Research Project: Molecular Characterization of Foodborne Pathogen Responses to Stress

Location: Characterization and Interventions for Foodborne Pathogens

2019 Annual Report

Objectives
1: Molecular characterization of Shiga-toxin producing Escherichia coli (STEC) and extra-intestinal pathogenic E. coli (ExPEC) with specific emphasis elucidating the responses to food-related stresses, and genomic and proteomic studies to assess virulence and to identify genetic markers for detection and typing. 1A: Perform molecular characterization of acid tolerance in STEC. 1B: Perform molecular characterization of ExPEC. 1C: Develop molecular genoserotyping and pathotyping platforms for E. coli. ID: Characterization of STEC isolates from swine. 1E: Develop and evaluate immunologic-based methods for detection of STEC. 2: Genomic and proteomic analysis of Campylobacter with emphasis on virulence and the molecular characterization of the effects of acidification and other food-processing related stresses on survival Campylobacter in poultry products. 2A: Determine composition and effects that different poultry exudates play in the survival of the contaminating Campylobacter species. 2B: Investigate attachment and formation of biofilms by Campylobacter species on poultry skin in the presence of different poultry exudates. 2C: Investigate practical methods, chemical and microbiological based, for acidification of poultry exudate and their effects on the survival of contaminating Campylobacter spp. 3: Functional and molecular characterization of L. monocytogenes serotypes with emphasis on elucidating responses to food-related stresses through functional genomics; and determining virulence differences among L. monocytogenes strains and serotypes through comparative genomics. 3A: Determine strain variations in growth/survival with exposure to weak organic acids and olive leaf extracts among different L. monocytogenes serotypes. 3B: Determine genes that are essential for the survival and growth of L. monocytogenes under weak organic acid conditions in RTE meat. 3C: Investigate molecular responses of L. monocytogenes exposed to the olive leaf extracts using transcriptomics.

Approach
The goal of this project is to use omic technologies (proteomic, genomic, and transcriptomics methods) and bioinformatics in a systems approach to understand how pathogens become resistant to food-related stresses, to determine their pathogenicity, and to identify markers for detection and typing. Pathogens that will be investigated include: Shiga toxin-producing Escherichia coli (STEC) and extraintestinal pathogenic E. coli (ExPEC), Campylobacter species, and Listeria monocytogenes. We will use omic technologies to analyze a large variety of strains of each of the pathogens to identify genes and proteins necessary for pathogens to survive stresses encountered in food environments and cause human illness. Research on pathogenic E. coli will focus on examining the association between acid tolerance in STEC and virulence potential, curli expression, biofilm formation, and persistence. This work will provide information to understand the virulence characteristics of STEC and how food environment-related conditions may impact the virulence and persistence in the food environment. We will examine poultry and swine as reservoirs for food-borne infections linked to ExPEC and STEC, respectively, and characterize isolated strains to determine their virulence. The omic data will also reveal genetic markers for identification, molecular typing, and detection of these pathogens. In previous work, we found that the use of certain polyphosphates commonly used during poultry processing increased the survival of Campylobacter by causing subtle changes in pH. Building on our previous research, we will investigate strain diversity and mechanisms of tolerance to stresses, including acid and exposure to antimicrobial compounds, as well as investigate factors affecting attachment and biofilm formation of Campylobacter. In addition, there has been limited effort to identify the microbial makeup of poultry and the processing environment and how these may provide a survival advantage for Campylobacter. Thus, we will investigate environmental stresses that affect the survival and persistence of Campylobacter during poultry processing and the role that the microbial ecology of this environment plays in this process. Finally, we will examine stress responses in L. monocytogenes and explore novel approaches to control this pathogen and determine the genes and proteins that help the pathogen overcome stresses. Genes that are essential for the survival and growth of L. monocytogenes under weak organic acid conditions in RTE meat will be determined. We will also investigate the effect of olive leaf extracts on inactivation of L. monocytogenes, and using transcriptomics, we will determine the molecular responses of this pathogen when exposed to the olive leaf extracts. The research will expand the knowledge on the survival mechanisms of important food-borne pathogens, provide insight into the evolution of pathogens, as well as tools to detect, identify, and type food-borne pathogens, and will assist in the development of practical preservation systems that minimize health risks and assist regulators in making science-based food safety decisions.

Progress Report
This report documents progress for the parent project 8072-42000-082-00D, Molecular Characterization of Foodborne Pathogen Responses to Stress, which falls under NP108. The project plan was approved (from 2016 to 2021), and this past year, progress was made on all three main objectives. The research focuses on using omics technologies to understand how food-borne pathogens tolerate stresses encountered in food environments and how food processing conditions may induce resistance to stresses. The research is also focused on identifying food and animal reservoirs for emerging foodborne pathogens, and work will provide information for understanding how these pathogens cause disease in humans and identification of genetic markers for detection and typing. Related to Objective #1 of the project plan, significant progress was made in developing a diagnostic method for molecular serotyping of E. coli. Traditionally, serotyping has been used to distinguish >180 different E. coli O-serogroups (O-antigen) and 53 H-types (H-flagellar antigen) based on cell surface structures). However, this procedure, while laborious and often inaccurate, can only be performed in specialized laboratories. In our research, more than 70 genomes of E. coli reference O-group strains were analyzed to determine the DNA sequence of the cluster of genes involved in production of cell surface polysaccharides that define the different E. coli O-serogroups. This research was intended to develop more rapid and simple methods for detection, typing, and identification of different serogroups of pathogenic E. coli. Working with CRADA partners, we determined the unique genes that can be targeted to identify the different O- and H-groups. Based on this genetic information, a molecular DNA sequencing-based platform known as AmpliSeq was developed to determine the presence of O- and H-group genes, as well as virulence genes (involved in causing disease) in E. coli strains. The specificity of the method was tested with all the E. coli reference strains and the other field strains isolated from humans, animals, and the environment. The new developed molecular method is inexpensive and will greatly enhance the ability to identify, detect, and type pathogenic E. coli and will eliminate the use of the labor-intensive and inaccurate traditional serotyping procedure. Other work focused on characterizing hundreds of extraintestinal pathogenic E. coli (ExPEC) strains that were isolated from human and poultry in collaboration with another ARS scientist at Wyndmoor, Pennsylvania. ExPEC that are present in produce, poultry and meat can cause illness in humans. Molecular techniques were used to trace the association between foodborne ExPEC and human diseases. Significant progress was made on determining the prevalence of ExPEC in poultry. Genetic-based PCR methods were used to characterize different ExPEC strains. The genome sequence of several ExPEC isolates from human and chicken was determined, which is a critical first step to understand the epidemiology of ExPEC in humans and chicken and their potential to cause illness. The information is important for development of strategies to control ExPEC and prevent contamination from poultry products. Work pertaining to Objective #2 continues to progress well and all the subobjectives for the 36-month milestones have been at least substantially met. Specifically, the effectiveness of irradiation, high pressure processing and cold storage interventions for reducing Campylobacter numbers in poultry products was determined resulting in two publication. Campylobacter strains with different biofilm forming potential were identified and a direct relationship between the amounts of biofilm a Campylobacter strain can produce and the bacteria’s ability for movement using their flagellar organelle was established. Campylobacter strains that were strong swimmers also produced greater amounts of biofilm compared with Campylobacter that had poor swimming movement and produced less biofilm material. Reducing the bacteria’s ability to swim using flagella with a specific chemical also reduced the Campylobacter’s ability to form biofilms. Finally, we have described the potential use of acidic whey as a wash to reduce Campylobacter numbers on poultry products. Whey is an edible byproduct resulting from the manufacture of cheese. Whey is known to be acidic and is rich in a variety of probiotics including lactic acid bacteria. Initial studies have shown that whey solutions were able to produce large reductions (>5 logs) in Campylobacter numbers, even when applied as a minor component. Related to Objective #3 of the project plan, significant progress was made towards understanding the survival mechanisms of L. monocytogenes after exposure to olive leaf extract (OLE). Beneficial to human health, OLE is an herbal supplement with antimicrobial properties. Therefore, OLE was explored as a natural antimicrobial to control foodborne pathogens in food. Our results showed that OLE inhibited growth of L. monocytogenes in milk. The survival of L. monocytogenes in milk with different concentrations of OLE was conducted to determine the inactivation rates and the optimal dose of OLE to use for gene expression analyses. The RNA-Seq method was used to measure the level of gene expression in L. monocytogenes after exposure to sub-lethal dose of OLE. Increased or decreased expression of several genes have been identified, which provides the information essential to understanding the specific mechanisms and genes required for growth /survival in food-related stress conditions. This information is also necessary for the design of interventions that will allow complete inactivation of L. monocytogenes. Additional research showed that OLE can be used as an antimicrobial film to inhibit the growth of foodborne pathogens, indicating the potential use of OLE as food packaging material, which will be further explored in the future. Significant progress was also made in search for new antimicrobials for use as food additives and preservatives to inactivate pathogens and to increase the shelf life of foods. Compared to synthetic compounds, plant extracts are generally more likely to be accepted as generally recognized as safe (GRAS), may have a lower cost, and are typically more eco-friendly. In this project cycle, over 1000 plant extracts provided by the Baruch S. Blumberg Institute through a Material Transfer Research Agreement (MTRA) were screened for inhibition of growth of L. monocytogenes. A plant extract from Stenotus armerioides (Thrift mock golden weed) was identified with antimicrobial activity to L. monocytogenes. This plant extract was further fractionated, and an active compound was purified, identified, and characterized. The Minimal Inhibition of Concentration (MIC) of this compound was comparable to the known antibiotics used in the clinical studies. To further confirm this result, the compound was synthesized in vitro, which showed the same antimicrobial activity as the natural plant extract. In addition, results from Ames test showed that this compound did not possess mutagenic activity. Taken together, our results showed that thrift mock golden weed extract has the potential to be used a sanitizer or packaging material in the food industry to control foodborne pathogens such as L. monocytogenes. Further research will focus on antimicrobial screening for growth inhibition of more foodborne pathogens such as Shiga toxin-producing E. coli (STEC), and Salmonella spp. This research is being conducted in collaboration with Blumberg Institute and Villanova University.

Accomplishments
1. Whole genome sequencing for understanding the antibiotic sensitivity of Shiga toxin-producing E. coli (STEC). Shiga toxin-producing E. coli (STEC) can cause serious outbreaks and sporadic cases of food-borne illness. STEC strains belonging to serogroup O111 are harmful pathogens associated with food. The current methods for detection of STEC in food need novobiocin as a selective agent in the enrichment media and selective agars to prevent the growth of background bacteria. However, novobiocin also inhibits the growth of some STEC, particularly STEC O111, which may lead to false-negative detection results. ARS scientists at Wyndmoor, Pennsylvania, first determined the genome sequence (an organism’s complete set of DNA) of seven STEC O111 strains with different sensitivities to novobiocin. The genes or alterations in the genome involved in novobiocin sensitivity were identified, and the genome sequences were deposited to GenBank, a public gene sequence database. This information is important for understanding the characteristics of this pathogen at the molecular level for development of enrichment media and selective agars that will improve detection of STEC O111 and prevent the release of contaminated food products to the consumers.

2. Understanding the nature and behavior of L. monocytogenes strains through DNA sequencing. Listeria monocytogenes is an important foodborne pathogen that causes listeriosis associated with high mortality rates. L. monocytogenes is very difficult to control in the food industry since it can survive under very harsh conditions such as high salt, low pH, and low temperature. ARS scientists in Wyndmoor, Pennsylvania determined the genome sequence of seven strains of L. monocytogenes that varied in their ability to cause disease and response to stresses to gain a better understanding about how to control this pathogen. Thus, these sequences were also compared to determine the genes involved in virulence (disease causing ability) and stress responses. The genome sequences were deposited in a GenBank sequence database that can be accessed by the public. The information obtained from this research helps in the design of strategies to control L. monocytogenes in food, and potentially in the development of more effective therapeutic approaches.

3. Olive leaf extract as a natural antimicrobial compound for the food industry. There is a need for novel methods to control pathogenic bacteria in the food industry. Olive leaf extract (OLE) is often used as an herbal supplement and is considered beneficial to human health. ARS researchers in Wyndmoor, Pennsylvania studied the application of OLE as an antimicrobial agent for controlling major foodborne pathogenic bacteria, such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis, and Staphylococcus aureus. The research showed that an antimicrobial film prepared with OLE was able to inhibit growth of foodborne pathogens. Therefore, OLE has the potential to be used as a natural antimicrobial food packing material to control foodborne pathogens in food and the food processing environment.

4. Biofilm formation and flagella for Campylobacter spp. Campylobacter bacteria form biofilms that allow them to persist on foods and in food processing environments increasing their potential to cause human disease. We identified Campylobacter strains that formed either strong or weak biofilms. Next we determined that the ability to form strong biofilms correlated with the ability of Campylobacter to move using hair-like appendages called flagella. A chemical was identified that stopped the bacteria’s movement using the flagella. The same chemical was also shown to reduce Campylobacter biofilm formation. Our work had demonstrated that flagella play an important role in Campylobacter biofilm formation and have identified a chemical that can interfere with the functionality of flagella and reduce biofilm formation. Our research has provided a new approach for reducing Campylobacter biofilms which could lead to reductions in the numbers of Campylobacter found on food products and in food production facilities. Fewer Campylobacter will mean fewer sick consumers.

Review Publications
Smith, J., Gunther, N.W. 2019. Commentary: Campylobacter and hemolytic uremic syndrome. Foodborne Pathogens and Disease. 16(2):90-93. https://doi.org/10.1089/fpd.2018.2513.
Gunther, N.W., Abdul Wakeel, A.Y., Scullen, O.J., Sommers, C.H. 2019. The evaluation of gamma irradiation and cold storage for the reduction of Campylobacter jejuni in chicken livers. Food Microbiology. 82:249-253. https://doi.org/10.1016/j.fm.2019.02.014.
Accumannola, G.M., Richards, V.A., Gunther, N.W., Lee, J. 2018. Purification and characterization of the thermostable metalloprotease produced by Serratia grimesii isolated from channel catfish. Journal of the Science of Food and Agriculture. 99:2428-2437. https://doi.org/10.1002/jsfa.9451.
Bobokalonov, J., Liu, Y., Shahrin, T., Liu, L.S. 2018. Transcriptomics analysis on the regulation of tomato ripening by the ethylene inhibitor 1-methylcyclopropene. Journal of Plant Studies. 7(2):49-60. https://doi.org/10.5539/jps.v7n2p49.
Liu, Y., Xu, A., Fratamico, P.M., Sommers, C.H., Rotundo, L., Boccia, F., Jiang, Y., Ward, T.J. 2018. Draft whole genome sequence of seven L. monocytogenes strains with variation in virulence and stress responses. Microbiology Resource Announcements. 7(13). https://doi.org/10.1128/MRA.01038-18.
Rotundo, L., Boccia, F., Fratamico, P.M., Xu, A., Sommers, C.H., Liu, Y., Bono, J.L., Pepe, T. 2018. Draft genome sequences of seven strains of Shiga toxin-producing Escherichia coli O111 with variation in their sensitivity to novobiocin. Microbiology Resource Announcements. 7(10). https://doi.org/10.1128/MRA.01030-18.
Smith, J., Fratamico, P.M. 2018. Emerging and re-emerging zoonotic food-borne pathogens. Foodborne Pathogens and Disease. 15(12). https://doi.org/10.1089/fpd.2018.2493.
Gunther, N.W., Abdul Wakeel, A.Y., Ramos, R.V., Sheen, S. 2019. The evaluation of hydrostatic high pressure and cold storage parameters for the reduction of Campylobacter jejuni in chicken livers. International Journal of Food Microbiology. 82:249-253. https://doi.org/10.1016/j.fm.2019.02.014.
Andreozzi, E., Gunther, N.W., Reichenberger, E.R., Cottrell, B.J., Rotundo, L., Nunez, A., Uhlich, G.A. 2018. Pch genes control biofilm and cell adhesion in a clinical serotype O157:H7 isolate. Frontiers in Microbiology. 9(2829). https://doi.org/10.3389/fmicb.2018.02829.
Patel, I.R., Gangiredla, J., Lacher, D.W., Mammel, M.K., Bagi, L.K., Baranzoni, G., Fratamico, P.M., Roberts, E.L., Debroy, C., Lindsey, R.L., Stripling, D., Martin, H., Smith, P., Strockbine, N.A., Elkins, C.A., Scheutz, F., Feng, P.C. 2018. Interlaboratory evaluation of the FDA-ECID microarray for profiling Shiga toxin-producing Escherichia coli. Journal of Food Protection. 81:1275-1282.