Development of Assays for the Identification of Virulent Enterohemorrhagic Escherichia Coli Strains
Bibliographic record
Abstract
Escherichia coli (E. coli) is a diverse Gram-negative, rod-shaped, facultatively anaerobic, and non-sporing bacterium belonging to the family Enterobacteriaceae. Strains of E. coli can be found in the environment and the intestinal microflora of mammals. While many E. coli strains have developed an essential mutualism relationship in our colonized gastrointestinal tract, some have evolved with the ability to cause diarrheal disease in humans. Diarrheal disease-causing strains are categorized as intestinal pathogenic Escherichia coli (IPEC). IPEC can be further classified into five phenotypes: enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enteropathogenic Escherichia coli (EPEC), enteroaggregative Escherichia coli (EAEC), Shiga toxin-producing Escherichia coli (STEC), and Enterohemorrhagic Escherichia coli (EHEC). While Shiga toxin-producing Escherichia coli (STEC) and enterohemorrhagic Escherichia coli (EHEC) are interchangeably in scientific literature. However, based on the Food and Agriculture Organization (FAO), STEC is defined as strains that harbor the stx gene that produces Shiga toxins but may not carry other virulent genes. Similarly, EHEC is defined as strains that harbor the stx and intimin (eae) genes (FAO, WHO, 2018). Strains that have both these genes are considered virulent, while strains that have only one or neither are considered potentially avirulent. Virulent strains with stx and eae are responsible for significant foodborne outbreaks, are a major foodborne pathogen implicated in red meat outbreaks, and are considered adulterants in beef products. On the contrary, potentially avirulent strains are not considered a significant concern for the red meat industry. Currently, MLG 5.03 is the standard method for the detection of virulent strains. However, this method suffers from high false-positive rates. The false-positive test results arise from this method as it uses separate primer pairs to detect stx and eae independently within a sample. The presence of multiple avirulent strains or other background genera that may also contain stx or eae found in meat samples result in the false detection of strain(s) containing both of these genes. The purpose of this study will be to initially standardize and validate four high-resolution melt (HRM) assays, followed by a conversation of these assays to industry-friendly hydrolysis probe (HP) assays for the differentiation of potentially virulent strains of EHEC serogroups belonging to O26, O103, O111, and O121. The designed primers and probes will target a conserved single nucleotide polymorphism (SNP) located on the serogroup-specific fnl1, wbdK, wbtD, and vioA genes of O26, O111, O103, and O121, respectively. The assays will be validated using pure cultures strains from Canada, France, Switzerland, and the United States, laboratory-inoculated beef and spinach samples, laboratory fractionally inoculated ground beef samples, and DNA samples from the federal red meat surveillance program. This study will provide significant insight into the accurate identification of E. coli strains present in tested samples. The work will allow the food industry to continue preventing foodborne outbreaks while increasing the volume of raw food products brought to the market.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".