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:
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. 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 second year of the project. In collaboration with ARS scientists in Beltsville, Maryland, we obtained a number of ExPEC isolated from dairy cattle which have already been subjected to whole genome sequencing. We completed whole genome sequencing of twenty ExPEC clinical and food isolates, and published three Genome Announcements. In cooperation with our collaborators at National Taiwan University (Taipei, Taiwan) and the Department of Veterans Affairs (Minneapolis, Minnesota) we have determined the high pressure processing, thermal processing, and ionizing radiation inactivation kinetics for those twenty isolates suspended in ground chicken meat. We found that, in general, the Uropathogenic E. coli clinical isolates to be the most heat, high pressure, and irradiation resistant while the meningitis-causing E. coli tended to be the most sensitive, with the food isolates covering a wide range of sensitivity and resistances to the processing technologies. Inactivation kinetics are typically described as a D-10 value, or the processing condition needed to kill 90% of a microorganism. Overall, for the ExPEC tested, the thermal D-10 at 55 degrees Centigrade ranged from 0.5 to 7.6 min, the HPP D-10 at 400 MPa (4 degrees Centigrade) ranged from 0.5 to 9.6 min, and the ionizing radiation D-10 from 0.18 to 0.68 kGy. Of the ca. 53 ExPEC isolates characterized to date, over 45 were resistant to multiple antibiotics. In cooperation with our collaborators at National Taiwan University (Taipei, Taiwan) and the Department of Veterans Affairs (Minneapolis, Minnesota) 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. Substantial progress has been made on determining the incidence and prevalence of ExPEC in retail chicken parts. We found that ca. 12% of the E. coli isolated from retail chicken skin were ExPEC. Those ExPEC (ca. 33) were then characterized by whole genome sequencing. This will enable food processors to apply appropriate thermal processing conditions to meat and poultry to control ExPEC. 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, USDA-FSIS, USDA-Economic Research Service, consumer groups, and the food processing industry through presentations at scientific meetings, published manuscripts and 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. In addition, we are now collaborating as part of an ExPEC working group with scientists from the Pennsylvania Department of Health (Harrisburg, Pennsylvania), Penn State University (State College, Pennsylvania), University of Pennsylvania (Philadelphia, Pennsylvania), the Centers for Disease Control and Prevention (Atlanta, Georgia) and the US-FDA (Food and Drug Administration) (Washington, District of Columbia).
1. Heat kills E. coli in chicken meat that infects women. Uropathogenic Escherichia coli (UPEC) causes approximately 11 million cases of urinary tract infections (UTI) each year, primarily in women. UPEC are found mostly in retail chicken meat and are linked to UTI in women. ARS scientists at Wyndmoor, Pennsylvania, working with scientists in the Department of Veterans Affairs (Minneapolis, Minnesota) and National Taiwan University (Taipei, Taiwan) determined that cooking at 65 degrees centigrade for 30 seconds, or 60 degrees for 150 seconds, killed almost all UPEC in ground chicken meat. These cooking conditions define the time and temperature needed to kill UPEC in chicken meat.
Xu, A., Hertrich, S.M., Needleman, D.S., Sheen, S., Sommers, C.H. 2018. Draft genome sequences of four uropathogenic escherichia coli 04:H5 isolates (ATCC 700414,700415,700416 and 700417). Genome Announcements. 6(11):e00134-18.
Xu, A., Johnson, J., Sheen, S., Needleman, D.S., Sommers, C.H. 2018. Draft genomic sequencing of six potential extraintestinal pathogenic Escherichia coli isolates from retail chicken meat. Genome Announcements. https://doi.org/10.1128/genomeA.00449-18.
Xu, A., Johnson, J., Sheen, S., Sommers, C.H. 2018. Draft genome sequences of six neonatal meningitis-causing escherichia coli isolates (SP-4, SP-5, SP-13, SP-16, SP-46, and SP-65). Genome Announcements. https://doi.org/10.1128/genomeA00091-18.