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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Molecular Characterization of Foodborne Pathogens Research » Research » Research Project #430258

Research Project: Molecular Characterization of Foodborne Pathogen Responses to Stress

Location: Molecular Characterization of Foodborne Pathogens Research

2018 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
This report documents progress for 8072-42000-082-00D, Molecular Characterization of Foodborne Pathogen Responses to Stress, which falls under National Program 108. The research focuses on using omics technologies and systems biology to understand how foodborne pathogens tolerate stresses encountered in food environments and how food processing conditions may induce resistance to stresses. The research also focuses on identifying food and animal reservoirs for emerging foodborne pathogens. The research will provide information for understanding how these pathogens cause disease, as well as for identification of genetic markers for detection and typing. Related to Objective 1, progress was made in gaining a better understanding of the mechanism of acid tolerance in Shiga toxin-producing E. coli (STEC) O157:H7 and non-O157 STEC. The ability to survive low pH conditions results in a low infectious dose and the ability to overcome low pH conditions in food that normally should inactivate the pathogens. To gain a better understanding of genes involved in acid tolerance, the genomes of >25 strains of E. coli O157:H7, consisting of both acid tolerant and acid sensitive strains, were sequenced. Gene expression studies were also performed on a representative set of acid sensitive and acid tolerant strains. Overall, results showed that a complex mechanism regulates acid tolerance in E. coli O157:H7. For example, a comparison of gene expression analyses of acid sensitive and acid resistant strains indicated that a gene called csg (curli adhesive fimbriae) and another called hde (acid induced chaperone) were responsible for the characteristics of the strains when exposed to acid conditions. These genes may serve as suitable targets for development of interventions to control STEC. In addition, E. coli strains that caused a laboratory infection were sequenced using three different DNA sequencing platforms, and the sequence data were combined and analyzed. The strains included those that were used in the laboratory experiments, as well as the same strains isolated from the scientist/patient. It was determined that the antibiotics that were administered to the patient during hospitalization caused an increase in the production of the dangerous Shiga toxins, and this led the patient to acquire hemolytic uremic syndrome and encephalitis. For this work, an enzyme-linked immunoassay developed in collaboration with a CRADA partner was used to quantify the level of Shiga toxins, and with collaborators at the University of Texas, the results were confirmed by using a Shiga toxin gene expression assay. In collaboration with CRADA partners, other research focused on developing a DNA sequence-based platform known as AgriSeq, using PCR primers specific for each E. coli O-group and H-type, as well as several virulence genes (stx, eae, and extra-intestinal E. coli virulence genes) as a method for molecular serotyping and virulotyping (presence of specific set of virulence genes) of E. coli. Related to Objective 2, the research is currently focused on three separate areas: the primary work detailed in the five-year plan, work in collaboration with scientists at the Food Safety Inspection Service (FSIS) addressing outbreaks of Campylobacter infection associated with chicken liver, and work in collaboration with researchers at Villanova University under a Material Transfer Research Agreement, developing new surface disinfecting products. Work pertaining to the 5-year plan continues to progress well, and the sub-objectives for the 24th month milestones have been at least substantially met. Specifically, a method for investigating Campylobacter attachment onto poultry skin under processing and storage conditions has been fully developed and utilized. Intervention techniques for reducing Campylobacter numbers in chicken liver have been investigated and reported in the literature. This has produced specific recommendations for the FSIS to consider for mitigation of disease outbreaks associated with chicken liver. Finally, several novel multi-cationic quaternary ammonium compounds have been identified that produce equal or greater reductions in Campylobacter numbers as compared to commercial products at the same concentrations. Listeria monocytogenes is a major foodborne pathogen that causes a serious human illness known as listeriosis, and the pathogen is relatively resistant to commonly used inactivation treatments. Related to Objective 3, significant progress was made towards understanding the survival mechanisms of L. monocytogenes with exposure to organic acids. The level of gene expression in L. monocytogenes was measured with exposure to organic acids using a technology known as RNA-Seq. Global profiling of gene expression of L. monocytogenes allowed us to to identify highly expressed and repressed genes, which reflect the pathogen’s response to the experimental conditions (exposure to organic acids). This information provides information essential to determine specific mechanisms and genes required for growth or survival in the selected food-related stress conditions. Progress was also made towards understanding the survival mechanisms of L. monocytogenes with treatment with olive leaf extract (OLE). OLE is a natural material that has antimicrobial properties, and it is also used as an herbal supplement due to its health promoting properties. Thus, due to these characteristics, OLE is a novel material that can be utilized to control pathogenic bacteria in food. It was demonstrated that OLE inhibited growth of foodborne pathogens, as well as formation of biofilms (aggregates of bacteria attached to a surface), and therefore, OLE has the potential to be used as a natural antimicrobial to control foodborne pathogens in food and the food environment. Bacterial cells treated with sub-lethal doses of olive leaf extract were used to study the gene expression profile of L. monocytogenes. In addition, through a Material Transfer Research Agreement with the Baruch S. Blumberg Institute, over 1,500 plant extracts were screened for their ability to inactivate L. monocytogenes. Several extracts showed antimicrobial activity, and the active compound in one extract was purified and characterized. This compound and others that are being examined can be used in formulations of antimicrobial products/disinfectants for the inactivation of L. monocytogenes in food environments.

4. Accomplishments
1. A method to identify E. coli strains based on specific genetic sequences. Traditionally, a procedure called serotyping has been used to distinguish among the >180 different E. coli O-serogroups and 53 H-types (O-polysaccharide antigen and H-flagellar antigen are E. coli cell surface structures); however, this procedure can only be performed in specialized laboratories, and it is laborious and often inaccurate. To develop more rapid and simple methods for detection, typing, and identification of E. coli belonging to all of the different types and to identify the specific virulence genes (genes associated with causing disease) the strains carry, ARS researchers at Wyndmoor, Pennsylvania sequenced the genomes of E. coli reference O-group strains, determined the DNA sequence of the cluster of genes involved in production of cell surface polysaccharides that define the different E. coli O-serogroups, and the sequences were deposited in the GenBank DNA sequence public database. Working with CRADA partners, unique genetic regions that can be targeted in methods to identify the different O- and H-groups have been determined. Based on this genetic information, a molecular DNA sequencing-based platform known as AgriSeq was developed to test for the presence of O- and H-group genes, as well as virulence genes in E. coli strains. The accuracy of the method was tested with all of the reference strains, as well as with other strains isolated from humans, animals, and the environment. This new molecular method is inexpensive, will greatly enhance the ability to identify, detect, and type E. coli, and will eliminate the use of the labor-intensive and inaccurate traditional serotyping procedure.

2. Understanding the survival mechanism of Listeria monocytogenes with exposure to organic acids. L. monocytogenes is an important foodborne pathogen that causes listeriosis associated with high mortality rates, and furthermore, this pathogen can survive antimicrobial treatments that are normally used during food processing. Organic acids such as lactic acid and diacetic acid have been applied to control L. monocytogenes in ready-to-eat (RTE) meat; however, the mechanism used by L. monocytogenes to adapt to exposure to organic acids remains unclear. ARS researchers in Wyndmoor, Pennsylvania used a procedure known as RNA sequencing (RNA-Seq) to determine the genes that are affected in L. monocytogenes with exposure to levels of sodium lactate normally used during food processing. Genes involved in transport of nutrients into the cell and bacterial movement and attachment, as well as a number of other genes were affected. These results reflect the pathogen’s response to the environmental conditions and provide information essential to determine specific mechanisms required for growth or survival in the selected food-related stress conditions. This study provides insight on the adaptation mechanism of L. monocytogenes with treatment of sodium lactate and will aid in developing more effective strategies to control L. monocytogenes in RTE meat.

3. The effectiveness of gamma irradiation to inactivate Campylobacter jejuni in chicken liver. Recent outbreaks linked to undercooked chicken liver contaminated with C. jejuni necessitate the development of a safer liver product. Intervention methods to reduce the numbers of C. jejuni present on an uncooked chicken liver would reduce the number of disease cases resulting from a contaminated product that may be consumed undercooked. ARS researchers in Wyndmoor, Pennsylvania showed that application of gamma irradiation was successful in reducing Campylobacter numbers on or in the liver without any visual or undesirable changes to the liver product. When irradiation was followed by cold storage, the level of irradiation needed to decrease C. jejuni numbers to undetectable levels was significantly reduced. This work represents the first time that irradiation has been used to inactivate C. jejuni present in chicken liver and therefore has produced novel information with regards to treatment dose and bacterial reductions, as well as the interaction this treatment has with typical food storage conditions. This work is of considerable interest and value to poultry producers, as well as consumers. Since the current methods for processing chicken liver is likely to continue to result in disease outbreaks, this new inactivation method is essential for producing a liver product that is safe for consumers.

4. The effectiveness of high hydrostatic pressure to inactivate Campylobacter jejuni in chicken liver. Recent outbreaks of disease caused by C. jejuni contamination in undercooked chicken liver has necessitated the development of methods to reduce C. jejuni numbers in processed chicken liver. High hydrostatic pressure processing has been used successfully for reducing bacterial counts in a variety of different food products. ARS researchers in Wyndmoor, Pennsylvania showed that a range of high pressure treatments of chicken liver produced only modest reductions in C. jejuni levels. Additionally, the pressure treatments produced undesired changes in the appearance of the liver at the highest pressures tested; therefore, high pressure treatments were also performed in conjunction with reduced temperature storage. With this combination treatment, the reductions in C. jejuni numbers increased; however, they still did not reach the desired levels. This work demonstrated that high pressure treatment in conjunction with cold storage is more effective than high pressure treatment alone; however, this combination treatment will not be sufficient to significantly reduce the number of outbreaks. This work is of considerable interest and value to poultry producers and food safety researchers who will need to include additional technologies in conjunction with high pressure/cold storage treatment if this method is to be utilized as a primary intervention for increasing the safety of chicken liver.

Review Publications
Smith, J., Fratamico, P.M. 2018. Escherichia coli pathotypes. Pathogenic E. coli: Evolution, Omics, Detection, and Control – Caister Academic Press, United KingdomBook Chapter.
Gehring, A.G., Fratamico, P.M., Lee, J., Ruth, L., He, X., He, Y., Paoli, G., Stanker, L.H., Rubio, F.M. 2017. Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples. Food Control. 77:145-149.
Rotundo, L., Fratamico, P.M., Amagliani, G., Carloni, E., Omiccioli, E., Magnani, M. 2018. Comparison of the Diatheva STEC FLUO with BAX system kits for detection of O157:H7 and Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) in ground beef and bean sprout samples using different enrichment protocols. Journal of Food Analytical Methods.
Baranzoni, G., Fratamico, P.M., Kim, G., Reichenberger, E.R., Funk, J., Manning, S. 2017. Genome sequences of 34 Shiga toxin-producing E. coli isolated from swine and other sources. Genome Announcements.
Gurtler, J., Doyle, M.P., Kornacki, J.L., Fratamico, P.M., Gehring, A.G., Paoli, G. 2017. Advantages of virulotyping foodborne pathogens over traditional identification and characterization methods. Foodborne Pathogens Virulence Factors and Host Susceptability. New York, NY: Springer Publishing. p. 3-40.
Uhlich, G.A., Reichenberger, E.R., Cottrell, B.J., Fratamico, P.M., Andreozzi, E. 2017. Whole-genome sequence of Escherichia coli serotype O157:H7 strain B6914-ARS. Genome Announcements.
Amagliani, G., Rotundo, L., Carloni, E., Omiccioli, E., Magnani, M., Brandi, G., Fratamico, P.M. 2018. Detection of Shiga toxin-producing Escherichia coli (STEC) in food: evaluation of culture enrichment conditions. Food Research International. 103:398-405.
Zhang, X., Ashby, R.D., Solaiman, D., Liu, Y., Fan, X. 2017. Antimicrobial activity and inactivation mechanism of lactonic and free acid sophorolipids against Escherichia coli O157:H7. Biocatalysis and Agricultural Biotechnology. 11(C):176-182. doi: 10.1016/j.bcab.2017.07.002.
Liu, Y., Yoo, B., Hwang, C., Suo, Y., Sheen, S., Khosravi, P., Huang, L. 2017. LMOf2365_0442 encoding for a fructose specific PTS permease IIA may be required for virulence in L. monocytogenes Strain F2365. Frontiers in Microbiology. 8:01611.
Suo, Y., Gao, S., Xie, Y., Liu, Y., Qu, Y., Shen, Y., Zhou, C. 2017. A multi-pathogen selective enrichment broth for simultaneous growth of Salmonella enteria, Escherichia coli O157:H7 and Shigella flexneri. Journal of Food Safety. doi:10.1111/jfs.12388.
Gunther, N.W., Reichenberger, E.R. 2017. Complete genome sequence of Campylobacter jejuni RM1246-ERRC that exhibits resistance to Quaternary Ammonium Compounds. Genome Announcements.
Suo, Y., Gao, S., Baranzoni, G., Xie, Y., Liu, Y. 2018. Comparative transcriptome RNA-Seq analysis of Listeria monocytogenes with sodium lactate adaptation. Food Control. 91:193-201.