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:
The goal of this project is to use molecular technologies to understand how STEC, Campylobacter, and Listeria monocytogenes, become resistant to food-related stresses and to uncover the genetic basis of their disease causing potential (virulence). Researchers at the ARS in Wyndmoor, PA are examining strains of each pathogen isolated from various sources by comparative analyses to understand which genes and proteins are necessary to cause disease and to survive stresses, including acid stress, encountered in food environments. Also, genes that are biomarkers of virulence and genes specific to E. coli serogroups are being utilized to develop methods for STEC detection and for developing methods for molecular serotyping of E. coli. The USDA FSIS declared six non-O157 STEC serogroups of major public health significance as adulterants in beef. Methods were developed for detection and isolation of the non-O157 STEC, which are being used for regulatory testing by the FSIS. Additionally, platforms for molecular serotyping of E. coli are being developed since the DNA sequences 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 comparative analyses using DNA microarrays and by using bioinformatics techniques. The effect of food-grade polyphosphates, added to processed chickens to enhance moisture retention, on survival of Campylobacter was assessed. Polyphosphates caused changes in pH and enhanced the survival of Campylobacter during typical poultry processing and storage conditions. Thus, use of polyphosphates increases the probability of developing infection via cross contamination or by consumption of improperly cooked chicken. This work provides information to the poultry industry to help in understanding how to control growth and survival of Campylobacter. 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 the growth of L. monocytogenes in apple juice and other foods and in determining the dose response of nisin (compound known as a bacteriocin that inactivates L. monocytogenes). Molecular methods were used to monitor the regulation of genes in L. monocytogenes in response to high pressure and nisin treatments. Results demonstrated that the expression of certain genes was appreciably altered by pressure and nisin treatments, and these data will provide insight into the molecular mechanisms of survival of L. monocytogenes in food. Finally, a novel workflow technique that allows for enhanced separation and identification of bacterial proteins was developed. This proteomic technique will become an essential part of research projects aimed at understanding how Campylobacter, E. coli, or other food-borne pathogens are able to overcome the environmental stresses relevant to food settings.
1. A method to detect harmful bacteria in beef. In addition to E. coli O157:H7 certain similar types of harmful E. coli have also been declared as adulterants (zero tolerance) in beef by the USDA Food Safety and Inspection Service (FSIS). Since methods to detect these pathogens in beef that can be used for regulatory testing were needed, ARS researchers at Wyndmoor, Pennsylvania worked with the FSIS to develop a method that is currently being used by in FSIS laboratories to test for these important pathogens in beef samples collected from federally inspected establishments and retail stores. Use of these methods will ensure that beef contaminated with these harmful E. coli types will not be sold to the consumers, thus preventing serious illness and deaths caused by these pathogens.
2. A new and improved method to study bacterial proteins. To understand how harmful bacteria survive and persist within the food supply, it is necessary to be able to identify and measure the products that the bacteria produce, their proteins. This field of research is referred to as proteomics and can be studied using various techniques. However, a bacterium produces a very large number of proteins and studying them can be complicated. Thus, ARS researchers at Wyndmoor, Pennsylvania developed a novel and easy to perform proteomics method that allowed analysis of a large number of proteins produced by bacteria grown under different conditions, and the method was sensitive and reproducible. The workflow developed from this research will become an essential part of different research projects that will increase the understanding of how pathogens are able to overcome environmental stresses inherent in food settings.
3. Studying genes involved in the ability of harmful bacteria to cause illness. Harmful bacteria often carry genes on plasmids (non-chromosomal circular DNA), that are important for the disease process (virulence). ARS researchers at Wyndmoor, Pennsylvania determined the DNA sequence of a large plasmid in a pathogenic E. coli strain to gain a better understanding of how this important pathogen causes illness and to discover diagnostic markers for its identification. The virulence plasmid carried several of the same key virulence genes that are carried on the plasmid of a similar harmful bacterium, E. coli O157:H7; therefore, these genes are expected to be important for virulence. Related studies compared all of the genes in pathogenic E. coli strains to identify genes important for causing illness. Knowledge of the virulence gene profiles of E. coli strains that cause severe disease and understanding the stepwise evolution of these pathogens provide the foundation for developing strategies for detection of highly pathogenic strains and for their control in food.
4. A rapid screening method for detection of harmful E. coli in food. Rapid and simple screening methods that can be used by the food industry for detection of types of E. coli that were recently declared as adulterants in beef by the Food Safety and Inspection Service (FSIS) are needed. The food industry cannot hold perishable foods for long periods; therefore, the availability of rapid methods for detection of contamination with these pathogens is critical. ARS researchers at Wyndmoor, Pennsylvania worked with a company to develop a rapid, simple, and reliable method that allows screening of beef for these pathogens with fewer hands-on steps. This method was developed into a commercially available assay kit that will be particularly useful for the food industry in the U.S. and throughout the world, since the FSIS also requires that beef that is imported into the U.S. be tested for these pathogens.
5. Use of specific compounds during poultry processing increases the survival of Campylobacter. Campylobacter species are responsible for a large number of cases of food-borne illness annually in the developed world; 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. 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 during normal poultry processing and storage conditions. It was determined that the certain types of polyphosphates more than others were responsible for changing the acidity level during processing of poultry. This research identified specific polyphosphates that could be used during poultry processing that are less of a food safety risk since they do not enhance Campylobacter survival.
6. Understanding how Listeria monocytogenes is able to survive treatments used to inactivate it. 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 provided information to show that a combination of high pressure and nisin can be applied to effectively control L. monocytogenes in food. This study also revealed the mechanism of this interaction, and this knowledge may contribute to the design of safe and economically feasible treatments to inactivate L. monocytogenes during food processing.
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