Research Project: Advance the Development of Technologies for Detecting and Determining the Stability and Bioavailability of Toxins that Impact Food Safety and Food Defense

Location: Foodborne Toxin Detection and Prevention Research

2020 Annual Report

Objectives
The overall objective of this project is to advance the development of technologies for detecting toxins that impact food safety and food defense and to determine their stability and bioavailability. Specifically, the project will focus on the following four objectives: Objective 1: Advance the development of structure- and activity-based detection methods for protein toxins. Subobjective 1A: Develop new antibodies (Abs) to botulinum neurotoxin (BoNT) serotype F, with serotypes C, D, and G as secondary priorities. Subobjective 1B: Determine the impact of different types of accessory proteins on the detection of BoNTs. Subobjective 1C: Develop activity-based detection methods for staphylococcal enterotoxin (SE) serotype E. Subobjective 1D: Develop monoclonal antibodies (mAbs) to Shiga toxin (Stx) subtypes and variants, including those from non-E. coli. Objective 2: Advance the development of detection methods for non-bacterial toxins. Subobjective 2A: Develop new detection methods for plant-derived protein toxins such as abrin. Subobjective 2B: Develop new detection methods for mushroom toxins such as amatoxins. Objective 3: Assess foodborne risks through examination of toxin stability and bioavailability in relation to intrinsic and extrinsic stresses. Subobjective 3A: Use activity-based assays to assess impact of food processing, matrices and accessory proteins on toxin activity. Subobjective 3B: Determine the factors that affect the bioavailability of toxins using rodent bioassay. Objective 4: Advance the development of instrumental, portable, and field-deployable testing methods. Subobjective 4A: Develop platforms such as optical array technologies to detect toxins. Subobjective 4B: Utilize instrumental methods to detect toxins based on mass spectra and/or other physicochemical characteristics.

Approach
Objective 1 has 2 general approaches: (1) Exploit ELISA and related technologies because of their versatility, robustness, and sensitivity. The mAbs developed for immunoassay will also be useful for sample preparation and for establishing design criteria for protective antibodies with clinical utility. (2) Develop activity assays for Stx2 variants and SEs. Activity assays will be especially useful to measure toxins in the presence of thermally inactivated and degraded proteins that are expected in processed food samples. Both approaches address practical analytical problems. The following hypotheses will be tested:(a) High affinity mAbs and recombinant Abs for BoNT serotypes and NAPs as analytical targets will provide useful reagents for ELISA and sensor methodology. (b) Neurotoxin-associated proteins (NAPs) influence the physicochemical properties of BoNT complexes, and their ease of detection in food matrices. These effects depend on the types of accessory proteins present. (c) A cell line can be engineered to provide a cell-based activity assay for SEE to measure active toxin in food matrices to replace bioassay and improve upon structure-based immunoassay. (d) New mAbs will be able to distinguish new subtypes of Stx1 or Stx2 produced by non-E. coli bacteria such as Enterobacter cloacae. Objective 2 will exploit immunoassays, especially ELISA and related technologies (as in Objective 1) and also develop and utilize activity assays. The mAbs developed for immunoassay will also have important utility for sample preparation. The following hypotheses will be tested: (a) High affinity variant-specific mAbs will provide useful reagents for ELISA and sensor methodology for detecting nonbacterial protein toxins like abrin. (b) High affinity toxin-specific and group-specific mAbs will provide useful reagents for ELISA and sensor methodology for mushroom toxins. Objective 3 will exploit activity assays and rodent bioassay to better define the vulnerabilities of our food supply and the analytical needs. The following hypotheses will be tested: (a) Food processing conditions, food matrices, and accessory proteins impact toxin activity. (b) The oral bioavailabilities of BoNT and abrin vary among subtypes of toxin and state of the toxin (pure, complexed, crude) and depend on the food matrix. Objective 4 will exploit instrumental and portable technologies for toxin detection. Some of these technologies will will utilize binding molecules and activity assays developed under Objectives 1 and 2. To advance the development of instrumental and field-deployable testing, the following hypotheses will be tested: (a) A robust cell-based activity assay for SEE in a food matrix can be developed using a small fluorescence-detecting charge-coupled device to read data. (b) Mass spectral data and other physicochemical properties are useful for detection of toxin proteins and peptides. Contingency plans are built into the Approach for each objective and sub-objective. For example, contingencies for Objective 1 include use of alternative immunogens, sample preparation strategies, and assay formats.

Progress Report
Under Sub-objective 1A, previous attempts to produce mouse monoclonal antibodies (mAbs) for botulinum neurotoxin serotype F (BoNT/F) did not result in highly sensitive reagents that could be used in immunoassays. ARS researchers at Albany, California, used BoNT/HA (a BoNT/F and BoNT/A chimera) to make new antibodies against BoNT/F. Nine monoclonal antibodies were obtained that recognized the BoNT serotypes F and A. These new antibodies will be further characterized for toxin binding and use in immunoassays. Under Sub-objective 1A, ARS researchers, in collaboration with researchers at the University of California, San Francisco, and the California Department of Public Health, developed novel humanized recombinant antibodies to BoNT/HA produced from a Clostridium botulinum strain isolated from an infant botulism case. These antibodies specifically recognize BoNT/H and not the other seven BoNT serotypes (A-G). The current equine anti-toxin made against BoNT serotypes A-G is not effective against BoNT/H and these new recombinant antibodies would be important for toxin detection as well as used as potential therapeutic treatment. Under Sub-objective 1D, ARS researchers, in collaboration with scientists from the Chinese Centers for Disease Control and Prevention (CDC), isolated nine bacterial strains that produce a novel subtype of Shiga toxin 2 (Stx2), designated Stx2k. Molecular features, pathogenic capacity, and possible mechanism of emergence of new Shiga toxin-producing E. coli (STEC) strains were investigated. Additionally, ARS researchers developed a new polyclonal antibody capable of neutralizing Stx2k and a sandwich enzyme linked immunosorbent assay (ELISA) that can detect as little as 48 pg/mL of Stx2k in saline buffer. The potential virulence of Stx2k was evaluated by comparing its biological properties including cytotoxicity, thermostability, acid tolerance, and receptor binding with the archetypical Stx2a. These studies are important for reducing public risk of infection by less-known STECs. Under Sub-objective 2A, ARS researchers developed new mAbs against the B subunit of abrin toxin. These also recognized the whole abrin toxin. Researchers also showed that most of these mAbs can significantly neutralize abrin toxin’s cytotoxicity in the Vero cell culture model for toxicity. These mAbs will be tested with previously developed antibodies against the A subunit of Abrin to further improve on toxin detection sensitivity and neutralization. Under Sub-objective 2A, ARS researchers investigated the prevalence of mobile colistin-resistance in 5,169 domestic animal-origin samples randomly collected by the USDA Food Safety and Inspection Service (FSIS). A novel ELISA developed in house combined with culture-based antibiotic susceptibility tests and real-time polymerase chain reaction (PCR) were used to screen samples. One colistin-resistant Escherichia coli strain harboring a plasmid-borne mcr-1 gene was isolated from a pork sample, designated EC2492. The phenotypic and genomic features of EC2492 were studied by antimicrobial susceptibility test and whole genome sequence analysis. The transfer ability of the mcr-1 mediated colistin-resistance in E. coli was confirmed by conjugation experiments. This research suggests that continued vigilance to minimize further spread of mobile colistin resistance is warranted. Under Sub-objective 2B, ARS researchers developed an antibody-based dipstick lateral flow immunoassay (LFIA) that sensitively and selectively detects amatoxins. This LFIA was successfully used for the detection of amatoxins from mushroom extracts and in human and dog urine samples. From testing 110 wild mushrooms, the LFIA identified six out of six species that were known to contain amatoxins, while other poisonous mushrooms known not to contain amatoxins tested negative. The detection of amatoxins in urine samples correlated very well with the gold standard liquid chromatography-mass spectrometry (LC-MS) detection methods performed by researchers at the CDC and the University of California, Davis. Under Sub-objective 3B, in collaboration with researchers at Framingham State University in Framingham, Massachusetts, and the Marche Polytechnic University in Ancona, Italy, ARS researchers showed that witch hazel extract interfered with bacterial quorum sensing, biofilm formation and inhibited Staphylococcus enterotoxin A (SEA) toxin production. Furthermore, researchers showed that witch hazel extract can be used as an additive or alternative to antibiotics to inhibit bacterial growth and pathogenesis, even in antibiotic resistant bacteria. Importantly, resistance to witch hazel extract has not been demonstrated. In addition, researchers showed that witch hazel extract, when used in combination with BacStop iodine-based teat dip (DIP) significantly enhanced the antibacterial effect of DIP against bacteria that can cause bovine intramammary infections that play a crucial role in spreading of foodborne bacteria among dairy farms. Under Objective 4, ARS researchers built a simple and low-cost charged-coupled device (CCD) camera to detect active abrin toxin. This cell-based method detected the presence of active abrin as either a color change or a change in the fluorescent glow of cells. The fluorimetric method detected as little as 0.1 pg/mL of active abrin.

Accomplishments
1. Identification and characterization of a new subtype of Shiga toxin 2. Shiga toxin (Stx) is the major virulence factor of Shiga toxin-producing Escherichia coli (STEC). STEC evolve rapidly and new toxin subtypes continue to emerge and thus pose challenges for effective disease management and surveillance strategies. A new Stx, Stx2k, was recently identified in E. coli isolated from a wide range of sources including diarrheal patients, animals, and raw meats, and was poorly detected by existing immunoassays. ARS researchers at Albany, California, elucidated the crystal structure of Stx2k, developed new antibodies and immunoassays that detected as little as 48 pg/mL of Stx2k. Researchers also evaluated the potential virulence of this new toxin, by determining its cytotoxicity, thermal stability, acid tolerance, and receptor-binding, in comparison with the archetypical Stx2a. This information is invaluable in the early detection and control of emerging STECs, which will result in better treatment outcomes.

2. Investigation of mobile colistin-resistance in U.S. animal-origin food. Recently, a mobile colistin resistance gene, mcr-1, was discovered in clinical and animal samples. Occurrence of horizontally transferable antibiotic resistance to one of the last-resort antibiotics could directly threaten human health. However, the prevalence of mcr-1-mediated colistin resistance has never been investigated in U.S. animal products. To fill this gap, ARS researchers at Albany, California, screened over 5,000 domestic food samples, including chicken rinse, ground beef, beef trim, poultry, raw pork and catfish randomly collected by the USDA Food Safety Inspection Service for the presence of mcr-1 using a novel enzyme-linked immunosorbent assay (ELISA) combined with real-time polymerase chain reaction methods. Very low prevalence of mcr-1 gene, 0.24% in raw pork and less than 0.02% in all samples tested was observed. Subsequent whole genome sequence analysis revealed that the mcr-1 gene resided on an IncI2 plasmid. Two other plasmids harboring aminoglycosides and tetracycline resistance and virulence genes, respectively, were also found in the same strain. This is the first systemic and large-scale investigation of mobile colistin-resistance in U.S. food animal products, and the information obtained is useful for trade and food safety risk assessments.

3. Simple and portable test for amatoxins. Amatoxins are lethal toxins found in certain mushrooms. ARS researchers at Albany, California, developed an antibody-based dipstick lateral flow immunoassay (LFIA) that sensitively and selectively detected as little as 10 ng/ml of amatoxins in both mushroom and urine samples without the need for specialized equipment. The detection of amatoxins in human and dog urine samples correlated very well with the gold standard liquid chromatography-mass spectrometry detection methods. The speed of analysis and lack of requirement for trained personnel and expensive instrumentation means that this patent-pending test strip method could be a very useful tool in the diagnosis of amatoxin-specific mushroom poisonings. It is currently being used in animal clinics.

4. New recombinant antibodies against a novel chimeric botulinum neurotoxin serotype HA. Botulinum neurotoxins are some of the most potent toxins known to man and are both public health and food safety concerns. A new botulinum neurotoxin serotype HA (BoNT/HA) has been identified recently from a Clostridium botulinum strain isolated from an infant botulism case and cannot be neutralized with existing anti-toxin countermeasures. Researchers at Albany, California, along with researchers at the University of California, San Francisco and the California Department of Public Health, developed new recombinant antibodies to BoNT/HA. These antibodies specifically recognized serotype H and not any of the other seven BoNT serotypes (A-G). Sensitive and neutralizing antibodies against this new BoNT are important for the development of detection methods as well as potential therapeutics.

5. Simple, low-cost CCD camera system for active abrin detection. Abrin is an extremely potent toxin produced in the seeds of the Rosary Pea (Abrius precatorius) with similar mode of action as the select agent, Ricin. There are many ways to test for the presence of abrin, but few detection assays can distinguish between the active and inactive form of the toxin. ARS researchers at Albany, California, built a simple and low-cost charge-coupled device (CCD) camera and applied it to cell-based assays, that when in presence of active abrin, either produced a color change or a change in the fluorescent glow of cells. The fluorimetric method was able to detect as little as 0.1 pg/mL of active abrin. Simple and inexpensive methods add to the arsenal of tools in the accurate detection of abrin contaminations in cases of intentional food adulterations.

6. Use of witch hazel extract to inhibit bacterial pathogens and enhance the efficacy of current antibiotics. WhISOBAX (WH) is a witch hazel extract that contains high levels of phenolic compounds such as hamamelitannin. ARS researchers in Albany, California, and researchers in Framingham State University in Framingham, Massachusetts, and Marike Polytechnic University in Ancona, Italy, showed that both gram-positive and gram-negative bacteria are sensitive to WH, including those that are notoriously resistant to antibiotic treatment, like Methicillin-resistant Staphylococcus aureus (MRSA). WH was also shown to have anti-biofilm and anti-toxin production properties Additionally, WH enhances the effect of commonly used antibiotics while preventing bacterial pathogenesis, thus enabling eradication of infection while reducing the need of excessive antibiotic use. This discovery provides new options for improved treatment of bacterial infections.

7. Witch hazel dramatically improves the efficacy of commercially available teat dips. Bovine intramammary infections (IMIs) are the main cause of economic loss in milk production. Antibiotics are often ineffective in treating these infections due to antimicrobial resistance and the formation of bacterial biofilms that enhance bacterial survival and persistence. Teat dips containing germicides are often used to prevent IMIs and improve udder health and milk quality. ARS researchers in Albany, California, tested the use of witch hazel extracts with BacStop iodine-based teat dip (DIP) and found that the minimal inhibitory concentration or bactericidal concentrations of DIP against planktonic S. epidermidis cells decreased up to 160-fold and was 10-fold more effective against biofilms. While both DIP and WH are effective in inhibiting the growth of S. aureus, only WH inhibited staphylococcus enterotoxin A production. Importantly, WH also significantly enhanced the antibacterial effect of DIP against gram-negative bacteria that cause IMIs, like Escherichia coli and Pseudomonas aeruginosa. All together, these results showed that WH significantly enhanced the antibacterial activity of DIP, and thus have potential in eradicating bacterial infections, both in acute (planktonic-associated) and in chronic (biofilm-associated) conditions.

Review Publications
Bever, C.R., Swanson, K.D., Hamelin, E.I., Filigenzi, M., Poppenga, R., Kaae, J., Cheng, L.W., Stanker, L.H. 2020. Rapid, sensitive, and accurate point-of-care detection of lethal amatoxins in urine. Toxins. 12(2):123. https://doi.org/10.3390/toxins12020123.
Hughes, A.C., Zhang, Y., Bai, X., Xiong, Y., Wang, Y., Yang, X., Xu, Q., He, X. 2019. Structural and functional characterization of Stx2k, a new subtype of Shiga toxin 2. Microorganisms. 8(1):4. https://doi.org/10.3390/microorganisms8010004.
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. https://doi.org/10.1089/fpd.2019.2767.
Wang, Y., Hou, N., Johnston, J., Sarreal, C., Jarosh, J., Hughes, A.C., Gu, Y.Q., He, X. 2020. Low prevalence of mobile colistin-resistance in U.S. meat, catfish, poultry and genomic characterization of a mcr-1 positive Escherichia coli strain. Food Control. 118. https://doi.org/10.1016/j.foodcont.2020.107434.
Wang, B., Park, B., Chen, J., He, X. 2020. Rapid and label-free immunosensing of Shiga toxin subtypes with surface plasmon resonance imaging. Toxins. https://doi.org/10.3390/toxins12050280.
Tam, C.C., Land, K.M., Cheng, L.W. 2019. Prebiotics, probiotics, and bacterial infections. In: Franco-Robles, E. and Ramírez-Emiliano, J., editors. Prebiotics and Probiotics, Potential Benefits in Nutrition and Health. IntechOpen. https://doi.org/10.5772/intechopen.89052.
Fan, Y., Barash, J.R., Conrad, F., Lou, J., Tam, C.C., Cheng, L.W., Arnon, S.S., Marks, J.D. 2019. The novel Clostridial neurotoxin produced by strain IBCA10-7060 is immunologically equivalent to BoNT/HA. Toxins. 12(1):9. https://doi.org/10.3390/toxins12010009.
Henderson, E., Tam, C.C., Cheng, L.W., Ngono, A.E., Nguyen, A., Shresta, S., McGee, M., Padgett, H., Gill, L., Martchenko, M. 2020. Investigation of the immunogenicity of Zika glycan loop. Virology Journal. 17:43. https://doi.org/10.1186/s12985-020-01313-1.
Yang, X., Bai, X., Zhang, J., Sun, H., Fu, S., Fan, R., He, X., Scheutz, F., Matussek, A., Xiong, Y. 2020. Escherichia coli strains producing a novel Shiga toxin 2 subtype circulate in China. International Journal of Medical Microbiology. 310(1):151377. https://doi.org/10.1016/j.ijmm.2019.151377.
Rasooly, R., Choi, H., Do, P.M., Morroni, G., Brescini, L., Cironi, O., Giacometti, A., Apostolidis, E. 2020. whISOBAXTM inhibits bacterial pathogenesis and enhances the effect of antibiotics. Antibiotics. 9(5):264. https://doi.org/10.3390/antibiotics9050264.
Bever, C.R., Adams, C.A., Hnasko, R.M., Cheng, L.W., Stanker, L.H. 2020. Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms. PLoS One. 15(4). https://doi.org/10.1371/journal.pone.0231781.
Bever, C.R., Hnasko, R.M., Cheng, L.W., Stanker, L.H. 2019. A rapid extraction method combined with a monoclonal antibody-based immunoassay for the detection of amatoxins. Toxins. 11(12). https://doi.org/10.3390/toxins11120724.
Rasooly, R., Molnar, A., Choi, H., Do, P.M., Racicot, K., Apostolidis, E. 2019. In-vitro inhibition of staphylococcal pathogenesis by witch-hazel and green tea extracts. Antibiotics. 8(4):244. https://doi.org/10.3390/antibiotics8040244.