended in ASW approximately 15 minutes prior to plati ng onto cell culture media. If significant die-off did occur by denying CFU nutrients in ASW for approximately 16-18 hours, then there would have been an overestimation of viable cell numbers seeded into enrichment broth. An experiment was performed to determine if die-off was occurring, by enumerating CFU suspended in ASW using cu lturable plate counts before and after suspensions were incubated at room temperat ure for 18 hours. There were approximately 4.76 CFU present before incubation, and approximately 4.46 CFU present after incubation, suggesting a 4.4% (~25 CFU) loss in viable cells This suggests that the sensitivity of the enrichment assays may have been slightly underestimated; however the 4.4% decline is well within the generally accepted reproducibility of plate count methods (10%).

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119 This research demonstrates the abilit y of the Analyte 2000 biosensor to detect V. vulnificus in several matrices. They assay remained specific when tested against a variety of closely related non-target Vibrio spp. Although there was a loss of sensitivity when detection was attempted in estuarine water, the addition of an enri chment step reduced the limit of detection significantly (from 13 to 100 CFU for cell extracts). Even with the addition of an enrichment step, the assa y is still able to detect low levels of V. vulnificus, with a high degree of specificity, in less than seven hours when using pretreated waveguides. As rapid sensor met hods for foodborne and waterborne pathogens are improved, these methodologies have the pote ntial to significantly improve protection of public health.

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Development of a sensitive and specific biosensor assay to detect Vibrio vulnificus in estuarine waters
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ABSTRACT: Biosensor development has the potential to meet the need for rapid, sensitive, and specific detection of pathogenic bacteria from natural sources. An antibody-based fiber-optic biosensor assay to detect low levels of Vibrio vulnificus in estuarine waters following an enrichment step was developed. The principle of the sensor is based on an immuno-sandwich assay where an anti-V. vulnificus polyclonal capture antibody preparation was first immobilized on a polystyrene fiber-optic waveguide using a biotin-avidin association. The capture antibody is responsible for binding the target cells to the waveguide. Cyanine-5-conjugated anti-V. vulnificus polyclonal antibodies are subsequently allowed to bind to immobilized cells, and detection occurs when a photodetector collects emitted light (670-710 nm) from the fluorophore, which is excited with 635-nm laser light produced by the Analyte 2000 biosensor.Any detection signal greater than a pre-determined threshold signal is considered to be a positive detection event, while any signal lower than the threshold is considered no detection. This immunosensor assay proved highly specific when tested against whole cells and cell extracts from V. cholerae, V. parahaemolyticus, V. alginolyticus, and E. coli. isolates. Following a four hour enrichment in PNCC broth, and in a total of less than seven hours, the assay was able to detect cell extracts from as few as 100 V. vulnificus colony forming units suspended in sterile water. This method holds promise for detection of low numbers V. vulnificus and other autochthonous pathogens in estuarine waters.
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