Location: Microbial and Chemical Food Safety
2025 Annual Report
Objectives
Objective 1. Explore the use of a cocktail containing phages and predatory bacteria to kill Vibrio parahaemolyticus in market oysters.
Objective 2. Compare and contrast Halobacteriovorax and phage levels in oysters, seawater, and sediment as a prerequisite to the development of future prediction and forecast models for pathogenic vibrios in market oysters.
Objective 3. Probe the biology, host range, and infectivity of predatory bacteria to optimize their potential use as treatment against shellfish-borne vibrios.
Objective 4. Develop novel and comprehensive methods for virus detection in shellfish that may also have potential for other foods.
Objective 5: Identify novel in vitro propagation methods for human norovirus and hepatitis E virus.
Objective 6: Evaluate inactivation technologies for virus-contaminated shellfish and other foods.
Approach
Under objective 1, a cocktail of phages and predatory bacteria will be formulated from isolates collected during surveys of Delaware Bay oysters. Cocktail effectiveness in eliminating V. parahaemolyticus (VP) from seawater will be tested followed by efficacy testing of the cocktail against VP in naturally-contaminated, market-size oysters. Under objective 2, a quantitative Halobacteriovorax (HBX) assay will be developed using a most probable number (MPN) based approach to quantify HBX in seawater, oysters and marine sediments. Positive tubes will be determined by plaque assay. Alternative, enzyme-based assays will also be explored. In the second phase of this objective, information will be collected on HBX and total and pathogenic VP abundances in oysters, seawater and sediments for the development of future prediction and forecast models for pathogenic vibrios in oysters. Phage abundances will also be monitored by plaque assay. The goal of objective 3 is to further our understanding of factors that affect the biology, host range and infectivity of predatory bacteria under various environmental conditions and how HBX impact pathogenic VP levels in oysters and their environment. Among questions to be answered are: whether HBX replicates within oyster gut or gill tissues; what is the generation time for HBX in VP; do HBX persist or die in the absence of host vibrios, do environmental conditions (temperature, salinity, or pH) affect HBX infection and replication within host cells, and what is the host range of HBX isolates. Under objective 4, metagenomics will be used to detect viruses in shellfish. The principal challenges and limitations will be sample preparation and sensitivity, so several virus extraction procedures will be investigated. All methods will be evaluated for purity and yield of virus RNA using shellfish samples seeded with surrogate viruses. Laboratory-spiked shellfish and wild shellfish impacted by sewage outfalls or from other areas prone to contamination will be evaluated. The goal of objective 5 is to identify novel in vitro propagation methods for human norovirus (HuNoV) and hepatitis E virus (HEV). Two established embryonic cell lines from zebrafish will be investigated for HuNoV replication. After incubation for up to 2 weeks, virus yields will be determined by RT-qPCR. The feasibility of a surrogate trout HEV assay will also be investigated as a potential model system for HEV inactivation. Trout HEV will also be evaluated in nonthermal virus inactivation studies. Other potential HEV cultivation techniques will be investigated to assess the infectivity and inactivation of genotype 3 zoonotic HEV including a 3-dimensional, microgravity culture system. Under objective 6, inactivation technologies for virus-contaminated shellfish will be evaluated including: high pressure processing (HPP) of frozen oysters to reduce or eliminate HuNoV; the use of X-rays with and without singlet oxygen enhancers to inactivate surrogates for HuNoV, hepatitis A virus and HEV; and targeted heating with infrared or radiofrequency to eliminate viruses and bacteria in specific shellfish tissues.
Progress Report
This report is for project 8072-42000-090-000D entitled “Innovative Detection and Intervention Technologies Mitigating Shellfish-borne Pathogens”. The project involves bacterial and viral pathogens which are particularly problematic to the molluscan shellfish industry and to regulatory agencies charged with protecting the safety of the food supply and consumers. Oysters are particularly problematic, especially when eaten raw, which is a common practice in the United States. Oysters, clams, and mussels can concentrate bacteria and viruses within their edible tissues and transmit diseases to unsuspecting consumers, usually through raw or lightly cooked shellfish. Vibrio parahaemolyticus (VP), the most frequently acquired bacterial illness among seafood consumers, is a major obstacle to seafood safety. Some naturally occurring bacteria in the marine environment are predators of vibrios and other bacteria. Predators include Halobacteriovorax (HBX) which ARS researchers under this project showed can effectively kill a broad range of human pathogenic strains of VP. Growth characteristics of HBX were explored and the HBX were found to replicate slowly in the marine environment. Another bacterium, first shown by this lab to be predatory toward vibrios, is Pseudoalteromonas piscicida (PAM). Under Objective 3, “to complete studies on the generation time for Halobacteriovorax infection of host cells”, ARS researchers at Dover, Delaware, better characterized HBX and PAM predatory bacteria by cultural methods and electron microscopic observation. HBX replicated much more slowly than PAM, suggesting that PAM may be a better alternative treatment to reduce VP in shellfish. An earlier paper published by ARS scientists showed scanning electron microscopic images of PAM bacteria and surface vesicles (sacks of digestive enzymes), which they physically transfer to the surface of the vibrios. The digestive enzymes in the vesicles digest holes in the VP cell walls killing the VP (and other bacterial prey) in the process. PAM feeds on the nutrients released by the dead vibrios. Electron micrographs showed that some PAM have these surface vesicles, while others don’t. ARS scientists performed and published a study showing that the PAM could convert from having no vesicles to becoming highly vesiculated. The transformation to the vesiculated state occurred in both low-nutrient seawater and seawater supplemented with growth media even in the absence of prey cells. The PAM cells also formed unusual, highly elongated, cell structures that were densely vesiculated. The reason for these structures remains to be determined. The size of these enzyme-containing-vesicles also varied greatly from small to large. Some vesicles were tethered to the surface of the PAM with string-like connectors (pili) of varying lengths ranging from short to extremely long. It is not known if these different morphologies affect PAM’s ability to attack and kill their prey; however, we hypothesize that the longer tethers allow the PAM’s vesicles to extend into the matrix of biofilms to attack and digest holes in prey bacteria that would otherwise be out of reach.
Other research investigations centered around the challenge of oysters with PAM to determine if PAM could reduce levels of VP and Vibrio vulnificus (VV). VV is a serious human pathogen causing high mortalities in infected shellfish consumers. Together, VP and VV, contribute to considerable illness from the consumption of seafoods and can be found at high levels in the stomachs of oysters. Consequently, the development of an effective commercial treatment to reduce or eliminate vibrios in oysters would require three things: 1) effective uptake of the treatment organism within the oysters’ stomachs, 2) the ability of the treatment organism to survive within the stomach, and 3) the ability of the treatment organism to kill the vibrios within the stomach. To determine whether PAM can achieve these three requirements, naturally contaminated oysters from Delaware Bay were placed in 10- gallon tanks of seawater and treated with high levels of PAM. Negative control tanks consisted of seawater and oysters not treated with PAM. After 2 days, testing for PAM and Vibrio levels were performed on the control and treated seawater and the oysters’ stomachs where vibrios are known to concentrate. Oyster stomachs and their contents were dissected from the oysters for both the negative controls and PAM inoculated samples. Tests of tank seawater did not reveal any PAM naturally present in the negative controls. The seawater from duplicate treatment tanks to which PAM were added revealed 5,500 and 1.1 million PAM per milliliter of seawater. Oyster stomachs from treated tanks contained high levels of PAM (15 million per gram and 200 thousand per gram for the two treated tanks). This demonstrated for the first time that PAM can accumulate and remain viable within the oysters’ stomachs. Additionally, tests for Vp and V. vulnificus within the seawater and oysters gave surprising results. In negative control tanks, initial vibrio counts for both vibrio species were less than 5,000 per milliliter of seawater but were 300,000 per gram for both vibrio species in the negative control oyster stomachs, indicating a vibrio bloom had occurred during the 2-day incubation period. For the PAM-treated oysters, Vibrio counts from the stomachs were high (500,000 per gram) for both the Vp and Vv from one tank. The duplicate treated tank had 300,000 Vp per gram and 400,000 Vv per gram of stomach material. Thus, the very high levels of viable PAM and vibrios within the oysters’ stomachs indicate that the PAM and vibrios can co-exist at high levels within the stomach. Consequently, the third requirement for an effective treatment (the ability of the treatment organism to kill the vibrios within the stomach) was not achieveable. Results suggest that PAM are not effective in eliminating vibrios from the gut of the oyster. Reasons could include the possibility that PAM shuts down its predatory function when ample food is available in its environment.
Collaborative studies were also completed to evaluate the presence of Shewanella species in seawater and oysters. In the past, ARS scientists were the first to isolate large numbers of Shewanella in Delaware Bay oysters and seawater. Some species of Shewanella are known to be flesh-eating human pathogens. ARS collaborated with two universities to extend our knowledge on the levels and species of Shewanella present in the Chesapeake Bay and Maryland Coastal Bays, Maryland, and in Apalachicola Bay, Florida. High levels of Shewanella of varying species were similarly detected. Antibiotic sensitivity tests were also performed on the Shewanella isolates and many were found to be resistant to antibiotics, including species known to be pathogenic.
Accomplishments
1. Bacterial predators effectively kill pathogenic vibrios. The bacterial genus Vibrio consists of a wide range of human, fish, shellfish, and coral pathogens. They are the most common bacterial source of seafood-related illnesses in the U.S. Predatory bacteria, known as Pseudoalteromonas (PAM), were shown to effectively kill vibrios in seawater by transferring “bags” of digestive enzymes (known as outer membrane vesicles) from their surface to the surface of unsuspecting vibrios. These enzymes digest holes in the vibrios, readily killing them and allowing the PAM to feed on nutrients released from their prey. Although PAM can kill other types of bacteria, their preferred prey is pathogenic vibrios, strains commonly found in the marine environment and in biofilms. ARS researchers in Dover, Delaware, identified PAM strains capable of killing human, fish and shellfish-associated vibrios. These PAM isolates were provided to a biotech company to upscale production and to field test them as a potential new technology to combat vibrios and other pathogens in commercial aquaculture settings. Effective treatments would reduce Vibrio-associated contamination in aquaculture as well as larval oyster mortalities in shellfish hatcheries.
Review Publications
Johnson, T., Richards, G.P., Jacobs, J., Townsend, H., Almuhaideb, E., Rosales, D., Chigbu, P., Dasilva, L., Parveen, S. 2025. Prevalence and pathogenic potential of Shewanella species in oysters and seawater collected from the Chesapeake Bay and Maryland Coastal Bays. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2025.1503443.
Boas Lichty, K., Bhide, S.M., Thomas, H.E., Richards, G.P., Boyd, E. 2024. Draft genome sequence of the marine halophile Vibrio diabolicus strain 3098. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00940-24.
Richards, G.P., Uknalis, J., Watson, M.A. 2025. Highly pleomorphic strains of the Vibrio predator Pseudoalteromonas piscicida and their outer membrane vesicles: A scanning electron micrographic study. Microorganisms. 13(2). https://doi.org/10.3390/microorganisms13020365.
Thomas, H., Boas Llchty, K.E., Richards, G.P., Boyd, F.E. 2025. Dual roles of glycine betaine (GB), dimethylglycine, and sarcosine as osmoprotectants and nutrient sources in Vibrio natriegens. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.00619-25.
Sher, S., Richards, G.P., Parveen, S., Willimas, H.N. 2025. Characterization of antibiotic resistance in Shewanella species: an emerging pathogen in clinical and environmental settings. Microorganisms. https://doi.org/10.3390/microorganisms13051115.