1a. Objectives (from AD-416):
The overall goal of this project is to determine the growth and inactivation kinetics of foodborne pathogens suspended in foods treated using thermal and nonthermal process interventions, with a strong emphasis on ExPEC. 1. Develop and validate models to simulate pathogen behavior under both growth and inactivation conditions. 2. Developing and validating non-thermal and thermal intervention technologies to inactivate pathogens and spoilage microorganisms in raw and ready-to-eat foods and food contact surfaces. 3. Examine any relationship between genotype (virulence factors) and pathogen resistance to interventions. The results of this research will be transferred to regulatory agencies (USDA Food Safety Inspection Service (FSIS), US Food and Drug Administration (FDA)) to develop genomic-based risk assessments. In addition, results will be transferred to women’s health groups, commercial entities, and the meat and poultry industry. This approach may be ultimately expanded to include other thermal and nonthermal intervention technologies and extraintestinal foodborne pathogens.
1b. Approach (from AD-416):
Extraintestinal Escherichia coli (ExPEC) are common contaminants in food which includes fresh produce, fish, meat and poultry. Illness occurs after contaminated food is consumed, the ExPEC colonize the gastrointestinal tract, and are then accidentally transferred to the urethra. They then cause urinary tract infections (UTI), sepsis, and meningitis. Approximately 6-8 million cases of UTI and 23,000 deaths annually are attributed to ExPEC. ExPEC and other extraintestinal foodborne pathogens which are found in meat and poultry have been directly traced to illness in humans. In addition, these emerging foodborne pathogens are resistant to multiple antibiotics and are considered a national research priority as noted in the President’s Council of Advisors on Science and Technology (PCAST, 2014). As specifically noted by regulatory agencies this project addresses “an area of growing concern to FSIS and the public health community” which will help: 1) improve the ability to develop safe processing procedures and to evaluate the impact of processing deviations on pathogen growth in the impacted products; 2) provide insights into mechanisms that contribute to the survival of pathogens to commonly used microbial intervention and mechanisms that affect the severity of illness in humans, and antibiotic resistance in outbreak strains; and 3) provide a scientific foundation for the development of new Agency food safety policies. The Centers for Disease Control and Prevention (CDC) recommends the use of foods treated with appropriate intervention technologies to lessen the risk of foodborne illness for “at risk” individuals. There is little if any information about growth or inactivation kinetics of the ExPEC in food using both thermal and nonthermal food safety intervention technologies or how pathogen genotype affects their resistance to intervention technologies, hence we will generate such data to fill the informational void regarding these emerging pathogens as part of this unique food safety project.
3. Progress Report:
Progress was made on all objectives, all of which fall under National Program 108 – Food Safety, Component I Foodborne Contaminants. Progress on this project focuses on Problem Statement 5, Intervention and Control Strategies Including Mycotoxins; and Problem Statement 6, Predictive Microbiology/Modeling: Data Acquisition. This project focuses on assessing, characterizing, and killing the emerging pathogen extraintestinal pathogenic E. coli (ExPEC) in meat and poultry, with a genomics component to investigate the role that virulence factors and antibiotic resistance play in pathogen resistance to intervention technologies to aid in metagenomic risk assessments conducted by USDA Food Safety Inspection Service. ExPEC are a diverse set of emerging pathogens that are present in poultry and red meat, in addition to produce. The association of ExPEC with disease in humans can be traced directly from food animals, produce, and food to humans through modern genetic analysis techniques. They are associated with illnesses such as sepsis (the 6th leading cause of death), ulcerative colitis and Crohn’s Disease (ca. 1 million cases), urinary tract infections (10.5 million case annually, and 23,000 deaths), and meningitis (ca. 500 deaths) annually. The ExPEC that cause illness in humans are categorized as Uropathogenic E. coli (UPEC), Sepsis-Associated Pathogenic E. coli (SEPEC) and Neonatal Meningococcal E. coli (NMEC). There is now very strong evidence these disease conditions are a form of foodborne illness. Many ExPEC are also resistant to multiple antibiotics, including the antibiotics of last resort. In contrast, there were only six confirmed cases of deaths associated with Shiga toxin-producing E. coli such as O157:H7 in 2016. There has been substantial progress in the first year of the project. In collaboration with scientists at Penn State University (State College, PA) we obtained a number of ExPEC clinical isolates associated with disease in humans and others from the American Type Culture Collection (Manassas, VA.) We obtained a number of food and animal ExPEC isolates from our collaborators at the Veterans Administration (St. Paul, MN). Substantial progress has been made on isolation of ExPEC from ground chicken and chicken parts, determining their status as ExPEC by polymerase chain reaction (PCR)-based phylogrouping and virulence factor profiling, and subjecting those isolates to genetic analysis. Isolates have been subjected to whole genome sequencing with our collaborators from Drexel University (Philadelphia, PA). Food processing technologies used by industry include thermal processing (cooking), and US Food and Drug Administration approved nonthermal processing technologies including high pressure processing, ionizing radiation, and ultraviolet light. Each of these technologies is commercialized and used by the food processing industry. High pressure processing (HPP) kills E. coli by crushing it. The D10 value, the HPP time and pressure parameters required to reduce ExPEC levels in ground chicken by 90%, were determined to be 30.6, 8.37, and 4.43 min at 300, 400, and 500 MPa, respectively. Furthermore, in experiments conducted in collaboration with scientists from National Taiwan University, we determined that natural antimicrobials mixed in with ground meat such as thymol essential oil and other essential oil mixtures substantially enhanced the killing of ExPEC and E. coli O157:H7, and a predictive model was developed to describe the essential oil/HPP killing process. Gamma radiation kills bacteria by damaging their DNA and proteins. We determined the gamma radiation inactivation kinetics for ExpEC suspended in ground chicken, with the gamma radiation D10 value being 0.28 and 0.36 kGy at 4 degrees C and -20 C, respectively. Ultraviolet light also kills bacteria by damaging their DNA and proteins. The ultraviolet light D10 for ExPEC suspended in poultry purge and placed on stainless steel and plastic food contact surfaces ranged from 11.4 to 12.9 mJ/cm2. We also completed a growth model for ExPEC suspended in ground chicken in collaboration with scientists from National Taiwan University. Data from the studies listed above have been shared with stakeholders and customers including the Food and Drug Administration, USDA Food Safety Inspection Service, consumer groups, and the food processing industry through meeting presentations scientific manuscripts, working groups. As a result, we have been asked to participate in a USDA FSIS working group which will address the risk posed from ExPEC in foods.
1. Inactivation of Uropathogenic Escherichia coli (UPEC) in ground chicken or chicken purge by high pressure processing, gamma radiation, and ultraviolet light. High pressure processing, gamma radiation, and ultraviolet light are sustainable food safety technologies that can kill harmful bacteria in meat and poultry. Uropathogenic Escherichia coli are an emerging and common contaminant in poultry meat and are associated with urinary tract infections which affect over 10 million people, primarily women, each year including 23,000 deaths. ARS researchers at Wyndmoor, Pennsylvania found that High pressure processing (500 MPa, 4 C, 4.43 min), gamma radiation (1.3 kGy at 4 C or 1.6 kGy at -20 C), or ultraviolet light (125 mJ/cm2) killed 99.999% of UPEC in chicken meat or chicken purge. The results of this study will allow regulatory agencies and food processing industries to conduct risk analysis and provide safer poultry meat to consumers. Consumers, especially those who are immuno-compromised (e.g. women, cancer patients, diabetics, and the HIV/AIDS population) will benefit from having more information about foods treated with alternative processes which kill harmful bacteria such as UPEC.
2. Growth kinetics for Uropathogenic Escherichia coli in ground chicken meat. Uropathogenic Escherichia coli (UPEC) are an emerging and common contaminant in poultry meat and are associated with urinary tract infections which affect over 10 million people, primarily women, each year including 23,000 deaths. ARS researchers at Wyndmoor, Pennsylvania, in collaboration with scientists from National Taiwan University, completed a growth model to describe the ability of UPEC to grow in ground chicken. UPEC was unable to grow at proper refrigeration temperature (4C), but was able to grow significantly at the mild abuse temperature of 10C. Food processors and risk assessors will be able to provide safer ground poultry meat to consumers. Consumers, especially those who are immuno-compromised (e.g. women, cancer patients, diabetics, and the HIV/AIDS population) will benefit from having more information about foods treated with alternative processes which kill harmful bacteria such as the UPEC.
Chien, S., Sheen, S., Sommers, C.H., Sheen, L. 2016. Modeling the inactivation of Escherichia coli 0157:H7 and uropathogenic E.coli in ground chicken by high pressure processing and thymol. Frontiers in Microbiology. 7(920):1-11.
Sommers, C.H., Scullen, O.J., Mackay, W. 2016. Inactivation of Staphylococcus saprophyticus in chicken meat and exudate using high pressure processing, gamma radiation, and ultraviolet light. Food Control. 75:78-82.
Li, C., Hsu, H., Wang, Y., Cassidy, J.M., Sheen, S., Liu, S. 2017. Effects of heat treatment on antioxidative and anti-inflammatory properties of orange by-products. The Royal Society of Chemistry. doi: 10.1039/C7FO00188F.
Sommers, C.H., Gunther, N.W., Sheen, S. 2016. Inactivation of foodborne pathogens in chicken purge or skin using a 405-nm LED array. Food Microbiology. 64:135-138.