Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Research Project #430338

Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

2020 Annual Report

Objective 1: Elucidate biological factors and molecular mechanisms that enhance or reduce fitness characteristics related to survival and growth of enteric pathogens in the produce production continuum. Sub-objective 1A-1I (Refer to uploaded Project Plan) Objective 2: Identify environmental factors that affect the persistence and transmission of enteric pathogens in the produce production environment for risk assessment. Sub-objective 2A-2H (Refer to uploaded Project Plan) Objective 3: Develop methods for the detection and subtyping of enteric bacterial and viral pathogens from produce production environments; to aid epidemiological investigations and to distinguish pathogenic from non-pathogenic strains. Sub-objective 3A-3E (Refer to uploaded Project Plan) Objective 4: Study the ecology of Shiga toxin-producing E. coli (STEC) bacteriophages and its association with bacterial hosts. Objective 5: Development of immuno-, bacteriophage-, and mass spectrometry-based methods for rapid detection of foodborne pathogens.

Plant-microbe model systems in combination with population studies, ecology, molecular methods, genomics, and microbiology will be used to investigate the interaction of human bacterial and viral pathogens with plants and plant-associated bacteria, as well as to develop improved methods for detection and subtyping of human on produce. Pathogenic E. coli is a foodborne pathogen that has been linked to numerous outbreaks of foodborne illnesses, and the illnesses are primarily attributed to the ingestion of Shiga toxin-producing E. coli (STEC). Previous research has indicated the virulence markers such as stx genes, of STEC strains are conferred to stx-encoding bacteriophages and can be transduced into the susceptible bacterial hosts. In order to understand the interplay between STEC-specific phages and their bacterial hosts in the environment to enhance the safety of food products and the prevention of new emerging foodborne pathogens, the initial focuses of the phage research are to isolate, collect and characterize STEC phages and to understand the relationship between phages and their hosts in the environment. Efficient methods for isolation of STEC bacteriophages will be utilized. Characterization of STEC bacteriophages will be established using genomic sequencing and proteomic analyses. The association of fecal contamination with the population of STEC bacteriophages in the environment will be determined. Environmental factors that influence the geographical distribution of STEC bacteriophages will be identified. This will establish a foundation to study biological interactions between phages and their hosts and the association of phages with bacterial evolution as well as to utilize collected phages to develop biosensors and pre-harvest biological controls for STEC to improve the microbiological food safety of the food supplies.

Progress Report
Salmonella serovar Senftenberg is more fit than Typhimurium in growth inside lettuce cells (apoplast), and in the phyllosphere under growth-promoting conditions (Sub-objective 1A). We discovered that S. Senftenberg utilizes ammonium, a common nitrogen source for bacteria on plants, better than S. Typhimurium. Comparative genomics of strains of both serovars isolated from produce and other foods are ongoing to identify differences in nitrogen assimilation pathways. In collaboration with University of California (UC) Davis and Russell Ranch, work continued analyzing microbial activity and metagenome analysis of organic vs. conventional soils (Sub-objective 1D). The organic soils, which suppressed growth of six Shiga toxin-producing Escherichia coli (STEC) serotypes on lettuce plants, demonstrated differential microbial activity from indigenous microflora, suggesting that elevated microbial activity in organic soils was responsible for pathogen suppression. Various methods to extract DNA from soil were tested. Metagenome analysis supports the hypothesis that bacteria more abundant in organic soils are from species closely related to the pathogens and thereby reduce pathogen survival. A manuscript is in preparation. In a project to identify genes of Listeria monocytogenes involved in the attachment to and growth on cantaloupe and lettuce (Sub-objective 1F), high quality DNA preparations were made from 20 different transposon library mutant pools. The pools are enriched for strains that are poor in attachment and growth in three different outbreak strain backgrounds. The DNA preparations are ready for Illumina Mi-Seq testing and analysis. The effect of lettuce accession, growing season, agricultural practices, and storage temperature on the microbiome of stored cut lettuce was determined (Sub-objective 1G). Important microbiome trends were identified, and results are being written for publication. Additionally, we tested the effect of systemic plant resistance on S. Senftenberg and S. Typhimurium colonization inside lettuce cells (apoplast). Treatment with both chemical elicitors and microbial root colonists that trigger hypersensitivity responses in plants inhibited Salmonella in the lettuce apoplast. This correlates with enhanced and sustained expression of plant basal defense genes in the lettuce. A manuscript was submitted for publication. Candidate ligands for Human Norovirus (HuNoV) binding to Romaine lettuce were isolated using bacteria engineered to display HuNoV capsid proteins (Sub-objective 1I). Ligand structures were analyzed by Electrospray Ionization-Mass Spectrometry, and the predicted structure is a chimera of Human Blood Group Antigen (HBGA) type A and Lewis Antigen Type A with a molecular weight of 1042 Daltons. HBGA-like molecules were also identified in some commensal bacteria strains isolated from lettuce. Expression of HBGA in bacteria could be enhanced when the bacteria were cultured in a low nutrient medium. Two manuscripts were published. Metagenome analysis on six sediments from the Salinas region that contained STEC (Sub-objective 2A) were completed in collaboration with Georgia Institute of Technology. The results were comparable to similar metagenomic surveys in sediments from various parts of the world. We calculated that the six metagenomes were significantly different from one another and the content was moderately correlated with rainfall. The metagenomes also contained large numbers of antibiotic resistance and production genes, indicating that the sediment acts as a reservoir of these genes. A manuscript was published. Research continued on performing comparative genomics analysis of environmental STEC strains (Sub-objective 2D). We completed genome sequences of two environmental STEC O121 strains carrying a non-H19 antigen (O121:H10 and O121:H7), as well as completing draft genomes for 34 environmental STEC O121:H19 strains. Comparative analysis of clinical STEC O121:H19 genomes with the environmental STEC O121 genomes is in progress to identify important genetic loci that can define the virulence lineage of O121:H19. A manuscript is in preparation. Salmonella and L. monocytogenes isolates from the Salinas region continue to be characterized (Sub-objective 2E). Whole Genome Sequencing (WGS) of nearly 1000 L. monocytogenes strains were completed in collaboration with the Food and Drug Administration, and there is genomic subtype information for 1,231 L. monocytogenes strains isolated from the region. The second most common subtype is ST382, isolated 223 times in a five-year survey of public access waters of the Salinas region. ST382 was responsible for three produce-related outbreaks in the last six years. A match to one of those outbreak strains (the 2014 stonefruit outbreak) was identified from a sample taken in 2014. ST382 is widespread and endemic in the region. A manuscript is in preparation. The genomes of 120 STEC strains isolated from the Salinas region were sequenced and used for Whole Genome Multi-Locus Sequence Typing (wgMLST) analysis (Sub-objective 3B). The strains were isolated from a variety of environmental locations, sources, and species. Results from the wgMLST subtyping method gave improved resolution compared to our previous subtyping methods, but in most cases wgMLST confirmed our previous genetic clustering data. With the additional WGS data, proper criteria for new wgMLST analysis of newly described strain clusters is being determined. Using the wgMLST data we demonstrated pathogen transport in the environment, and it appears that long-range transport of STEC appears to occur in the Salinas region, primarily through domestic and wild animals. Research continued to examine bacterial factors that impact genome and virulence evolution of STEC (Sub-objective 3C). We are studying evolution of virulence by measuring different strains’ abilities to express dormant, lysogenic shiga toxin converting (stx)-phages in the genomes and transfer them to stx-negative recipient strains. These phages are induced by stresses, and we are using mitomycin C in this work. We have encountered technical difficulties in this approach and are screening for additional stx-phage donor strains. We found additional strains in our work under Sub-objective 2D and are marking these new strains with antibiotic resistance to use in new experiments. There were technical difficulties in using droplet digital polymerase chain reaction (ddPCR) and quantitative reverse transcription PCR (RT-qPCR) in the assays developed to measure infectivity and inactivation of Tulane Virus (TV) and HuNoV (Sub-objectives 3D and 3E). Therefore, we developed a new assay based on DNA aptamers and immunochromatography. Aptamers were developed to target the S-domain of VP1 proteins, which varies in different strains of HuNoV. Primers and probes for RT-qPCR were re-designed based upon updated data of viral genome sequences in GenBank. Using WGS, we subtyped HuNoV strains from clinical samples. One manuscript was published, and three additional manuscripts were submitted. Research continued to establish a quick screening method via spectrophotometer to facilitate phage isolation and to determine the antimicrobial activities of isolated phages (Objective 4). Approximately 28 new phages lytic against STEC were isolated from water, soil, sediment, and fecal samples collected from an organic farm. Phages with strong antimicrobial activity were characterized and sequenced. A lyophilization method was developed for long-term phage storage. In a lysogenic phage study, seven different STEC strains (including O45, O103, and O157 strains) were sequenced to find virulence factors common to Stx-converting prophages, and based on high sequence similarities of virulence factors, we found that clinical E. coli O45:H2 was genetically close to E. coli O103:H2 in comparison with environmental E. coli O45:H16 strains. We found that disodium ethylenediaminetetraacetic acid (EDTA) and ultraviolet (UV) irradiation could induce both Stx1 and Stx2 prophages from these new strains. Additionally, both Stx-converting phages could infect non-pathogenic E. coli and form new lysogens. WGS characterization of both Stx1 and Stx2 converting phages showed that the Stx2-converting bacteriophage contained more genes associated with induction and transduction processes, contributing to a higher self-induction rate of Stx2-converting bacteriophages from their STEC hosts. We continued the development and enhancement of phage-based detection technologies (Objective 5). A patent for the portable phage-based electrochemical biosensor for the detection of viable Shiga toxin-producing Escherichia coli (STEC) was issued. To optimize for a suitable commercial platform, the phage-based liquid reagent was converted into a pre-made, dry format by freeze-drying without compromising viability. Once rehydrated, the reagent can be directly used with biofunctionalized disposable chips for screening STEC strains (O26, O157, O179). The modified biosensor chips were also preserved by using simple sugars and air-drying, resulting in glassy sugar films that maintain the viability and stability of phages on the chips for two months. The detection system was wirelessly connected via Bluetooth to an Android device and application for data processing and readouts. Both approaches, which aimed to enhance commercialization potential, were recognized by USDA as a winner of an Innovation Fund Award. Another method developed is a prototype of a closed-enrichment system that can simultaneously enrich and colorimetrically detect low-level foodborne pathogen contaminants in food samples. The leak-proof container prototype allows the addition of colorimetric reagents for the detection of foodborne pathogens and a biocide for minimizing risks of contamination and infection for end-users in the field. Further optimization is needed.

1. Induced systemic resistance inhibits Salmonella colonization of lettuce. Outbreaks of foodborne disease linked to lettuce continue to impact public health as well as U.S. lettuce production, which is valued annually at nearly $2 billion. Mitigation strategies to prevent microbial contamination of crops are lacking. Evidence is increasing that enteric pathogens can reach the interior space of leaves where they confront broad plant immune responses. In collaboration with scientists at The Volcani Center and the University of Tel Aviv, Israel, researchers at ARS, Albany, California, demonstrated that root treatment of lettuce and basil plants with chemical elicitors or microbial root colonists that induce plant systemic resistance also reduced internal colonization by two Salmonella serovars. These findings reveal that enhanced plant defenses can act as an intrinsic hurdle against the establishment of foodborne pathogens in leafy vegetables. This technology provides sectors of the lettuce and basil industry with a sustainable approach to enhance the microbial safety of their products.

2. Prototype for enrichment and colorimetric detection of foodborne pathogens. Traditional in-house foodborne pathogen detection methods and biochemical analysis take more than a week to generate confirmatory results. Though currently available antibody and molecular-based methods on the market have shorter turn-around time, they are generally expensive and have issues such as cross-reactivity and insensitivity. ARS researchers in Albany, California, and a cooperative research and development agreement partner developed a prototype of a closed-enrichment system that can simultaneously enrich and colorimetrically detect low-level foodborne pathogen contaminants in food samples. By incorporating bacteriophages and other essential components into a leak-proof prototype container, the time needed for enrichment and foodborne pathogen detection can be reduced to under three hours without expensive equipment. This system will assist regulatory agencies, food producers, and processors to ensure a safe food supply.

3. Reduced fitness of Shiga toxin-producing E. coli strains on lettuce growing in organic maintained soil. Manure-amended soils, commonly encountered with organic farming methods, continue to represent a potential hazard in leafy greens production. ARS scientists from Albany, California, and collaborators from the University of California, Davis, completed a three-year study that monitored the effect of organic vs conventional soils on the survival of six different serotypes of Shiga toxin-producing Escherichia coli (STEC) on contaminated lettuce. In soils collected from four time periods during the growing season, the lettuce grown in organic soils demonstrated a significant suppression of STEC levels on the lettuce. The organic soils have significantly higher microbial activity than the conventional soils, suggesting that elevated microbial activity in organic soils was responsible for pathogen suppression on lettuce. These results indicate that the presence of one or more bacteria, which occupy a similar niche with STEC in organic soils, are likely competitive with STEC and may effectively reduce the perception of hazards associated with use of animal manures with organic lettuce cultivation. This information is of importance to growers and regulators for promotion of food safety of produce.

4. Endemic subtypes of Listeria monocytogenes in public access waters of the Central California Coast. Listeriosis, a foodborne illness caused by Listeria monocytogenes, has a high fatality rate, and is a major concern in the United States. The last decade has seen an increase in the number of outbreaks and product recalls due to L. monocytogenes contamination of fresh produce. In a five-year survey of L. monocytogenes prevalence in public access waters of a major agricultural region of the Central California Coast, ARS researchers in Albany, California, found a 42% prevalence of a diverse population of L. monocytogenes strains. The second most common genomic subtype identified belonged to Sequence Type 382, which is an emerging strain in the United States, that has been responsible for three large produce-related outbreaks in the last five years. Clonal Complex 1 strains, a hypervirulent subtype responsible for outbreaks world-wide for many years, are the third most common genomic subtype in the region. This information is important for growers, food processors, regulators, and public health agencies to monitor locations and subtypes of pathogens in the region that that the potential to contaminate pre-harvest produce.

5. Impact of agricultural runoff on STEC and antibiotic resistance gene prevalence. Shiga toxin-producing Escherichia coli (STEC), which is transmitted to humans mainly by water or food, is an important group of foodborne pathogens implicated in over 270,000 cases of human illnesses annually in the United States. STEC naturally reside in cattle, and little is known about the human public health risks associated with it in natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. ARS researchers at Albany, California, applied metagenomics and culture-based tests to detect STEC from the water-sediment interface of two creeks in the Salinas River Valley of California. Shiga toxin-encoding genes were not detected directly in the metagenomes of samples that were culture-positive for STEC, indicating that STEC was present at very low levels in those sediments; however, a high number of antibiotic resistance genes were detected in all samples. Furthermore, there were no significant differences in the abundance of human or cow-specific microbiome sequences between the control and sampling sites, implying natural dilution of the human inputs. This study provides parameters including sampling volumes and sequencing depth for public health experts when using metagenomics for studies of STEC in environmental samples.

6. Detecting intact Shiga toxins in complex samples. Shiga toxin-producing Escherichia (E.) coli (STEC) are important foodborne pathogens and are responsible for the gastrointestinal illness of 270,000 American consumers yearly. Shiga toxins are large multicomponent proteins and the primary virulence factor of STEC. ARS scientists in Albany, California, developed a mass spectrometry-based method of detecting a characteristic portion of one subunit of Shiga toxins. This new method was combined with previously developed methods to detect intact Shiga toxins instead of using cell- or animal-based assays. This method allows researchers to rapidly detect the presence of intact Shiga toxins in complex materials, such as serum and bacterial growth media.

7. Lytic bacteriophages as biocontrol agents and alternative to antibiotics for STEC. Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains have been increasingly associated with foodborne outbreaks in the United States, resulting in 81% of all STEC gastroenteritis and 32% of Hemolytic Uremic Syndrome cases. ARS researchers in Albany, California, isolated and characterized three novel viruses (bacteriophage) that kill STEC strains of serotypes O45 and O103. These bacteriophages showed strong antimicrobial effects, high stability in a wide temperature range, and high stability in a wide pH range. Importantly, these bacteriophages lack virulence genes, and they effectively kill the target STEC host (O45 or O103) within 6 hours of treatment at room temperature. These bacteriophages provide an alternative solution for food producers and processors to control STEC in pre-harvest and post-harvest settings, and also an alternative to traditional antibiotics for clinical treatments.

8. Identification of HuNoV-binding substrates on lettuce leaves. Human Norovirus (HuNoV) is the most common cause of diarrheal illness in the United States, and HuNoV-contaminated lettuce has caused several outbreaks and sporadic illness. In humans, HuNoV binds to Histo Blood Group Antigens (HBGA), but it is unclear how the virus attaches to and accumulates on pre-harvest lettuce leaves, since HuNoV is difficult to grow in the laboratory for direct studies. ARS researchers in Albany, California, in collaboration with scientists at Shanghai Jiao Tong University and Shanghai Institute of Technology in China, showed that bacteria altered to carry the HuNoV capsid protein (P protein) could be used to capture candidate HuNoV receptors present both on lettuce and on bacteria that live on the surface of lettuce. The scientists identified a HBGA-like oligosaccharide on the lettuce leaves as well as a substance produced by natural lettuce-resident bacteria, indicating that HuNoV has multiple binding sites on pre-harvest lettuce surfaces. This new information on HuNoV binding to produce surfaces provides information on these binding compounds provide protection and a means of transmission of HuNoV to consumers, and this research is important to public health professionals, growers, and researchers developing interventions to reduce HuNoV contamination.

9. “Mis-matched” anti-repressor can silence the expression of Shiga toxin genes in E. coli. Foodborne infections cause a huge burden to global public health, and Shiga toxin-producing E. coli (STEC) causes over two million acute illnesses annually worldwide. Strains of STEC vary in pathogenicity, and genetic markers of hyper-virulent, adulterating strains are critically needed for rapid detection and risk assessment of STEC by the food industry and regulatory agencies. ARS researchers in Albany, California, identified a previously unknown mechanism controlling the expression of Shiga toxin in E. coli. A swap of the gene encoding an anti-repressor and mutations in the DNA sequence for binding of that anti-repressor to the regulatory region of Shiga toxin production genes led to a block in the production of Shiga toxin. This study suggests that not only virulence genes, but also regulatory factors associated with virulence genes, are important in STEC risk assessment. This information is essential for public health risk assessments for food safety.

10. An effective lyophilization method for long-term storage of bacteriophages. Lytic bacteriophages have economic value as an alternative to antibiotics for combatting bacterial pathogens. After isolation of bacteriophages, it is very important to have an efficient storage system to prevent degradation and maintain their original viability for downstream analysis and applications. ARS researchers in Albany, California, developed an effective lyophilization (freeze-drying) method to preserve bacteriophages. A population of lyophilized (freeze-dried) phages, which were isolated as specific for Shiga toxin-producing Escherichia coli, was not significantly reduced after six months of storage regardless of the use of cryoprotectants (substances that prevent damage during freezing). This technology provides a valuable way to effectively preserve the phages for downstream development of novel biocontrol interventions for food safety.

11. Development of multiple assays for the detection of Human Norovirus. Human Norovirus (HuNoV) is the most common cause of diarrheal illness in the United States, and sensitive detection of different subtypes is needed for diagnostic tests and foodborne outbreak tracing. ARS researchers in Albany, California, in collaboration with scientists at Shanghai Jiao Tong University, developed several assays to detect (HuNoV). The first is a rapid lateral flow assay using antibodies to detect HuNoV, and this assay is sensitive and specific. The second assay is based on binding of HuNoV to a DNA molecule (called an “aptamer”), and primers and probes from an established assay were re-designed to detect more strains of HuNoV that have arisen due to natural changes in HuNoV surface components. Finally, a next generation sequencing method was used to fingerprint HuNoV from a foodborne outbreak and this method was able to detect multiple genotypes of HuNoV in the same patient. This study provides evidence of foodborne outbreaks with multiple types of HuNoVs and raises new concerns for diagnosis and vaccine development, which is important information for public health risk assessors.

Review Publications
Zhang, Y., Liao, Y., Sun, X., Wu, V.C.H. 2020. Is Shiga toxin-producing Escherichia coli O45 no longer a food safety threat? The danger is still out there. Microorganisms. 8(5):782.
Liu, F., Liao, Y., Li, R.W., Wu, V.C. 2019. Complete genome sequence of Escherichia coli phage vB_EcoM Sa157lw, isolated from surface water collected in Salinas, California. Microbiology Resource Announcements. 8:e00718-19.
Liu, D., Zhang, Z., Liao, N., Zou, S., Tang, H., Tian, P., Wu, Q., Wang, D. 2019. Culturable bacteria resident on lettuce might contribute to accumulation of human noroviruses. International Journal of Food Microbiology. 317.
McGarvey, J.A., Tran, T.D., Hnasko, R.M., Gorski, L.A. 2019. Use of phyllosphere associated lactic acid bacteria as biocontrol agents to reduce salmonella enterica serovar poona growth on cantaloupe melons. Journal of Food Protection. 82(12):2148-2153.
Carter, M.Q., Pham, A.C., He, X., Hnasko, R.M. 2020. Genomic insight into natural inactivation of Shiga toxin 2 production in an environmental Escherichia coli strain producing Shiga toxin 1. Foodborne Pathogens and Disease. 17(9):555-567.
Hnasko, R.M., McGarvey, J.A., Lin, A.V. 2019. Rapid detection of staphylococcal enterotoxin-B by lateral flow assay. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 38(5):209-212.
Munther, D.S., Carter, M.Q., Aldric, C.V., Ivanek, R., Brandl, M. 2020. Formation of E. coli O157:H7 persister cells in the lettuce phyllosphere and application of differential equation models to predict their prevalence on lettuce plants in the field. Applied and Environmental Microbiology. 86(2):e01602-19.
Suttner, B.J., Johnston, E.R., Orellana, L.H., Rodriguez, L.M., Hatt, J.K., Carychao, D.K., Carter, M.Q., Cooley, M.B., Konstantinidis, K.T. 2020. Metagenomics as a public health risk assessment tool in a study of natural creek sediments influenced by agricultural and livestock runoff: potential and limitations. Applied and Environmental Microbiology. 86(6):e02525-19.
Carter, M.Q., Pham, A.C., Carychao, D.K., Cooley, M.B. 2019. Complete genome sequences of two Shiga toxin-producing Escherichia coli strains isolated from crows. Microbiology Resource Announcements. 8(45):e01082-19.
Sun, X., Hao, L., Xie, Q., Lan, W., Zhao, Y., Pan, Y., Wu, V.C. 2019. Antimicrobial effects and membrane damage mechanism of blueberry (Vaccinium corymbosum L.) extract against Vibrio parahaemolyticus. Food Control. 111:107020.
George, A.S., Rehfuss, M.Y., Parker, C., Brandl, M. 2020. The transcriptome of Escherichia coli O157:H7 reveals a role for oxidative stress resistance in its survival from predation by Tetrahymena. FEMS Microbiology Ecology. 96(3).
Melotto, M., Brandl, M., Jacob, C., Jay-Russell, M.T., Micallef, S.A., Warburton, M.L., Van Deynze, A. 2020. Breeding crops for enhanced food safety. Frontiers in Plant Science. 11:428.
Zhang, Y., Liao, Y., Salvador, A., Sun, X., Wu, V.C. 2020. Investigating the whole-genome sequences of a new locus of enterocyte effacement-positive Shiga toxin-producing Escherichia coli O157:H7 strain isolated from river water. Microbiology Resource Announcements. 9(12):e00112-20.
Lennon, M., Liao, Y., Salvador, A., Lauzon, C.R., Wu, V.C. 2020. Bacteriophages specific to Shiga toxin-producing Escherichia coli exist in goat feces and associated environments on an organic produce farm in Northern California, USA. PLoS One. 15(6):0234488.
Milczarek, R.R., Vilches, A.M., Olsen, C.W., Breksa III, A.P., Mackey, B.E., Brandl, M. 2020. Physical, microbial, and chemical quality of hot-air-dried persimmon (diospyros kaki) chips during storage. Journal of Food Quality. 2020.
Gorski, L.A., Rivadeneira, P., Cooley, M.B. 2019. New strategies for the enumeration of enteric pathogens in water. Environmental Microbiology Reports. 11(6):765-776.
Liu, D., Zhang, Z., Wu, Q., Tian, P., Geng, H., Wang, D. 2020. Redesigned duplex RT-qPCR for detection of GI and GII human noroviruses. Engineering. 6(4):442-448.
Zhang, Z., Liu, D., Wu, Q., Lu, Y., Tian, P., Wang, Z., Wang, D. 2019. Characterization of a histo-blood group antigen-like substance in romaine lettuce that contributes to human norovirus attachment. Journal of Agricultural and Food Chemistry. 68(5):1207-1212.
Zhang, Y., Liao, Y., Salvador, A., Sun, X., Wu, V.C. 2020. Prediction, diversity, and genomic analysis of temperate phages induced from Shiga toxin-producing Escherichia coli strains. Frontiers in Microbiology. 10:e3093.