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United States Department of Agriculture

Agricultural Research Service

Research Project: Genomic and Proteomic Analysis of Foodborne Pathogens

Location: Molecular Characterization of Foodborne Pathogens

2013 Annual Report


1a.Objectives (from AD-416):
1. Conduct a functional and molecular characterization of Shiga-toxin producing Escherichia coli (STEC) with specific emphasis elucidating the responses to food-related stresses, and genomic and proteomic studies to assess changes in virulence and pathogenicity. 1A: Comparative phylogenomics and phenomics of non-O157 STEC. 1B: Examine and compare stress responses, including acid tolerance, in E. coli O157:H7 and non-O157 STEC. 1C: Role of SdiA in acid tolerance of STEC O157:H7 and non-O157 STEC. 1D: Molecular serotyping of E. coli. 1E: Methods for detection and identification of non-O157 STEC.

2: Conduct functional and molecular characterization of Campylobacter species with specific emphasis on responses to intrinsic and extrinsic stresses through genomic and proteomic studies, and examination of morphological and physiological changes. 2A: Determine the “mode of action” by which polyphosphates (extrinsic stress) enhance the survival of C. jejuni and C. coli strains. 2B: Use genomic and/or proteomic studies to molecularly characterize Campylobacter’s physiological response to food additives under poultry processing conditions. 2C: Determine if members of the microbial ecology of chicken exudates provide survival advantages/disadvantages to Campylobacter. 2D: Determine if common food additives change the composition of the microbial ecology of chicken exudate and if these changes are responsible for enhancing the survival of Campylobacter under food processing and storage conditions.

3: Conduct functional and molecular characterization of Listeria monocytogenes serotypes with specific emphasis on elucidating responses to food-related stresses through proteomics and genomics; and determining virulence differences among L. monocytogenes serotypes through sequencing and comparative genomics. 3A: Determine genes that are essential for the survival and growth of L. monocytogenes under weak organic acid conditions. 3B: Determine genetic responses of a pressure-resistant L. monocytogenes mutant exposed to the food preservative nisin. 3C: Determine genes responsible for the differences in virulence and stress responses among L. monocytogenes serotypes through sequencing, gene expression, and comparative genomics.


1b.Approach (from AD-416):
The overall goal of this project is to apply comparative genomic/proteomic/phenomic technologies to understand how pathogens become resistant to food-related stresses and to uncover the genetic basis of their virulence. Three major food-borne pathogens will be investigated: Shiga toxin-producing Escherichia coli (STEC), Campylobacter species, and Listeria monocytogenes. A combination of “omics” techniques, including transcriptomics, comparative genomics, proteomics, and phenotypic arrays will be employed to analyze a large variety of strains of each of these pathogens to identify genes and proteins necessary for them to survive stresses encountered in food environments and to identify genes/mobile genetic elements necessary for them to cause human illness. Comparative genomic and gene expression techniques will be used to assess the virulence profiles of highly pathogenic non-O157 STEC strains and to determine genes responsible for the differences in virulence and stress responses among L. monocytogenes serotypes. STEC, Campylobacter spp., and L. monocytogenes will be exposed to food environments and food-processing related stresses, including acid, high pressure, exposure to antimicrobial compounds, and other stresses. In addition, we will investigate environmental stresses that affect the survival and persistence of Campylobacter spp. during poultry processing and the role that the microbial ecology of this environment plays in this process. The mechanism by which polyphosphates enhance the survival of C. jejuni and C. coli strains will be determined, and genomic and proteomic techniques will be used to molecularly characterize the physiological response of Campylobacter to food additives under poultry processing conditions. It will also be determined if members of the microbial ecology of chicken exudates provide survival advantages/disadvantages to Campylobacter. The microbiological and molecular data will aid in the development of practical preservation systems that minimize health risks and assist regulators in making science-based food safety decisions. The “omic” data will also reveal biomarkers useful for identification, molecular typing, and detection of the pathogens. Methods and platforms for molecular serotyping of E. coli and for detection and identification of non-O157 STEC will be developed. The research will expand our knowledge on the survival mechanisms of important food-borne pathogens, will provide insight into the evolution of pathogens, provide the tools to detect, identify, and type food-borne pathogens, and ultimately lead to better control strategies for STEC, Campylobacter, and L. monocytogenes in food.


3.Progress Report:
Progress was made on all three objectives and sub-objectives of this project (falls under National Program 108) that focuses on the use molecular technologies to understand how pathogens become resistant to food-related stresses, which may influence their disease causing potential (virulence). Researchers at the Eastern Regional Research Center are examining food-borne pathogens isolated from various sources by comparative analyses to understand which genes and proteins are important for the pathogens to cause disease and to survive stresses encountered in food environments. Genes that are biomarkers of virulence and genes specific to E. coli serogroups are being utilized for developing methods for molecular serotyping of E. coli and for detection of Shiga toxin-producing E. coli (STEC). The methods are being used for regulatory testing for STEC. Additionally, platforms for molecular serotyping of E. coli are being developed since the DNA sequence of sets of genes important for identification of the ca. 180 different serogroups of E. coli were determined. Substantial progress was also made in understanding the genes that are important for STEC virulence by DNA sequencing of STEC strains and by comparative analyses using bioinformatic techniques. Finally, a variety of STEC were isolated from swine, and the strains were characterized. A novel workflow technique that allows for enhanced separation and identification of bacterial proteins was developed and employed to compare the proteins expressed by an E. coli O157:H7 strain and a mutant of this strain that produces high levels of fibers important in biofilm formation (aggregates of bacteria that form on surfaces). This proteomic technique will be an essential part of research projects aimed at understanding how Campylobacter, E. coli, or other food-borne pathogens are able to overcome environmental stresses relevant to food settings. Furthermore, the effect of food-grade polyphosphates, added to processed chickens on survival of Campylobacter was assessed. It was determined that use of polyphosphate during poultry processing caused changes in pH and enhanced the survival of Campylobacter during typical processing and storage conditions. Although there has been considerable microbiological research aimed at understanding stress responses in L. monocytogenes in foods, there is only a rudimentary understanding of these responses at the molecular level, which is needed to design effective control strategies. Progress was made in understanding growth of L. monocytogenes in food and in determining the genes involved in regulating the response to nisin (compound known as a bacteriocin that inactivates L. monocytogenes) and high pressure treatments. Results demonstrated that the expression of certain genes was appreciably altered by high pressure and nisin treatments. Several genes that may be involved in stress responses and biofilm formation were identified through the production and analysis of strains lacking these genes (mutants). These data provide insight into the mechanisms of survival of L. monocytogenes in food.


4.Accomplishments
1. Development of tools to enhance the ability to detect non-0157 Shiga toxin-producing E, coli (STEC). Non-0157 Shiga toxin-producing E. coli (STEC) are import food-borne pathogens, and developing methods to rapidly and reliably detect these pathogens in food has been a challenge. To protect the public, six non-0157 E. coli known as 026, 045, 0103, 0111, 0121, and 0145 serogroups were declared as adulterants in raw beef by the USDA Food Safety and Inspection Service (FSIS). To comply with FIS testing protocols, components required for rapid and accurate testing were developed in collaboration with a company with which ARS researchers at Wyndmoor, Pennsylvania developed a CRADA. The materials that were developed and commercialized known as latex agglutination tests (LAT) and immunomagnetic separation (IMS) beads were used for efficient isolation, concentration, and identification of the target STEC from food samples. The developed LAT and IMS tests are rapid and sensitive and display a very high selectivity for the six STEC bacteria. Use of these tests will dramatically enhance the ability of regulatory agencies and the food industry worldwide to detect non-0157 STEC in food.

2. Method to detect and isolate Shiga toxin-producing (STEC) E. coli belonging to serogroup 0104. STEC belonging to serogroup 0104 caused a large serious outbreak in Germany in 2011 affecting over 4000 people and one of the first STEC outbreaks in the U.S. was caused by STEC 0104 associated with milk. Since methods for detection and isolation of STEC 0104 in food are not readily available, ARS researchers at Wyndmoor, Pennsylvania utilized materials developed with a CRADA partner (latex agglutination tests and immunomagnetic beads targeting STEC 0104) to design rapid and sensitive detection and isolation methodologies for STEC 0104. These methods can be used for detection of different subgroups of the pathogen in various types of food and will be useful for the food industry and regulatory agencies worldwide to monitor for this important STEC serogroup. Related studies involved DNA sequencing of two strains of STEC 0104, and the information obtained is useful in understanding how this pathogen causes illness and for identifying genetic markers for detection.

3. Use of specific compounds known as polyphosphates during poultry processing increases Campylobacter survival but does not affect inactivation of the pathogen. Campylobacter species are responsible for a large number of cases of food-borne illness annually worldwide; however, these pathogens do not survive well in food processing and storage environments. It is therefore important to understand what factors contribute to the ability of Campylobacter to survive in sufficient numbers to cause such a large amount of human illnesses and to develop technologies for inactivation of the pathogen in food. ARS researchers at Wyndmoor, Pennsylvania previously identified a food safety risk factor in the use of compounds known as polyphosphates during poultry processing for the primary purpose of enhancing moisture retention in the poultry product. Use of these polyphosphates increased the survival of Campylobacter species by changing the acidity level during poultry processing and storage conditions. Specific polyphosphates that are less of a food safety risk since they do not enhance Campylobacter survival were identified. Furthermore, it was determined that a technology known as flash freezing is effective for decreasing levels of Campylobacter in poultry, and the presence of polyphosphates do not interfere with the inactivation using this technology. This research provides valuable information for the design of control strategies to decrease the risk of human illnesses associated with Campylobacter.

4. Use of rapid detection methods to determine the prevalence of Shiga toxin-producing E. coli and Salmonella in beef. Rapid and simple screening methods that can be used by the food industry to simultaneously monitor for different food-borne pathogens can reduce testing costs. Shiga toxin-producing E. coli (STEC) and Salmonella are important food-borne pathogens commonly associated with beef, and rapid and sensitive methods for detection of these pathogens are needed to determine their prevalence in beef and to ensure food safety. In collaboration with CRADA partners, ARS researchers at Wyndmoor, Pennsylvania employed rapid polymerase chain reaction-based methods to test for the prevalence of these pathogens naturally present in retail ground beef. STEC were detected in beef samples and Salmonella was detected and isolated from over 9 percent of the ground beef samples. This study demonstrated the utility of the rapid test methods for simultaneous detection of STEC and Salmonella and provided information on the prevalence of these pathogens in ground beef.

5. Understanding the role of specific proteins in E. coli involved in responses to acid environments. E. coli strains that cause serious illness are generally very resistant to acid conditions that may be encountered in food and in the human host; therefore, understanding the mechanism of this resistance will help in the development of control strategies. A novel method was developed by ARS researchers at Wyndmoor, Pennsylvania to compare the proteins produced by E. coli O157:H7 strains (or other bacteria) in response to acid treatments. Various proteins were identified that played a role in the ability of this pathogen to respond to acid environments, and there was a relationship between expression of these proteins and production of surface structures on E. coli that are important for attachment to the human intestine and formation of biofilms (complex aggregates of microorganisms attached to surfaces). This research has uncovered a previously unknown pathway within the complex network of protein interactions in E. coli O157:H7 that allows the pathogen to survive in acid environments and to cause illness.

6. Understanding how Listeria monocytogenes is able to survive treatments used for its inactivation. Listeria monocytogenes is an important food-borne pathogen, and it is difficult to eliminate since it can form a biofilm (a complex aggregation of microorganisms growing on a solid surface), and it is generally resistant to treatments that are used for pathogen inactivation in food. ARS researchers at Wyndmoor, Pennsylvania performed genetic analyses of L. monocytogenes growing in food and identified several genes that may play a role in resistance of the pathogen to stresses, including acid and salt stress, encountered in food environments. To understand the function of these genes, they were deleted from L. monocytogenes, creating mutant strains. Our results showed that the mutant strains grew differently under different stress conditions compared to the parent strain. In addition, one of the mutant strains showed increased capacity for biofilm formation. Information from this study enhances the understanding of the role of specific proteins in L. monocytogenes under stress conditions and provides information to help in the development intervention strategies.

7. A strategy to control Listeria monocytogenes using nisin and high pressure. Listeria monocytogenes is an important food-borne pathogen, and it is difficult to eliminate since it can form biofilms (complex aggregates of microorganisms attached to surfaces). Furthermore, Listeria is generally resistant to treatments that are used for pathogen inactivation in food. Nisin is an antimicrobial compound, known as a bacteriocin, that can be used to control Listeria monocytogenes in food, and high hydrostatic pressure has also been used to inactivate L. monocytogenes in food. However, Listeria can become resistant to these inactivation treatments, and how this resistance occurs is not known. Studying a naturally-occurring strain of L. monocytogenes that was tolerant to high pressure treatment but showed increased sensitivity to nisin, ARS researchers at Wyndmoor, Pennsylvania demonstrated that a combination of high pressure and nisin can be applied to effectively control L. monocytogenes in food. This study revealed one of the mechanisms of how Listeria becomes resistant to nisin, and this knowledge will contribute to the design of safe and economically feasible treatments to inactivate L. monocytogenes during food processing.


Review Publications
Smith, J.L., Liu, Y., Paoli, G. 2012. How does Listeria monocytogenes combat acid conditions?. Canadian Journal of Microbiology. 59:141-152.

Feng, P., Strockbine, N., Fratamico, P.M. 2012. Methods of detection and characterization of pathogenic Escherichia coli. In: Eds.UNESCO-EOLSS Joint Committee. Encyclopedia of Life Support Systems. Oxford,UK: Eolss Publishers. Available: http://www.eolss.net.

Smith, J.L., Fratamico, P.M. 2012. Effect of stress on non-O157 Shiga toxin-producing Escherichia coli. Journal of Food Protection. 75(12):2241-2250.

Liu, Y., Morgan, S.L., Ream, A.R., Huang, L. 2012. Gene expression profiling of a nisin-sensitive Listeria monocytogenes Scott A CtsR deletion mutant. Journal of Industrial Microbiology and Biotechnology. 40(5):495-505.

Fratamico, P.M., Bagi, L.K. 2012. Detection of non-O157 STEC in ground beef using the GeneDisc real-time PCR system. Frontiers in Cellular and Infection Microbiology. DOI: 10.3389/fcimb.2012.00152.

Liu, Y., Ceruso, M., Pepe, T., Cortesi, M., Gunther, N.W., Fratamico, P.M. 2012. Construction of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette (ABC) transporters and analysis of their growth under stress conditions. Journal of Microbial and Biochemical Technology. 4(7):141-146.

Fang, T., Liu, Y., Huang, L. 2013. Growth kinetics of Listeria monocytogenes and spoilage microorganisms in fresh-cut cantaloupe. Food Microbiology. http://dx.doi.org/10.1016/j.fm.2012.12.05.

Suo, Y., Huang, Y., Liu, Y., Shi, C., Shi, X. 2012. The expression of superoxide dismutase (SOD) and a putative ABC transporter permease is inversely correlated during biofilm formation in Listeria monocytogenes 4b G. PLoS One. 7:e48467.

Tillman, G., Wasilenko, J.L., Simmons, M., Lauze, T., Miniocozzi, J., Oakley, B., Narang, N., Fratamico, P.M., Cray, W. 2012. Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121 and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment. Journal of Food Protection. 75:1548-1554.

Yan, X., Gurtler, J., Fratamico, P.M., Hu, J., Juneja, V.K. 2012. Phylogenetic identification of bacterial MazF toxin protein motifs among probiotic strains and foodborne pathogens and potential implications of engineered probiotic intervention in food. BioMed Central(BMC) Cell & Bioscience. 2:39.

Hu, Y., Meng, J., Shi, C., Hervin, K., Fratamico, P.M., Shi, X. 2013. Characterization and comparative analysis of a second thermonuclease in Staphylococcus aureus. Microbiological Research. 168(3):174-182.

Last Modified: 9/2/2014
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