GenEpi-BioTrain - Virtual training 23 - Virulence Profiling of E. coli Pathotypes

Topic outline

• Welcome to the virtual training on Virulence Profiling of E. coli Pathotypes!

  Welcome to the virtual training on Virulence Profiling of E. coli Pathotypes!

  Course description

  This two-half-day training course introduces participants to the major pathotypes of the diverse pathogen E. coli, and to the genetic virulence factors that characterise them. Differentiating among gastrointestinal (GI) E. coli pathotypes can be challenging, particularly when relying on phenotypic or conventional laboratory methods. Currently, many clinical laboratories use PCR panels that can confirm a range of pathogenic E. coli and additional GI bacterial pathogens in a single run, but provide no additional typing. In most clinical laboratories, there is still a need for an isolate to perform additional typing and further cluster analysis. Genomic analyses enable the detection of virulence genes and other pathotype-specific markers, as well as the application of other typing and clustering methods. There are, however, genomic challenges in distinguishing Shigella from Enteroinvasive E. coli (EIEC), which will also be addressed. The course will explore the application of virulence gene profiling to predict pathotypes and, hence, the pathogenicity of E. coli isolates using a combination of traditional typing methods and virulence profiling. Through guided analysis of E. coli genomes, participants will evaluate different virulence identification tools for E. coli in combination with subtyping methods and use them to interpret the pathogenic potential of E. coli isolates. Participants will go through a critical review of E. coli sequence QC data to evaluate potential outlying results, including intra-species contamination, and will then be introduced to online or graphical user-interface-based tools for typing and virulence profiling.

  The course includes an exercise on virulence profiling to reinforce practical understanding.

  
  

  Target Audience

  Microbiologists, clinicians, and epidemiologists with basic bioinformatics skills

  
  

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  Aim and Objectives

  Aim and Objectives

  Upon completion of this workshop, participants will be able to: 

  
  

  1. Understand the challenges in typing of E. coli and Shigella in the laboratory
  2. Understand the biological diversity within the E. coli pathotypes (incl. Shigella) and the significance for their virulence.
  3. Perform quality control of whole‑genome sequencing (WGS) E. coli data.
  4. Analyse and interpret the different sub-typing methods, including MLST, cgMLST, and serotyping of E. coli.
  5. Perform virulence profiling and typing using simple bioinformatics tools and determine/suggest the resulting pathotype by employing various virulence gene databases.
  6. Understand the application of command-line tools for virulence databases (KMA/BLAST).
  7. Identify real-life challenges in tracing emerging clones of pathogenic E. coli.
• Prepare for the training

  Prepare for the training

  Suggested Readings

  • Michelacci V, Tozzoli R, Arancia S, D’Angelo A, Boni A, Knijn A, et al. Tracing Back the Evolutionary Route of Enteroinvasive Escherichia coli (EIEC) and Shigella Through the Example of the Highly Pathogenic O96:H19 EIEC Clone. Front Cell Infect Microbiol. 2020;10:260.  https://doi.org/10.3389/fcimb.2020.00260 

  • Torpdahl M, White ED, Schjørring S, Søby M, Engberg J, Engsbro AL, Holt H, Lemming L, Lützen L, Olesen B, Coia JE, Kjelsø C, Müller L. Imported spring onions related to the first recorded outbreak of enteroinvasive Escherichia coli in Denmark, November to December 2021. Euro Surveill. 2023;28(15):pii=2200572. https://doi.org/10.2807/1560-7917.ES.2023.28.15.2200572 

  • Mudau KL, Ntobeng LR, Kalu CM, Tekere M, Pathogenicity and virulence factors of Escherichia coli discovered using next generation sequencing technologies and proteomics. 2025. Frontiers in Bacteriology 4.  https://doi.org/10.3389/fbrio.2025.1677775 

  • Robins-Browne RM, Holt KE, Ingle DJ, Hocking DM, Yang J and Tauschek M (2016) Are Escherichia coli Pathotypes Still Relevant in the Era of Whole-Genome Sequencing?. Front. Cell. Infect. Microbiol. 6:141. doi: 10.3389/fcimb.2016.00141 

  • Rapid risk assessment: Increase in OXA-244 -producing Escherichia coli in the European Union/European Economic Area and the UK since 2013, https://www.ecdc.europa.eu/en/publications-data/rapid-risk-assessment-increase-oxa-244-producing-escherichia-coli-eu-eea

  • Outbreak of gastroenteritis highlighting the diagnostic and epidemiological challenges of enteroinvasive Escherichia coli, County of Halland, Sweden, November 2017. Euro Surveill. 2020 Mar 5;25(9):1900466. doi: 10.2807/1560-7917.ES.2020.25.9.1900466
• Software Requirements

  Software Requirements

  Equipment and Tools

  To participate effectively in this course, ensure you have access to the necessary materials and that your machine meets the following requirements and has the appropriate tools installed.

  Equipment:

  • A laptop with internet access.
  • Sample datasets (provided later via Sciencedata.dk). 
  • For command-line usage: macOS or Linux recommended; Windows users should use WSL (Windows Subsystem for Linux) or Docker. 
  • Mamba or Conda for managing software environments and packages (installed, for example, through Miniforge: https://github.com/conda-forge/miniforge).

  Tool Setup:

  Participants may choose to use web-based (basic) tools or command-line (advanced) tools depending on their comfort and experience.

  • Pre-install the command-line tools before Session 1 for a smooth workflow.
  • Web tools require no installation — access via provided links.

  
  

  Web-Based Tools

  1. KmerFinder - Species identification using k-mer matching against bacterial genome databases.

   2. rMLST - Ribosomal MLST-based species identification using 53 ribosomal protein subunit (rps) genes for high-resolution bacterial taxonomy.

  3. PubMLST - Multilocus sequence typing (MLST), allele calling, and strain characterization for multiple bacterial species.

   4. SerotypeFinder - Serotype prediction for E. coli and Salmonella isolates from assembled or raw WGS data by detecting O and H antigen genes.

  5. MyDBFinder - Custom gene detection using user-defined FASTA databases and BLAST-based matching against uploaded bacterial genomes.

   6. PathogenFinder2 - Predict bacterial pathogenic capacity in humans using deep learning and protein language models; identify key proteins contributing to pathogenicity.

    7. VirulenceFinder - Identify virulence genes in bacterial isolates from total or partially sequenced genomes.

   8. VFDB Database - Virulence Factor Database (VFDB) — a comprehensive resource for bacterial virulence factors and associated genes.

  
  

  Command-Line Tools 

   9. MLST - Perform multilocus sequence typing (MLST) by scanning assembled contigs against PubMLST typing schemes.

  Installation (using Bioconda):

  conda config --add channels conda-forge
  conda config --add channels bioconda
  conda config --set channel_priority strict
  # Installation using mamba
  mamba install mlst
  # Verify installation
  mlst --version
  mlst --help

   10. VirulenceFinder - Identify virulence genes in bacterial genomes using BLAST or KMA alignment against the VFDB database.

  • Database: https://www.mgc.ac.cn/VFs/download.htm

  Installation (using Bioconda):

  # Installation using mamba
  mamba install virulencefinder 
  # Verify installation
  virulencefinder --help

   11. KMA - Ultra-fast k-mer-based alignment tool for mapping raw reads (short or long reads) directly against redundant gene databases.

  Installation (using Bioconda):

  # Installation using mamba
  mamba install kma
  # Verify installation
  kma -v
  kma -h

   12. BLAST+ - Sequence similarity searching using Basic Local Alignment Search Tool for nucleotide and protein database queries.

  Installation (using Bioconda):

  # Installation using mamba
  mamba install blast
  # Verify installation
  blastn -version

  
  

  Note: Make sure to download and index required databases (e.g., VFDB for VirulenceFinder, PubMLST schemes for MLST) before running the command-line tools. Refer to each tool's documentation for database setup instructions (e.g., VFDB for VirulenceFinder, PubMLST schemes for MLST) before running the command-line tools. Refer to each tool's documentation for database setup instructions.
• Session 1: Introduction to Escherichia coli pathotypes; evaluation of basic genomics, typing and virulence profiling

  Session 1: Introduction to Escherichia coli pathotypes; evaluation of basic genomics, typing and virulence profiling

  AGENDA FOR SESSION 1 – Tuesday, March 3, 2026

  
  

  
  

  • Opening and introduction to Session 1

     

  • 1.a. E. coli virulence

  • FilePathogenic E.coli: diagnostics, typing and when the infection is notifiable – an overview
  • 1.b. E. coli pathotypes

  • FileE coli pathotypes virulence
  • 2. E. coli genomics and quality control (QC)

  • FileE. coli genomics and quality control (QC)
  • 3. Taxonomical analysis and typing

  • FileTaxonomic analysis and typing v2
  • 4. Virulence gene databases

  • FileVirulence gene databases
  • 5 and 6. Introduction to the exercise on interpretation of virulence gene results

  • FileInterpretation of virulence gene results jetk v2
  • 7.  Q&A and Conclusion

  • SeminarOnline session 1 — Virtual training 23

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• Homework - between sessions

  Homework - between sessions

  You will receive a set of 17 E. coli/Shigella genomes (assemblies in FASTA format and raw FASTQ files) and the respective QC reports via ScienceData (View it!). Here you can also find a more detailed description of the homework.

  Your task is to evaluate the sequence quality (both fastq and assemblies, as well as the final QC report will be available), perform sub-typing (MLST, serotyping), and virulence profiling using freely available or recommended bioinformatics tools (you can use different tools). Use the obtained results to confirm species, check for contamination, and interpret E. coli pathotypes, including Shigella.
• Session 2: Interpretation of results and discussion on the application of subtyping and virulence profiling of different E. coli pathotypes

  Session 2: Interpretation of results and discussion on the application of subtyping and virulence profiling of different E. coli pathotypes

  AGENDA FOR SESSION 2 – Tuesday, March 10, 2026

  
  

  • SeminarOnline session 2 — Virtual training 23

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  • Opening of session 2

  • Guided Discussion 

    
    
  • FileECDC Presentation QC Analysis
  • Comparative and critical analysis

     

     
    
  • FileEIEC (Enteroinvasive E. coli) vs Shigella
  • FileEC pathotype prediction results¨
  • Confirmation of genus by typing 

    
    

  • FileConfirmation of genus by typing ¨
  • Command line approach (BONUS MATERIAL) 

     
    

  • FileCommandLine Approach
  • Real-life experience with E. coli outbreaks
  • FileReal-life experience with an E. coli outbreak
  • FileHybrid pathotypes outbreak
  •  Questions & answers and closing remarks

     

     

• Contacts

  Contacts

  For any questions regarding the this webinar series, please use the discussion forum.

  If you have questions regarding other training initiatives please contact: courses@ecdc.europa.eu.

  If you have technical issues please contact ECDC Helpdesk - ICT Front Office ICT.FrontOffice@ecdc.europa.eu.