1. Vik Dutta (Co-Chair)
2. KatieRose McCullough (Co-Chair)
3. Heather Carleton (CDC)
4. Anna Carlson
5. Hayriye Cetin-Karaca
6. Michael Hansen
7. Arie Havelaar
8. Janell Kause (FSIS)
9. Ramin Khaksar
10. Shannara Lynn (DOC)
11. Eric Moorman
12. Abani Pradhan
13. Marcos Sanchez-Plata
14. Kristin Schill
15. Nikki Shariat
16. Max Teplitski

The U.S. Department of Agriculture's (USDA) Food Safety and Inspection Service (FSIS) uses Whole Genome Sequencing (WGS) as the primary tool for subtyping and characterizing foodborne bacterial pathogens. FSIS first built capability to perform WGS, then increased capacity to sequence 100% of foodborne bacterial pathogens. Now, the agency envisions utilizing WGS and genomic analyses to further improve food safety by better understanding how genomic information can be used to rank microbial pathogen subtypes of public health significance. Reports in the published literature have described that certain foodborne microbial pathogen subtypes inherently have a higher risk of association with illness. For example, certain Shiga toxin-producing Escherichia coli (STEC) subtypes have genes for Shiga toxin that have been associated with hemolytic uremic syndrome in patients. FSIS acknowledges that certain foodborne pathogen subtypes pose an increased risk to public health and is seeking advice from NACMCF on how to strategically use genomic analyses, in addition to any other current or emerging technologies and strategies, to help the Agency rank and focus resources on foodborne microbial pathogen subtypes based on public health risk.

WGS is a laboratory technique that determines the deoxyribonucleic acid (DNA) makeup of an organism. Scientists analyze the genetic variation as determined through WGS to understand relationships between strains or among subtypes of bacteria and determine how genes can impart characteristics such as pathogenicity, virulence, persistence, or antimicrobial resistance. FSIS collaborated with the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA) and the National Institutes of Health (NIH) to implement WGS as the primary tool for tracking foodborne illness and characterizing foodborne bacterial pathogens more efficiently and in greater depth than was possible with the previous pulsed-field gel electrophoresis (PFGE)¹ technology. FSIS field service laboratories began implementing WGS to further characterize foodborne bacterial pathogens in 2014 and achieved 100% WGS laboratory capacity by 2016.

FSIS envisions that WGS will continue to play a significant role in enhancing food safety moving forward. The 2023-2026 FSIS Strategic Plan Outcome 2.1, Improve Food Safety Through the Adoption of Innovative Approaches and Technologies², describes how FSIS will follow advancements in genomics to "more efficiently and effectively detect, characterize, and track food safety hazards," including the improvement of food safety by incorporating analyses of pathogen genomics. The FSIS Strategic Plan also states, "As microbial characterization technology continues to evolve, FSIS will consider these new technologies that could potentially enhance and complement WGS to use resources more effectively, reduce time to results and expand policy options"².

FSIS and other public health authorities have actively sought to understand the role of specific virulence genes in the risk of illness and patient outcomes presented by certain foodborne pathogen subtypes. Examples include:

• In 2011, FSIS initiated the use of a screening test for Shiga toxin-producing Escherichia coli (STEC) using the stx and eae virulence genes, resulting in a new adulteration policy³. A charge was issued to NACMCF by FDA in 2015 to address how virulence and genomic attributes identify certain STEC as severe human pathogens⁴. The Committee recommended that genomic information could be used to identify STEC as well as a lineage or cluster more likely to cause serious disease. In 2018, the World Health Organization and the Food and Agriculture Organization of the United Nations released their report on STEC monitoring and surveillance. The report listed specific subtypes of STEC virulence factors that could lead to a more severe patient outcome⁵.
• Genomic variability within Listeria monocytogenes (Lm) has been associated with more virulent or hypervirulent strains⁶. Accounting for variability within the virulence of Lm could lead to targeted control strategies in food processing environments.
• Research in Campylobacter genomics has revealed the potential for identifying subtypes that pose greater threats to human health⁷. Strategies targeting Campylobacter subtypes of increased public health concern could lead to targeted mitigation policies.
• In 2019, NACMCF published a report on Salmonella Control Strategies in Poultry⁴. One question asked whether more virulent Salmonella subtypes could be differentiated from less virulent subtypes. At that time, NACMCF concluded that virulent Salmonella subtypes could not readily be differentiated from those that are less virulent. However, genomic and bioinformatic tools have since emerged with the potential to change this paradigm.

Objective:

FSIS wants to further utilize genomics to characterize and identify the foodborne pathogen subtypes isolated from regulated commodities that pose the greatest risk to public health. FSIS is seeking advice from NACMCF on the considerations, advantages, and disadvantages of using genomic analyses, as well as information on current or emerging technologies and strategies that would help to rank and focus resources on foodborne pathogen subtypes based on risk to public health. The charge questions below should be considered for Salmonella, STEC, Lm and Campylobacter routinely isolated from FSIS-regulated commodities. The information NACMCF provides will assist FSIS with decision-making to potentially reduce pathogen subtypes of public health significance through targeted risk management strategies.

¹ Stevens, E.L., Carleton, H.A., Beal, B., et al. Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States, Journal of Food Protection, Volume 85, Issue 5, 2022, Pages 755-772. Public Meeting on Whole Genome Sequencing (WGS) www.fsis.usda.gov/news-events/events-meetings/public-meeting-whole-genome-sequencing-wgs.

² FSIS FY2023-2026 Strategic Plan, www.fsis.usda.gov/sites/default/files/media_file/documents/Strategic%20Plan%202023-2026.pdf.

³ FSIS (2011), Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products, Federal Register 76(182).

⁴ NACMCF Advisory Committee Reports, www.fsis.usda.gov/news-events/publications/2015-2017-national-advisory-committee-microbiological-criteria-foods.

⁵ Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO): Report - Shiga toxin-producing Escherichia coli (STEC) and food: attribution, characterization, and monitoring. apps.who.int/iris/bitstream/handle/10665/272871/9789241514279-eng.pdf?sequence=1&isAllowed=y.

⁶ Vázquez-Boland J.A., Wagner M., Scortti M. Why Are Some Listeria monocytogenes Genotypes More Likely To Cause Invasive (Brain, Placental) Infection? mBio. 2020 Dec 15;11(6):e03126-20. doi: 10.1128/mBio.03126-20. PMID: 33323519; PMCID: PMC7774001; Cardenas-Alvarez, M. X., et al. (2022) Genome-Wide Association Study of Listeria monocytogenes Isolates Causing Three Different Clinical Outcomes. Microorganisms 10, DOI: 10.3390/microorganisms10101934; Kremer, P. H., et al. (2016). Benzalkonium tolerance genes and outcome in Listeria monocytogenes meningitis. Clin Microbiol Infect.

⁷ Hull D.M., Harrell E., van Vliet A.H.M., Correa M., Thakur S. (2021), Antimicrobial resistance and interspecies gene transfer in Campylobacter coli and Campylobacter jejuni isolated from food animals, poultry processing, and retail meat in North Carolina, 2018-2019. PLoS ONE 16(2): e0246571. doi.org/10.1371/journal.pone.0246571; Bandoy, D. D. R. and B. C. Weimer; (2020). Biological Machine Learning Combined with Campylobacter Population Genomics Reveals Virulence Gene Allelic Variants Cause Disease. Microorganisms 8(4): 549; Peters, S., et al. (2021). Campylobacter jejuni genotypes are associated with post-infection irritable bowel syndrome in humans." Communications Biology 4(1): 1015; Buchanan, C. J., et al. (2017). A Genome-Wide Association Study to Identify Diagnostic Markers for Human Pathogenic Campylobacter jejuni Strains. Front Microbiol 8: 1224.

1. Appropriate genomic and pathogen attributes: How can genomics be used to differentiate microbial pathogen (Salmonella, STEC, Lm, Campylobacter) subtypes by risk to public health in food products regulated by FSIS?
   
   1. What epidemiologic criteria should be used to rank subtypes by risk for each of the pathogens of concern to FSIS (Salmonella, STEC, Lm, Campylobacter), including but not limited to outbreak size and scope, link to sporadic illness, frequency of illness, severity of illness, and patient outcome?

   2. How can pathogen genomic data be incorporated into microbial risk assessments (i.e., hazard analysis, hazard identification, exposure assessment, hazard characterization and risk characterization)?

   3. In addition to putative or known virulence genes, what other genomic attributes of each of the pathogens of concern are associated with a higher risk to public health (e.g., antimicrobial resistance genes, plasmids or genes leading to persistence such as heat resistance or other tolerance attributes (metals, etc.))?

2. Available and applicable tools and analyses: What types of genomic-based approaches are currently used by U.S. and international entities to support food safety decisions?
   
   1. What tools and technologies (including but not limited to targeted metagenomics, shot-gun metagenomics and culture-independent diagnostic tests) are deployed and have they been validated to an accredited standard, including but not limited to Association of Official Agricultural Chemists or Clinical Laboratory Improvement Amendments?

   2. What analytical methods that integrate genomic data and metadata (including but not limited to genome-wide association, machine learning/random forest and artificial intelligence) are available for distinguishing strains based on likelihood of causing illnesses given exposure? Does the committee recommend a particular approach among available methods?

   3. What genomic databases, analytic criteria and information sharing mechanisms are harmonized domestically and internationally? How can genomic data or metadata currently publicly available in existing databases be improved to be more informative and meaningful in developing risk ranking models, tools, or analyses?

3. Knowledge gaps and research gaps: What research or knowledge gaps should be addressed to fully operationalize a genomics-based approach?
   
   1. Do current or emerging technologies rely on well-characterized genes to identify a riskier pathogen subtype? If further research is needed to link certain genomic factors with virulence and/or severe patient outcomes, how would the committee recommend focusing the research?
   2. How can currently available genomic information from Salmonella, STEC, Lm, and Campylobacter from FSIS-regulated products be leveraged to reduce time to subtype determination in a high throughput laboratory? How could rapid diagnostic tools be improved using genomic-based targets to identify riskier pathogen subtypes?
   3. Can genomic-based models or technologies be adapted to include emerging pathogenic subtypes; reoccurring, emerging or persisting strains; and plasmids or genes of public health concern in FSIS-regulated products? For example, how could the approach address the following?
      

      1. Non-monocytogenes Listeria spp.

      2. Polyphyletic Salmonella serotypes (including but not limited to the virulent clade of Salmonella Kentucky).

      3. Campylobacter species other than coli/jejuni/lari.

      4. STEC.

      5. Genes that are associated with virulence or multidrug resistance (MDR) such as the MDR plasmid in Salmonella Infantis.

      6. Other emerging pathogenic subtypes of the foodborne pathogens of importance to FSIS

   4. How can genomics be used to differentiate vaccine strains used in food safety (for example, vaccination of poultry against Salmonella) from wild-type, pathogenic bacterial strains?

4. Strategic Vision: Based on the risk management questions and tools being deployed, how might genomics inform FSIS and other Agency actions along the farm to fork continuum?

   1. How might regulatory agencies adjust sampling plans (both exploratory and routine verification testing) to optimize the use of pathogen genomic data?

   2. How might genomics be used to inform future risk management strategies?

   3. When implementing a risk management strategy, what are the benefits and considerations of using a genomic-based approach to identify and rank pathogen subtypes by risk to public health?

   4. How might U.S. regulatory entities interpret pathogen genomic information to support their agency regulatory actions?

• November 14-16, 2023, Subcommitee Meetings

1. Elisabetta Lambertini (Co-Chair)
2. Abby Snyder (Co-Chair)
3. Bledar Bisha
4. Ben Chapman
5. Betty Feng
6. Larry Figgs
7. Noel Kubat (DoD)
8. Indaue Mello
9. David Goldman
10. Shiv Rana
11. Bing Wang
12. Ben Warren (FDA)
13. Randy Worobo
14. Teshome Yehualaeshet

Cronobacter spp. (formerly Enterobacter sakazakii) are microorganisms present in the environment and can survive in dry foods, such as powdered infant formula. Cronobacter spp. infections among infants younger than 12 months have high case-fatality rates. Historical surveys of powdered infant formula have reported a relatively high prevalence rate, ranging from 2 to 15% of Cronobacter spp. contamination in these products. FDA regulations specify that manufacturers of infant formula must establish a system of production and in-process controls, covering all stages of processing, that is designed to ensure that infant formula does not become adulterated due to the presence of Cronobacter spp (see 21 CFR parts 106 and 107). In late 2021 and early 2022, a series of Cronobacter spp. illnesses among infants in the U.S. was associated with feeding powdered infant formula. In each illness, the formula was produced by a specific manufacturer at one facility. The resulting voluntary recall (and the temporary shutdown of the plant) was a major contributing factor to the infant formula shortage experienced across the U.S. in 2022. Better understanding of the factors that contribute to Cronobacter spp. contamination of powdered infant formula and the production environment is needed to increase the effectiveness of prevention and management strategies.

Cronobacter spp. (formerly Enterobacter sakazakii) are microorganisms present in the environment and can survive in dry foods, such as powdered infant formula. Cronobacter spp. infections among infants younger than 12 months have high case-fatality rates. Historical surveys of powdered infant formula have reported a relatively high prevalence rate, ranging from 2 to 15% of Cronobacter spp. contamination in these products. FDA regulations specify that manufacturers of infant formula must establish a system of production and in-process controls, covering all stages of processing, that is designed to ensure that infant formula does not become adulterated due to the presence of Cronobacter spp (see 21 CFR parts 106 and 107). In late 2021 and early 2022, a series of Cronobacter spp. illnesses among infants in the U.S. was associated with feeding powdered infant formula. In each illness, the formula was produced by a specific manufacturer at one facility. The resulting voluntary recall (and the temporary shutdown of the plant) was a major contributing factor to the infant formula shortage experienced across the U.S. in 2022. Better understanding of the factors that contribute to Cronobacter spp. contamination of powdered infant formula and the production environment is needed to increase the effectiveness of prevention and management strategies.

1. What is the current prevalence and level of Cronobacter spp. contamination in powdered infant formula in the U.S. market? What is known about Cronobacter spp. in other foods and in the home environment and the frequency with which these foods and environmental sources contribute to human infections?
2. What factors (e.g., virulence factors, host factors, dose of exposure) place an infant at greater risk for Cronobacter spp. infection and serious adverse health consequences or death?
3. What food safety management practices (e.g., facility and equipment design, hygienic zoning and packaging, preventive controls, verification activities) should manufacturers of powdered infant formula employ to further reduce the risk of Cronobacter spp. contamination of formula and/or the production environment?
4. Given that powdered infant formula is not sterile, how could food safety messaging be improved for infant care providers, with emphasis on use of sterile, ready-to-use formulas for infants at greatest risk and safe infant formula preparation and storage for infant formula in general?

• May 16-18, 2023, Subcommittee Meeting
• November 14-16, 2023, Subcommitee Meetings