Location: Food Safety and Intervention Technologies Research
2019 Annual Report
Objectives
The safety of aquaculture products, particularly molluscan shellfish, is jeopardized by Vibrio and enteric virus contamination and the lack of effective processing interventions. Among the foods of greatest concern are raw or lightly-cooked oysters and clams, which result in substantial health risks to consumers. The objectives of this project are designed to: identify practical intervention methods to eliminate vibrios in shellfish using bacteriophages and Bdellovibrio-and like-organisms (Vibrio predatory bacteria) and to develop and validate methods for enteric virus detection and elimination from shellfish.
1: Develop and evaluate intervention and control strategies for Vibrio species, with specific emphasis on the identification, characterization and application of bacteriophage to remediate shellfish mortalities in hatchery settings, and for use in commercial shellfish processing.
2: Evaluate a modified depuration process with marine Bdellovibrio and related bacteria to eliminate Vibrio in market oysters.
3: Develop and validate technologies to improve current virus detection and testing methods, including distinguishing infectious versus non-infectious virus; technologies for virus replication, for example, development of a cell culture propagation method for human norovirus; virus surrogates; and long-term virus persistence.
4: Develop and validate emerging technologies for inactivation of enteric virus-contaminated shellfish and other foods using novel applications of high pressure and laser-induced resonance energy.
Approach
Under objective 1, we will seek to reduce Vibrio-associated mortalities in larval oyster hatcheries by 50% using a mixture of bacteriophages (phages), and to reduce human pathogenic vibrios in market oysters using a second mixture of phages. Under a CRADA with Intralytix, Inc. and Oregon State University, ARS will continue efforts to commercialize this phage treatment against the larval shellfish pathogens V. tubiashii and V. coralliilyticus for use in shellfish hatcheries. Phages that we already isolated and identified will be further characterized genetically, morphologically, and mechanistically as potential candidates for commercialization. At the completion of these studies, efforts will shift to an evaluation of phages against the human pathogens V. parahaemolyticus and V. vulnificus in market-sized oysters. Oysters will be challenged with streptomycin-resistant strains of V. parahaemolyticus and V. vulnificus, allowed to bioaccumulate these vibrios in tanks of seawater and then treated with phages to determine the Vibrio reduction rates in the shellfish. Under objective 2, we will evaluate a modified depuration process with predatory bacteria known as Bacteriovorax species (recently renamed Halobacteriovorax) to reduce or eliminate Vibrio parahaemolyticus and Vibrio vulnificus in market-sized oysters. Work will be performed using Halobacteriovorax strains that we isolated and partially characterized from the U.S. Atlantic, Gulf, and Hawaiian coasts. Concurrent with the above research will be studies to better characterize Halobacteriovorax and related bacteria that inhibit pathogenic vibrios and other bacteria. Under objective 3, we will develop and validate our porcine gastric mucin-magnetic beads (PGM-MBs) assay to distinguish infectious from non-infectious human noroviruses (NoV), determine if a correlation exists between long-term persistence of NoV within oysters and MS-2 phages at different water temperatures, and attempt to develop an in vitro replication system for NoV. The degree to which MS-2 phages mimic NoV in their ability to remain viable within shellfish and survive chlorination levels found in sewage treatment will be determined. The persistence of NoV in shellfish, oyster hemocytes, and in sewage effluent will be evaluated along with potential interventions to eliminate viral contamination. Under objective 4, we will seek to overcome barriers to the widespread commercial use of high pressure processing (HPP) for oysters and identify substances, like ozone or copper ions, that may inactivate NoV during HPP treatment. We will also evaluate the use of modified atmosphere packaging for shellfish using “oxygen scavenger” technology to enhance freshness of HPP-treated oysters during transit. We will seek to understand how laser induced resonance energy can destroy small icosahedral viruses, like NoV. This work will be performed in collaboration with researchers at Delaware State University and the University of Maryland.
Progress Report
Bacteria within the genus Vibrio are commonly found in the marine environment. Some species, like Vibrio parahaemolyticus and Vibrio vulnificus are significant human pathogens most often transmitted by the consumption of raw or lightly cooked oysters and other seafoods. According to the Centers for Disease Control and Prevention (CDC), V. parahaemolyticus has been attributed to approximately 30,000 illnesses in the United States each year with a mortality rate around 1%. In contrast, V. vulnificus causes about 50 foodborne cases per year, but with a mortality rate around 35%, the highest mortality rate for any of the 31 foodborne pathogens monitored by the CDC. Processing interventions to reduce V. parahaemolyticus and V. vulnificus in raw shellfish have been only partially successful and new methods are needed to further enhance the safety of shellfish and other products. One method contemplated for potential commercial application is the use of bacteriophages (bacterial viruses) that can kill vibrios within seafoods, in general, and in oysters in particular. Oysters appear to be the leading cause of Vibrio-associated, shellfish-borne illnesses in the United States. Under the milestone for Objective 1, “to complete isolation and host specificity testing of phages against human vibrios”, we concluded a study to isolate, quantify, and characterize bacteriophages against vibrios (better known as vibriophages) from oysters collected from three sites along the Delaware Bay. Phages against three strains of V. parahaemolyticus were readily detected in summertime oysters from all three sites, but no phages were detected against V. vulnificus. Nine isolated phages were characterized for their ability to infect other strains of V. parahaemolyticus, as well as V. vulnificus, and other vibrios. All the phage isolates were relatively host specific, able to infect only one strain of V. parahaemolyticus or a strain of V. parahaemolyticus and a strain of Vibrio alginolyticus. Vibrio alginolyticus is associated with skin and ear infections in humans as well as disease in fish and shellfish. Electron microscopic studies allowed the phages to be tentatively identified. Results of this study were published in January 2019. These phages will be used in future research to determine their effectiveness in reducing V. parahaemolyticus levels in market-sized oysters.
Studies were conducted on an Objective 2 milestone, “to complete depuration trials on Vibrio parahaemolyticus in oysters using Bacteriovorax”, predatory bacteria isolated from the marine environment. Bacteriovorax was recently renamed Halobacteriovorax. Some strains of Halobacteriovorax that we isolated are predators of V. parahaemolyticus and may serve a role in shellfish depuration. Depuration is a commercial process where shellfish are placed in tanks of seawater that is recirculated through a battery of ultraviolet (UV) lights to disinfect the water. Over a 3-day period, shellfish purge contaminants, including bacteria, from their edible tissues into the seawater, and those bacteria are killed by the UV light. The shellfish may then be sent to market. Depuration works well for contaminants like E. coli and Salmonella species, which usually come from human sewage or from animal feces that enter the shellfish-harvesting areas. Unfortunately, naturally-occurring bacteria, like the vibrios, are poorly purged during depuration. Our objective was to modify conventional depuration practices by adding a pre-step to the depuration process, where predatory bacteria like Halobacteriovorax or a related predatory bacterium known as Pseudoalteromonas piscicida would be added to the tanks of shellfish for a period sufficiently long (1-2 h) for some of the predatory bacteria to be taken up by the oysters as they ingest food and bacteria from the water. In theory, once the predatory bacteria are inside the oysters, vibrios in the gut and tissues should be attacked and killed. Since these predatory bacteria are of no harm to humans, this step may provide a processing intervention that would render depuration more effective, subsequently reducing human illness from V. parahaemolyticus. In preliminary trials, pure cultures of Halobacteriovorax were difficult to obtain in sufficient quantities to conduct the planned studies. This is because Halobacteriovorax can only grow in the presence of host vibrios and filtration to remove the potentially dangerous host cells also removed many of the Halobacteriovorax. Consequently, we decided to replace Halobacteriovorax in these studies with the predatory bacterium Pseudoalteromonas piscicida, which can be grown in the presence or absence of host cells, making them more suitable and cost effective for commercial applications. We completely sequenced the genomes of three strains of P. piscicida, entered the sequences into the GenBank database, and published two papers on the sequence findings. These are the first and only fully sequenced P. piscicida genomes in GenBank. The DNA of the three strains contain between 5.3 and 5.4 million base pairs and the genomes each contain approximately 4700 genes. Initially, we challenged larval oysters with high levels of P. piscicida to determine if the bacterium had any toxic effects on the larvae. Results demonstrated no ill effects on the larvae. Consequently, the depuration studies on market-sized oysters were undertaken and are currently underway. It is necessary to conduct these studies over the summer months when V. parahaemolyticus levels are naturally high in oysters. As of this writing, results are pending completion of summertime analyses.
There were no milestones for Objective 3 this year, but the milestone for Objective 4, “to assess the effects of modified atmosphere packaging on high pressure process (HPP)-treated shellfish” was completed. In current HPP operations, pathogens in oysters are reduced by high pressure and then the shellfish are shipped country-wide to consumer markets. During shipping, there is a reduction in product quality due to the pressure treatment. Therefore, HPP-treated shellfish may have reduced pathogen loads but are less desirable to consumers after transport than fresh oysters. We evaluated whether modified atmosphere packaging would allow the fresh quality of HPP oysters to persist for several days after treatment. Unfortunately, there was no evidence that modified atmosphere packing improved the quality of HPP-treated oysters when stored under nitrogen at refrigeration temperatures. In fact, HPP-treated oysters subjected to modified packaging rapidly deteriorated in odor and, to a lesser extent, in appearance. Consequently, attempts to develop an improved method to store HPP-treated oysters was not successful.
Accomplishments
1. Discovery of novel predatory bacteria and their mechanisms of action. Newly discovered bacteria make Oysters safer. Vibrio parahaemolyticus (VP) is a naturally occurring marine bacterium that is transmitted to humans through the consumption of raw oysters. Researchers at ARS in Dover, Delaware, identified strains of another marine bacterium, Pseudoalteromonas piscicida (PP), that attack and kill VP by the direct transfer of enzyme-containing vesicles from PP to VP. Once transferred, enzymes in the vesicles kill VP by digesting holes in their cell walls. PP added to oysters reduced natural VP levels by greater than 5-fold. Using ARS isolates, collaborators in Zurich, Switzerland are identifying PP enzymes that may form commercially valuable bioactive products, and scientists at the Lawrence Berkeley National Laboratory in California are planning to test the ability of PP to control microbial communities.
Review Publications
Richards, G.P., Chintapenta, L.K., Watson, M.A., Abbott, A.G., Ozbay, G., Uknalis, J., Oyelade, A., Parveen, S. 2019. Bacteriophages against pathogenic vibrios in Delaware Bay oysters (Crassostrea virginica) during a period of high levels of pathogenic vibrio parahaemolyticus. Food and Environmental Virology. https://doi.org/10.1007/s12560-019-09365-5.
Richards, G.P., Kingham, B., Shevchenko, O., Watson, M.A., Needleman, D.S. 2018. Complete genome sequence of Vibrio coralliilyticus RE22, a marine bacterium pathogenic toward larval shellfish. Microbiology Resource Announcements. 7(17):e01332-18. https://doi.org/10.1128/MRA01332-18.
Richards, G.P., Needleman, D.S., Watson, M.A., Poulson, S.W. 2019. Whole genome sequences of two Pseudoalteromonas piscicida strains DE1-A and DE2-A, which exhibit strong antibacterial activity against Vibrio vulnificus. Microbiology Resource Announcements. 8:e01451-18.
Edlind, T., Richards, G.P. 2019. Development and evaluation of polymorphic locus sequence typing for epidemiological tracking of Vibrio parahaemolyticus. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2019.2649.
Kingsley, D.H., Chen, H., Annous, B.A., Meade, G.K. 2019. Evaluation of a male-specific DNA coliphage persistence within Eastern oysters (Crassostrea virginica). Food and Environmental Virology. 11:120-125.
Lacombe, A., Niemira, B.A., Gurtler, J., Kingsley, D.H., Li, X., Chen, H. 2018. Surfactant-enhanced disinfection of the human norovirus surrogate, Tulane virus, with organic acids and surfactant. Journal of Food Protection. 81(2):279-283.
Kingsley, D.H., Johnson, A., Robinson, K., Perez, R., Basaldua, I., Burkins, P., Marcano, A. 2018. Oxygen-dependent laser inactivation of murine norovirus using visible light lasers. Virology Journal. 15:117. https://doi.org/10.1186/s12985-018-1019-2.
