Location: Microbial and Chemical Food Safety
2024 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 for project 8072-49000-090-000D entitled “Innovative Detection and Intervention Technologies Mitigating Shellfish-borne Pathogens” involves bacterial and viral pathogens which are particularly problematic to the molluscan shellfish industry, particularly the oyster industry. Oysters are susceptible to contamination by Vibrio parahaemolyticus (Vp), a bacterium that is native to the marine environment and found at high levels in seawater and shellfish during the warm summer months. Vp are responsible for the greatest number of bacterial seafood-borne illnesses in the United Sates. Marine predatory bacteria, like Halobacteriovorax (HBX) and Pseudoalteromonas (PAM) can attack and kill Vp which serve as their prey. ARS studies with HBX and PAM will be further described below. Oysters and other shellfish can also become contaminated by human enteric viruses, including hepatitis E virus (HEV), which can also be found occasionally in pork and some wild animals. HEV is an emerging pathogen in the U.S. It causes liver disease and deaths, especially in immunocompromised individuals, people with pre-existing liver disease, and pregnant women. Another virus, norovirus is the major cause of foodborne illness in the U.S. each year and is a significant threat to shellfish safety. Norovirus, HEV, and VP are transmitted by the consumption of contaminated foods including oysters, which concentrate these and other pathogens within their edible tissues. Under Objective 1, “to explore the use of a cocktail containing phages and predatory bacteria to kill Vibrio parahaemolyticus in market oysters”, we evaluated the potential use of predatory bacteria, namely HBX and PAM species, to reduce pathogenic Vp in market oysters obtained from commercial sites in the Delaware Bay. Phage viruses were not used against Vp in this portion of the study because phages were found to have narrow host specificity toward Vp. Broad specificity of HBX toward Vp strains was desirable and obtained by combining two of our well-characterized HBX strains for use as the treatment cocktail. Treatment of market oysters revealed technical limitations that prevented an accurate assessment of HBX effectiveness. They included the fact that: 1) background levels of HBX contaminated the oysters and could not be distinguished from HBX used in the treatment cocktail, 2) the purified (filtered) HBX cocktail could not be totally separated from their host vibrios, which are required to enrich the HBX for cocktail preparation, thus contaminating treated oysters with additional vibrios, and 3) HBX could not be detected at significant levels in oyster stomachs and digestive organs, which are where vibrios congregate in naturally contaminated oysters. Consequently, the evaluation of the effectiveness of HBX treatments to reduce or eliminate Vp within oysters could not be accurately measured. Nevertheless, studies have clearly demonstrated that HBX plays an important role in moderating Vp and other pathogen levels in marine waters, which in turn reduces the levels of Vp available for uptake by oysters and other shellfish to enhance overall shellfish safety. At the time of this writing, this summer’s research has just gotten underway and involves an evaluation of whether PAM isolates will be more effective and more easily administered and monitored than HBX as a treatment for Vp in market oysters. We demonstrated that PAM bacteria are predatory in nature by the fact that they find and attack their prey, kill their prey, and utilize nutrients released by their prey. Unlike HBX, the PAM bacteria do not enter or reproduce within their prey. Instead, PAM transfer surface vesicles containing digestive enzymes to their prey (Vp in this case). The enzymes digest holes within the Vp’s cell wall, effectively killing the Vp and allowing the release of nutrients upon which the PAM feed. Experimentation with PAM will continue through this summer since summers are the only time that Vp are naturally present in oysters in high enough levels to conduct these experiments. The use of PAM will have fewer limitations and has the potential to streamline the disinfection of Vp in oysters. PAM bacteria have been used previously as probiotics in fish aquaculture, so a short-term application of PAM to reduce Vp in market oysters could enhance overall oyster safety. Under Objective 3, “to probe the biology, host range, and the infectivity of predatory HBX bacteria under different salinities”, we showed that four strains of ARS-isolated and well-characterized HBX from widely divergent locations (Hawaii, the Gulf of Mexico, and the Delaware Bay) had broad specificity toward 26 strains of pathogenic Vp from around the United States. Further characterization was performed using these same HBX strains against two other human pathogenic species of Vibrio, namely V. alginolyticus and V. vulnificus. Results revealed that all four HBX attacked and killed V. alginolyticus, but none of them killed V. vulnificus. Studies on the generation time for HBX replication were also performed using scanning electron microscopic observations. Based on electron micrographs, the generation time of HBX appears shorter than anticipated. Results revealed that the HBX predators could locate their prey (the Vp), enter the Vp, replicate within the Vp, and release about 3 to 5-progeny from the dead Vp within 3 hours. A more standard technique (spectrophotometry) for determining generation time could not be used for HBX since HBX cannot be totally separated from the host Vp to obtain valid readings. Salt tolerance was also tested to determine whether HBX infected vibrios preferentially under various salt conditions (10, 20 and 30 parts per thousand [ppt] salinity), which represent low, moderate, and high salt environments for oyster growth and reproduction. At 10 ppt salinity, the Hawaiian strain of HBX did not kill any of the vibrios, while the other three HBX strains did. All the HBX were able to attack and kill Vp and V. alginolyticus, another pathogenic vibrio species, at 20 and 30 ppt salinity. Hawaiian seawater is typically of high salinity, so it is understandable that the Hawaiian HBX strain was not adapted to or functional in low salinity environments. The Gulf Coast and the two Delaware Bay HBX strains had been isolated from coastal waters which are typically subject to dramatic salinity fluctuations, accounting for adaptation of those strains to all three salinities. Metagenonics is defined as the study of DNA obtained from whole communities of microbes from environmental or clinical samples by DNA sequencing. Under Objective 4, “to develop novel and comprehensive methods for virus detection in shellfish that may also have potential for other foods”, we utilized a metagenomics approach, which has the potential to detect all known and unknown viruses within shellfish samples and to provide a comprehensive picture of human impacts on shellfish harvesting areas. Studies of viral genome sequences were performed in shellfish samples using several technologies. Sequencing using the Illumina platform provided a limited number of short or truncated reads less than 100 base pairs in length. Another method currently under evaluation involves Nanopore technology. Nanopore sequencing permits longer lengths of DNA to be obtained. In theory, this technology should allow a single read to provide ample information to identify each virus in a sample. To date, the Nanopore system has been marginally successful in detecting virus-seeded samples purified by a method previously developed by ARS. A whole virus purification method (U.S. FDA method) has also been shown to be marginally effective. Current challenges are in removing extraneous DNA prior to sequencing, and in obtaining measurable amounts of DNA for sequencing. Amplification, better purification methods, and selective hybridization methods are being evaluated. Under Objective 5, “to identify novel lab methods to propagate human norovirus and hepatitis E virus”, we evaluated trout hepatitis E virus (tHEV) as a potential surrogate for zoonotic hepatitis E virus (zHEV). The tHEV was obtained from the U.S. Fish and Wildlife Service. While purported to be propagable in salmon cells, we were unable to replicate this work. However, ARS scientists were able to obtain a continually expressing zoonotic HEV strain-3 cell line from Germany. Work with these cells has been successful. To date, the virus was propagated in these cells and used to infect host cells. A molecular (RT-PCR) detection method for zHEV was also shown to be effective. In other work, select strains of human norovirus were replicated in the laboratory using a 3-dimensional, microgravity cell culture system containing cell masses resembling organ tissues (called organoid cells), but overall, the replication was very labor-intensive and was deemed impractical for the evaluation of norovirus intervention techniques.
Accomplishments
1. Ultracold high pressure processing kills viruses. High pressure processing (HPP) is effective against norovirus and other genetically related calciviruses. Previously it was shown that HPP was a more efficient food processing technology at refrigeration temperatures than at room temperature (greater inactivation or lower pressures to achieve comparable inactivation). ARS researchers at Dover, Delaware, demonstrated that HPP performed at -35 degrees C gives even greater inactivation at much lower pressures (2000-3000 atmospheres (atm) at -35 degrees C versus 3500-4000 atm at 4 degrees C). Food samples can be pressurized either frozen or thawed. Of particular note, samples that were thawed (equilibrated to 1.5 degrees C) and then pressurized for 5 min at -35 degrees C had significantly greater virus inactivation than those that were already frozen at -35 degrees C and then pressure treated. Why ultracold HPP gives greater inactivation overall compared to above freezing temperatures is uncertain, but one hypothesis is that different molecular forms of ice are formed at high pressure and low temperature. This work has implications as a virus intervention for the commercial frozen oyster market as well as the frozen berry industry where HPP could be applied either after freezing or as a combined ultracold HPP-freezing process.
Review Publications
Richards, G.P., Watson, M.A., Williams, H.N., Jones, J.L. 2023. Predator-prey interactions between Halobacteriovorax and pathogenic Vibrio parahaemolyticus strains: geographical considerations and influence of Vibrio hemolysins. Microbiology Spectrum. 11(4). https://doi.org/10.1128/spectrum.02353-23.
Boas Lichty, K., Loughran, R.M., Ushijima, B., Richards, G.P., Boyd, F. 2024. Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: Acquisition of catabolism gene clusters for the compatible solute and signal molecule myo-inositol. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.00920-24.
Dewitt, C.A., Nelson, K.A., Kim, H., Kingsley, D.H. 2024. Ultralow temperature high pressure processing enhances inactivation of norovirus surrogates. International Journal of Food Microbiology. 408:110438. https://doi.org/10.1016/j.ijfoodmicro.2023.110438.
Marcano-Olaizano, A., Zerrad, A., Janneto, F., Kingsley, D.H. 2023. Confirming the stimulated Raman origin of singlet-oxygen photogeneration. Journal of Raman Spectroscopy. https://doi.org/10.1002/jrs.6615.