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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

2017 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 the parent project 8072-42000-082-00D, Molecular Characterization of Foodborne Pathogen Responses to Stress, which falls under NP108. A portion of this work continues research from project 8072-42000-070-00D, Genomic and Proteomic Analysis of Foodborne Pathogens. The project plan was approved in February 2016, and this past year, progress was made on all three main objectives. The research focuses on using omics technologies and systems biology 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, as well as for identification of genetic markers for detection and typing. Related to objective #1 of the project plan, progress was made in gaining a better understanding of the mechanism of acid tolerance in Shiga toxin-producing E. coli (STEC) O157:H7. 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 and compared. 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. For example, to survive under acid stress conditions, acid sensitive strains express curli fimbriae, which allows the cells to form biofilms, providing protection and facilitating survival. Gene expression analyses of acid-sensitive and acid-resistant strains indicated that csg (curli adhesive fimbriae) and hde (acid induced chaperone) genes positively correlated with the phenotypic difference between sensitive and resistant strains. Related work showed that 7 other types of STEC could also tolerate acidic conditions in food as well or better than O157:H7, thus, non-O157:H7 STEC can be as dangerous as O157:H7, and control strategies should have the ability to inactivate all highly acid tolerant STEC. This research enhances the understanding of the disease-causing potential of STEC and the mechanism of acid tolerance and provides information to develop strategies to control these harmful pathogens in food. In addition, E. coli strains that caused a laboratory infection were sequenced and analyzed (the two mutant strains used in the laboratory, the parent strains, and the same strains [the mutants] isolated from the 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 a university in Texas, the results were confirmed by using a Shiga toxin gene expression assay. Continuing with the research on DNA sequencing of the E. coli O-group reference strains, the O-antigen gene cluster sequences of all of the E. coli reference strains were identified from the genome sequences, and O-group-specific primers were designed in collaboration with CRADA partners. A molecular sequence-based O- and H-antigen typing kit was designed, and this method can also determine the presence of specific virulence genes in the E. coli strain. Other work focused on characterizing hundreds of STEC strains that were isolated from pigs through funded grants and a collaboration with scientists at Michigan State University. The strains were characterized using genetic-based methods, and the genome sequence of almost 100 swine, cattle, and human STEC isolates was determined. The results showed that some STEC carried by pigs were similar to STEC associated with human illness and some strains from cattle. Among the different E. coli serogroups isolated from swine, human pathogens STEC O157:H7 and O26:H11, carrying genes that are involved in human illness were also isolated from swine. This work represents the largest comprehensive study on swine STEC in the U.S., and the data provide a critical first step to enhance the understanding of the epidemiology of STEC in swine and of the potential of swine STEC to cause illness. The information is important for development of strategies to control STEC shedding on farms and prevent contamination of pork products. Finally, the performance of a commercial kit for detection of STEC was compared to methods used by the Food Safety and Inspection Service (FSIS) for regulatory testing. The ability of the new commercial kit to detect STEC in food was similar to that of the kits used as part of the FSIS method. Related to Objective #2, the only 12-month milestone was to complete the collection of poultry exudates at the production and retail levels. Two exudate samples of significant volume were collected from production sources and two were collected from retail sources. This collection of exudates should be sufficient for experimental use to meet the subsequent milestones in sub-objective 2A. Work was continued to expand and characterize the laboratory collection of Campylobacter jejuni, and Campylobacter coli strains isolated from environmental and clinical sources. For ten of these Campylobacter strains, whole genome sequencing and characterization was completed. A collaboration with researchers at Villanova University produced a material transfer research agreement (MTRA) focused on investigating the effectiveness on a new class of antimicrobial compounds against Campylobacter strains. The research succeeded in identifying a trait specific to a sub-group of the antimicrobial compounds that increased the compounds’ effectiveness in inactivating Campylobacter strains. Related to Objective #3 of the project plan, significant progress was made towards understanding the survival mechanisms of L. monocytogenes with exposure to organic acids. Organic acids such as lactic acid and diacetic acid have been applied to control L. monocytogenes in ready-to-eat (RTE) meat; however, at concentrations used, they are not fully effective in inactivating all of the Listeria. A growth study of L. monocytogenes in RTE meat with 4% sodium lactate was conducted to determine inactivation rates and also the optimal dose of lactic acid to use for gene expression analyses. Bacterial cells treated with 4% sodium lactate for two days were used to study changes in L. monocytogenes gene expression in RTE meat. Increased or decreased expression of a number of genes occurred, and this information is being used in systems biology analyses to determine how L. monocytogenes may survive in food with exposure to lactic acid. Additional work being planned will further allow the identification of highly expressed and repressed genes, which reflect the pathogen’s response to organic acids and will provide the information essential to determine specific mechanisms required for growth or survival in the selected food-related stress conditions. This information is necessary for the design of interventions that will allow complete inactivation of L. monocytogenes. Additional research showed that olive leaf extract and oleuropein, a major compound found in olive leaf extract had antimicrobial properties against L. monocytogenes and other foodborne pathogens. Further research will explore whether exposure to both organic acids and olive leaf extract will result in a synergistic effect for inactivating L. monocytogenes.

4. Accomplishments
1. Swine as a reservoir for Shiga toxin-producing E. coli (STEC) that cause human illness. Although cattle are considered an important reservoir for STEC, food products from other animal species, including swine have been linked to foodborne illness. However, little is known of the prevalence and fecal shedding of STEC in clinically healthy swine over time. In collaboration with scientists in academia, ARS researchers at Wyndmoor, Pennsylvania conducted longitudinal studies to fill this gap by investigating fecal shedding of STEC in swine raised on conventional farms during the finishing period. A majority of the fecal samples tested were positive for the presence of the STEC Shiga toxin genes, and STEC strains of various types were recovered from a large portion of the pigs. The virulence (disease-causing potential) genes carried by the strains differed, some of the STEC carried by the pigs were similar to those associated with human illness and strains from cattle.

2. Method to identify E. coli strains based on differences in genes involved in production of cell surface structures. Traditionally, a procedure called serotyping has been used to distinguish among the > 180 different E. coli O-serogroups and 53 H-types (O-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 serogroups, 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 genes that can be targeted in genetic-based 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 AmpliSeq was developed to test for the presence of O- and H-group genes, as well as virulence genes (involved in causing disease) in E. coli strains. This new molecular method is inexpensive and 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.

3. Olive leaf extract is a natural compound that has antimicrobial properties. There is a need for novel methods to control pathogenic bacteria in the food supply. Olive leaf extract (OLE) is an herbal supplement that is beneficial to human health. It has antioxidant, as well as antimicrobial properties. Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis, and Staphylococcus aureus are major foodborne pathogens that cause serious human illness. ARS researchers at Wyndmoor, Pennsylvania showed that OLE inhibited growth of these foodborne pathogens, as well as the 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. Oleuropein is the key compound in OLE that has antimicrobial activity.

4. Antimicrobials known as biscationic quaternary ammonium compounds (QACs) are effective at inactivating Campylobacter. Monocationic QACs have been commercially used as disinfectants for many years and represent a significant portion of the cleaning products market. However, resistance to the QACs has steadily developed over the years of usage. In an effort to address the developing resistance, new QACs were constructed with unique structural changes from currently commercially available QACs. Together with collaborators, ARS researchers at Wyndmoor, Pennsylvania showed that a subgroup of these new QACs proved to be more successful at inactivating a major human pathogen, Campylobacter jejuni, compared to currently commercially available QACs. The chemical structure of the commercially available QACs has only one negative charge, and they are classified as monocationic; however, the more successful sub-group of the experimental QACs have two negative charges, and these are known as biscationic. Additionally, the remaining experimental QACs, a mix of tricationic (three negative charges) and tetracationic (four negative charges) structures, were less successful at inactivating C. jejuni cells compared to the monocationic and biscationic QACs. This research has resulted in the development of a new class of disinfectants with effectiveness higher than commercially available QACs, which will help to reduce contamination by Campylobacter and potentially other foodborne pathogens.

Review Publications
Fratamico, P.M., Bosilevac, J.M., Schmidt, J.W. 2017. Methods for detecting pathogens in the beef food chain: an overview. In: Acuff, G., Dickson, J. Ensuring safety and quality in the production of beef. Volume 1: Safety. Cambridge, UK: Burleigh Dobbs Science. p.35-51.
Fratamico, P.M., Bosilevac, J.M., Schmidt, J.W. 2017. Methods for detecting pathogens in the beef food chain: detecting particular pathogens. In: Acuff, G., Dickson, J. Ensuring safety and quality in the production of beef. Volume 1: Safety. Cambridge, UK: Burleigh Dobbs Science. p.59-72.
Beier, R.C., Franz, E., Bono, J.L., Mandrell, R.E., Fratamico, P.M., Callaway, T.R., Andrews, K., Poole, T.L., Crippen, T.L., Sheffield, C.L., Anderson, R.C., Nisbet, D.J. 2016. Disinfectant and antimicrobial susceptibility profiles of the big six non-O157 Shiga toxin-producing Escherichia coli strains from food animals and humans. Journal of Food Protection. 79(8):1355-1370.
Smith, J.L., Fratamico, P.M. 2016. Escherichia coli as other Enterobacteriaceae: food poisoning and health effects. In: Caballero, B., Finglas, P., and Toldra, F. (eds.) Encyclopedia of Food and Health. Oxford: Acad. p. 539-544.
Baranzoni, G., Fratamico, P.M., Reichenberger, E.R., Kim, G., Breidt, F., Kay, K., Oh, D. 2016. Complete genome sequences of Escherichia coli O157:H7 strains SRCC 1675 and 28RC that vary in acid resistance. Genome Announcements. 4:4. doi: 10.1128/genomeA.00743-16.
Gunther, N.W., Reichenberger, E.R., Bono, J.L. 2016. Complete genome sequence of UV-resistant Campylobacter jejuni RM3194, including an 81.08-kilobase plasmid. Genome Announcements. 4(2):e00305-16.
Yu, Q., Niu, M., Yu, M., Liu, Y., Wang, D., Shi, X. 2016. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai. Food Control. 60:263-268.
Kim, G., Fratamico, P.M., Breidt, F., Oh, D. 2016. Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid. Journal of Applied Microbiology. doi: 10.1111/jam.13223.
Li, X., Liu, Y., Jia, Q., Lamacchia, V., O'Donoghue, K., Huang, Z. 2016. A systems biology approach to investigate the antimicrobial activity of oleuropein. Journal of Industrial Microbiology and Biotechnology. 43(12):1705-1717.
Liu, Y., McKeever, L., Malik, N.S. 2017. Assessment of the antimobial activity of olive leaf extract against foodborne bacterial pathogens. Frontiers in Microbiology. doi: 10.3389/fmicb.2017.00113.
Kong, Q., Patfield, S.A., Skinner, C.B., Stanker, L.H., Gehring, A.G., Fratamico, P.M., Rubio, F., Qi, W., He, X. 2016. Validation of two new immunoassays for sensative detection of a broad range of shiga toxins. Austin Immunology. 1(2):1007.
Yoo, B.K., Liu, Y., Juneja, V.K., Huang, L., Hwang, C. 2017. Effect of environmental stresses on the survival and cytotoxicity of Shiga toxin-producing Escherichia coli. Food Quality and Safety. 1(2):139-146. doi: 10.1093/fqsafe/fyx010.
Yoo, B.K., Liu, Y., Juneja, V.K., Huang, L., Hwang, C. 2016. Effects of stresses on the growth and Cytotoxicity of Shiga-Toxin producing Escherichia coli in ground beef and spinach. Journal of Food Science and Technology. 1:1-7.
Yan, R., Liu, Y., Gurtler, J., Fan, X. 2017. Sensitivity of pathogenic and attenuated E. coli O157:H7 strains to ultraviolet-C light as assessed by conventional plating methods and ethidium monoazide-PCR. Journal of Food Safety. doi: 10.1111/jfs.12346.
Edlind, T., Brewster, J.D., Paoli, G. 2017. Enrichment, amplification, and sequence-based typing of Salmonella enterica and other foodborne pathogens. Journal of Food Protection. 80(1):15-24.
Uhlich, G.A., Chen, C., Cottrell, B.J., Andreozzi, E., Irwin, P.L., Nguyen, L.T. 2017. Gene duplication and promoter mutation expand the range csgD-dependent biofilm responses in a STEC population. Microbiology. 163:611-621.
Uhlich, G.A., Paoli, G., Zhang, X., Dudley, E.G., Figler, H.M., Cottrell, B.J., Androzzi, E. 2017. Whole-genome sequence of Escherichia coli serotype O157:H7 strain PA20. Genome Announcements. doi: 10.1128/genomeA.01460-16.