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FDA Summer Fellowship in Shiga Toxin Pathogenicity

*Applications will be reviewed on a rolling-basis.

A research opportunity is currently available at the U.S. Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), located in College Park, Maryland.

Approximately 265,000 cases of foodborne illness per year can be attributed to Shiga toxin-producing Escherichia coli (STEC). Assessment of the potential pathogenicity of STECs requires characterization of the whole genome, particularly the presence of Shiga toxin (stx1 and/or stx2) and intimin (eae) genes, serotype, and other virulence factors (e.g. tir, esp, toxB). Current FDA reporting protocols for STECs require approximately two weeks of analysis time for detection by qPCR and microbiological techniques, including several rounds of selective plating to generate a single isolate, followed by classification by whole genome sequencing.

We propose development and dissemination of a pipeline that would detect, quantify, and classify STECs in enriched irrigation water in less than half the time (3-4 days) by using a combined qPCR and metagenomic sequencing approach using the Oxford Nanopore MinION and Illumina MiSeq. This analysis pipeline (STEC precision metagenomics) will also have the potential to distinguish several different STECs present in the same tested sample. By determining the limits of detection of each technique (qPCR, Nanopore and MiSeq sequencing), we can effectively tailor our proposed pipeline to obtain a closed or fragmented STEC genome(s). We can use the current qPCR detection method to quantify the STECs in the enrichment and determine the expected level of sequencing coverage to be obtained. Determining the limit of detection will establish the necessary concentration for obtaining a complete or fragmented STEC genome, which would allow us to conduct an in silico analysis of the recovered STEC strain(s) and predict the risk of those STECs to human health. Our findings and the methods proposed herein will have wide implications for food safety, including decreased time to STEC identification during outbreaks, characterization of the microbial community, and the potential to use these methods to determine the limits for other foodborne pathogens.

We expect to develop a sequencing and analysis pipeline that will allow rapid and real-time analyses of important potential STEC contaminated samples (metagenomic applications). The correct analysis and determination of the detection limits proposed in this grant could allow the generation of completely closed or fragmented STEC genomes from 24-hour enrichments using Chapter 4 of the BAM methodology for STEC isolation from foods, that would enable fast responses to threats posed by these organisms. However, the detection limit of the nanopore technique for metagenomic approaches must be determined empirically. Our proposed approach of using real-time PCR (qPCR) for determining the STEC level in the 24-hour enrichment combined with subsequent nanopore sequencing of samples that contain enough STEC level to produce a successful sequencing result might be useful for applications to other important foodborne pathogens in any sample. This culture independent approach would allow the study of STEC genomes before we are able to generate a pure culture isolate, reducing the reporting time from 2 weeks to 3-4 days and allowing our specialists to inform all stake holders, such as ORS, OFS, CORE, STEC council, etc.

Scope of Project:

Under the guidance of a mentor, the ORISE participant shall perform the following tasks during the above-specified period including:

  • 1. Testing the effectiveness of new MinION sequencing kits and flow cells using 8 genomes already closed
  • 2. Data captured in reports and presentation in meetings
  • 3. Perform experiments as designed by PI
  • 4. Maintaining a detailed report and present to PI
  • 5. Maintaining the MiSeq and MinION laboratory, inventories of chemicals and reagents
  • 6. Assisting the PI in growing cultures, inoculating food products with bacterial cultures, and extracting DNA using various protocols
  • 7. Participating in writing of publications
  • 8. Evaluating the utility of PromethION nanopore sequencing for deep sequencing analysis of metagenomic samples and determine its detection limit
  • 9. Test the probability of obtaining high quality bacterial closed genomes (that can be used for SNP analyses) by also using MiSeq data (hybrid assembly) from samples that were within STEC levels 1 and 2 from the nanopore detection limit experiment
  • 10. Validate these pipelines using new unknown samples. To improve accuracy, these samples will also be sequenced using the Illumina MiSeq

Anticipated Appointment Start Date: June 1, 2022; start date is flexible

This program, administered by ORAU through its contract with the U.S. Department of Energy to manage the Oak Ridge Institute for Science and Education, was established through an interagency agreement between DOE and FDA. The initial appointment is for three months in the summer, but may be renewed upon recommendation of FDA contingent on the availability of funds. The participant will receive a monthly stipend commensurate with educational level and experience. Proof of health insurance is required for participation in this program. The appointment is full-time at FDA in the College Park, Maryland, area. Participants do not become employees of FDA, DOE or the program administrator, and there are no employment-related benefits.

Completion of a successful background investigation by the Office of Personnel Management is required for an applicant to be on-boarded at FDA. OPM can complete a background investigation only for individuals, including non-US Citizens, who have resided in the US for a total of three of the past five years.

FDA requires ORISE participants to read and sign their FDA Education and Training Agreement within 30 days of his/her start date, setting forth the conditions and expectations for his/her educational appointment at the agency. This agreement covers such topics as the following:
  • Non-employee nature of the ORISE appointment;
  • Prohibition on ORISE Fellows performing inherently governmental functions;
  • Obligation of ORISE Fellows to convey all necessary rights to the FDA regarding intellectual property conceived or first reduced to practice during their fellowship;
  • The fact that research materials and laboratory notebooks are the property of the FDA;
  • ORISE fellow’s obligation to protect and not to further disclose or use non-public information.