Location: Food Safety and Intervention Technologies Research
2019 Annual Report
Objectives
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.
Approach
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.
Progress Report
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 (FSIS). 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 (11 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. The total cost of ExPEC in Foods may be as high as $25 billion, annually. In contrast, there were only six confirmed cases of deaths associated with Shiga toxin-producing E. coli such as O157:H7 in 2016.
In continuing research on the presence of ExPEC in retail chicken meat we isolated ExPEC Sequence Type (ST) 131 and ST 117 isolates which are involved in urinary tract infections. Both isolates are resistant to multiple antibiotics. We also isolated Klebsiella pneumoniae isolates which contained virulence factors necessary for infection in humans which are resistant to multiple antibiotics as well as sanitizers used by both the hospital and food industries. Microbial Resource Announcements were published on these isolates and inactivation kinetic data presented at multiple scientific meetings and distributed to our stakeholders in FSIS as well as women’s health groups. We conducted a survey of E.coli types isolated from fresh herbs (e.g. mint, cilantro, basil, parsley) and found the E. coli ST were those associated with disease in humans, carried multiple ExPEC virulence factors, were resistant to multiple antibiotics, and industrial sanitizers. It appears E. coli on produce are being transferred there through animal waste as they are typically associated with both food and wild animals. The incidence of E. coli on the retail herbs ranged from zero to >10,000 per gram. The presence of E. coli with these virulence factors and antibiotics are concerning as each of the fore-mentioned fresh herbs are typically eaten raw.
In cooperation with our collaborators at National Taiwan University (Taipei, Taiwan) and the Department of Veterans Affairs (Minneapolis, Minnesota) we have determined the thermal inactivation kinetics of 18 individual ExPEC isolates and correlated the thermal resistance with the presence or absence of virulence factors. We found that ExPEC isolate difference in D-10 correlates with possessing or lacking the fdeC, sinH, cnf1, gad, ompT, iha, fimH and sat genes which are required for illness in humans and agricultural animals (e.g. avian colibacillosis). This may help some ExPEC survive cooking. We determined the thermal inactivation kinetics for multi-isolate cocktails of UPEC, NMEC, and food-source isolates at multiple temperatures and determined the thermal D-10 values or each ExPEC class, as well as the thermal z-values to generate mathematics based predictive equations that describe the heat resistance of the ExPEC over many temperatures. The mean D-10 values of the UPEC group were 7.34, 0.56, and 0.05 minutes at 55, 60, and 65 degrees Centigrade, respectively. The mean of the D-10 values of the NMEC group were 4.13, 0.47, and 0.08 minutes at 55, 60, and 65 degrees Centigrade, respectively. The z-values were 4.69, 5.89, and 5.53 degrees Centigrade/min, respectively. Each of those processes is used commercially in the U.S. for food safety and shelf-life extension. This work was published in Food Control (Impact Factor = ca. 4) in 2019.
In collaborative research with physicians and scientists at the Department of Veterans Affairs (Minneapolis, Minnesota) ARS scientists in Wyndmoor, Pennsylvania determined the ionizing radiation resistance of 25 ExPEC isolates suspended in refrigerated ground chicken meat. Irradiation of red and poultry meat is an FDA approved and commercialized process. We inoculated ground chicken meat with UPEC and NMEC, isolates from retail chicken meat (CM), as well as retail chicken-skin isolates identified in our laboratory (CS). We then determined their gamma radiation inactivation kinetics (D-10). The mean D-10 for all isolates (n=25) was 0.30 kGy. The mean D-10 for the UPEC, NMEC, CM, and CS isolates were 0.25, 0.29, 0.29, and 0.39 kGy, respectively. The mean D-10 for the clinical isolates was 0.27 kGy vs. 0.34 kGy for the non-clinical isolates. Unlike the results for thermal processing of ExPEC mentioned previously there was no correlation between presence of virulence factors, antibiotic resistance, and radiation resistance. This research has been accepted for publication in Food Microbiology (Impact Factor = ca. 4) and will be published in 2019.
Data from the studies listed above have been shared with stakeholders and customers including the Food and Drug Administration, the Centers for Disease Control and Prevention, FSIS, USDA-Economic Research Service, consumer groups, women’s health groups and the food processing industry through presentations at scientific meetings, published manuscripts and working groups. We participate in a FSIS working group which will address the risk posed from ExPEC in foods. Because of the high-profile nature of research on ExPEC in foods, ARS scientists in Wyndmoor, Pennsylvania organized and chaired the first ever symposium on the topic at a food safety meeting (Extraintestinal Pathogenic E.coli: Urinary Tract Infections and Sepsis)which was held at the International Association of Food Protection Meeting (Louisville, Kentucky) in July of 2019.
Accomplishments
1. Radiation kills E. coli found in chicken meat that infect humans. Uropathogenic Escherichia coli (UPEC) and other Extraintestinal Pathogenic E. coli (ExPEC) cause approximately 10 million cases of urinary tract infections and 1 million cases inflammatory bowel disease (e.g. Crohns Disease) each year, disproportionately in women. UPEC and ExPEC are readily found in retail poultry meat. ARS scientists at Wyndmoor, Pennsylvania, working with physicians in the Department of Veterans Affairs in Minneapolis, Minnesota determined the radiation resistance, a Food and Drug Administration approved process, for UPEC and ExPEC suspended in ground chicken meat. They found a very modest radiation dose of 0.30 kGy kills 90% of UPEC and ExPEC in chicken meat. Irradiation of chicken meat will help food processors eliminate UPEC and ExPEC from retail poultry products and protect consumers, primarily women, from these deadly antibiotic resistant bacteria.
Review Publications
Xu, A., Mackay, W., Scullen, O.J., Ramos, R.V., Sheen, S., Sommers, C.H. 2019. Draft genomic sequence of Escherichia coli ST131 B7S75 isolated from retail chicken skin. Microbiology Resource Announcements. https://doi.org/10.1128/MRA.01533-18.
Xu, A., Johnson, J., Scullen, O.J., Chuang, S., Sheen, L., Sheen, S., Sommers, C.H. 2019. Thermal inactivation kinetics of extraintestinal pathogenic escherichia coli suspended in ground chicken meat. Food Control. 104:269-277. https://doi.org/10.1016/j.foodcont.2019.05.001.
Chien, S., Sheen, S., Sommers, C.H., Sheen, L. 2018. Effects of combined treatments of high pressure processing, single-and multi-antimicrobial (Melissa officinalis extract)on the reduction of pathogenic Escherichia coli in ground beef. Food and Bioprocess Technology. 12:359-370. https://doi.org/10.1007/s11947-018-2211-5.
Xu, A., Abdul Wakeel, A.Y., Gunther, N.W., Sommers, C.H. 2019. Draft genomic sequence of Campylobacter coli isolated from chicken carcasses. Microbiology Resource Announcements. 8(28):e00564-19. https://doi.org/10.1128/MRA.00564-19.
Xu, A., Mackay, W., Sommers, C.H. 2019. Draft genomic sequence of multi-drug resistant Klebsiella peneumoniae B8S35, isolated from retail chicken skin. Microbiology Resource Announcements. 8(28):e00502-19. https://doi.org/10.1128/MRA.00502-19.
Xu, A., Scullen, O.J., Sheen, S., Johnson, J., Sommers, C.H. 2019. Inactivation of extraintestinal pathogenic E. coli clinical and food isolates suspended in ground chicken meat by gamma radiation. Food Microbiology. https://doi.org/10.1016/j.fm.2019.103264.
