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Reduction of Vibrio Vulnificus in Oysters by Treatment with Bdellovibrio and Like Organisms and Viruses


V. vulnificus causes fulminating diseases in susceptible populations of humans: septicemia after ingestion of raw oysters and wound infection from contact with sea water. Although the numbers of cases are low, V. vulnificus is the leading cause of reported death due to seafood in the U.S. In both diseases the bacteria replicate extremely rapidly in host tissues and are highly invasive, leading to high numbers of bacteria with extensive tissue damage. Even with antibiotic treatment, the mortality rates for septicemia and wound infection can be 77% and 15%, respectively, and patients can die within 24 hours of onset of symptoms. The infamous association of vulnificus disease with shell fish costs the industry millions of dollars per year in lost revenue. Because of the rapid and fulminating nature of the disease process, the best chances of reducing morbidity and mortality and increasing consumer confidence are in reducing contamination of oysters by V. vulnificus. <P>Therefore, based on concerns over health of oysters and health of consumers of shell fish, effective methods to reduce and control the numbers of harmful bacteria in oysters are needed. BALO and bacteriophage have the potential to be used for this purpose. The BALO are small, predatory, Gram-negative, motile bacteria that invade and consume other Gram-negative bacteria. They have been used to reduce bacteria in experimental eye infections in rabbits. No adverse effects were apparent when BALO alone was inoculated into the eyes. Bacteriophage are viruses that infect and frequently kill certain specific bacteria. They have been shown in a number of studies to reduce infections in animals.<P> Broad, long-term objectives: The long-term goal of the proposed research is to exploit the use of Bdellovibrio and like organisms (BALO), both alone and in combination with bacteriophage, as a new approach to eradicate pathogenic microbiota associated with oysters. BALO are a group of prokaryotes that prey on and kill many Gram-negative bacteria. Achievement of this goal would introduce a new and different mode of reducing some of the major human and/or seafood pathogens and make the nation's seafood safer and better protected. In the longer term, the procedure could be patented and commercialized. <P>Hypothesis: The hypothesis to be tested in the proposed study is that strains of BALO can be used alone and in combination with bacteriophage to reduce or eliminate V. vulnificus in oysters. <P>Our Specific Aims to test the hypothesis are: <ol> <LI>To identify and characterize HEK BALO strains that prey on V. vulnificus commonly found in oysters and to deposit them in a national culture collection to be available to other investigators<LI>To establish that HEK BALO as a single treatment and in combination with bacteriophage can significantly reduce V. vulnificus in oysters <LI>To show that various strains of HEK BALO can be introduced into oysters and mammals without apparent adverse side effects

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Non-Technical Summary: Vibrio species occur in various types of seafoods including raw oysters where they may be a potential health risk to consumers. It would be beneficial to develop effective methods to reduce the numbers of Vibrio species in sea food such as oysters. In this study a novel approach will be tested to eliminate Vibrio vulnificus strains from live oysters. A group of predatory bacteria known as the Bdellovibrio and like organisms (BALO) attacks and kills many V. vulnificus strains and other pathogenic Gram negative bacteria. The BALO will be tested for their effectiveness in reducing numbers of the V. vulnificus in oysters to safe levels. The BALO have been shown in previous studies to prevent or cure bacterial infections in rabbit eyes. Our first tasks will be to select from our collection of over 100 BALO isolates, a group of 10 to 15 strains that show the greatest kill rate against different V. vulnificus strains. These high-efficiency killer (HEK) BALO will be characterized by molecular and phenotypic methods to reveal distinctive properties. To demonstrate BALO's efficacy in reducing numbers of the bacteria in oysters each of the HEK strains will be introduced into pans of water with oysters experimentally infected with V. vulnificus. Following incubation for 2 hrs the oyster tissue will be homogenized and the numbers of V. vulnificus and BALO will be counted. BALO will be used both singularly and in combination with viruses. The expected outcome is that the Vibrio will be reduced to safe levels. Achievement of this goal would make the nation's seafood safer. <P> Approach: The experimental approaches is described below. Aim 1. To determine BALO isolates showing the greatest killing power against V. vulnificus strains. Thirty BALO isolates will be tested for ability to kill strains of V. vulnificus. BALO isolates that prey on the V. vulnificus strains will be further examined to identify those that exhibit highest rates of killing of V. vulnificus. This will be done by inoculating equal concentrations of the various BALO isolates into respective broth cultures of uniform densities of the different V. vulnificus strains and following the rate of predation by measuring the decrease in turbidity (OD) over time. BALO strains having the greatest kill potential will be those that decrease turbidity at the most rapid rate. The BALO showing the greatest rate of kill will be used for the oyster studies. Aim 2: To establish that HEK BALO can efficiently reduce V. vulnificus in oysters. Following overnight acclimation in autoclaved aerated seawater, live oysters will be treated with Rifampicin (Rif) (50 microgram per mL) overnight to kill the natural bacterial flora and potentiate infection by laboratory V. vulnificus strains. The seawater will then be inoculated with Rifr V. vulnificus FLA042 or FLA077 at a final concentration of 106 CFU per mL and oysters will be incubated with bacteria for 2 h. After the infection period, the infected oysters will be transferred to sterile seawater. BALOs will be added at concentrations varying from 5 x 107 to 5 x 109 PFU/ml for various time periods. After the BALO treatment period, the shells of the oysters will be rinsed with deionized water, the oysters shucked and the meat rinsed with sterile seawater and removed. Seawater will be added to the oyster tissue at 1 mL per g and the mixture homogenized, serially diluted and plated on LB-N plates containing Rif. The effects of BALO treatment to untreated, infected oysters by the Student t test of mean CFU per g tissue will be compared. Aim 3. Confirm that the HEK BALO identified in Experiment 1 will cause no harmful effects in oysters and mammals. A purified suspension of each BALO isolate will be added to water in the experimental holding tank for the rif treated oysters to achieve a finalconcentration of 109 PFU / ml. The oysters will be grossly examined following incubation for 2, 5, and 10 days. To examine potential detrimental effects of BALO on mammals, we will inoculate mice initially parenterally. Groups of 5 outbred ICR mice will be injected intraperitoneally with a BALO cell suspension at various PFU/mL. Mice will be examined for health (i.e., death, scruffy fur, lethargy, or change in rectal temperature) for one week then euthanized and the peritoneal cavity lavaged. The lavage fluid will be examined for BALO by plaquing. BALO will also be inoculated orally into mice to examine their pathogenicity in the intestines. The health of the mice will be monitored for one week, then they will be euthanized, the intestines removed, and the contents examined for BALO by plaquing. A section of intestines will be examined for histological damage by H&E staining. We will also examine a sample of blood for BALO.

Williams, Henry
Florida A&M University
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