What to expect of ExPEC: is the success of extraintestinal pathogenic E. coli linked to specific genomic and cellular properties?

Project Information

EXCELLGEN

Grant agreement ID: 101105027

DOI

10.3030/101105027

Project closed

EC signature date 29 June 2023

Start date 1 September 2023

End date 31 August 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 191 078,00

Coordinated by

KATHOLIEKE UNIVERSITEIT LEUVEN
Belgium

Periodic Reporting for period 1 - EXCELLGEN (What to expect of ExPEC: is the success of extraintestinal pathogenic E. coli linked to specific genomic and cellular properties?)

Reporting period: 2023-09-01 to 2025-08-31

Summary of the context and overall objectives of the project

The EXCELLGEN project aims to understand why some strains of Escherichia coli, normally harmless bacteria living in the intestines, can cause serious infections outside the gut. These so-called extraintestinal pathogenic E. coli (ExPEC) are a major cause of urinary tract infections, sepsis, and meningitis, and are often resistant to multiple antibiotics. Their growing prevalence represents a significant challenge for human and animal health and for the sustainability of healthcare systems.
The project investigates whether the success of ExPEC is linked to specific genetic or cellular features that distinguish them from harmless or intestinal pathogenic E. coli (IPEC). Combining large-scale comparative genomics with high-throughput single-cell analysis, EXCELLGEN bridges molecular biology, microbiology, and data science to reveal how particular genes and cellular behaviours contribute to ExPEC’s pathogenic potential.
Ultimately, EXCELLGEN contributes to the EU’s priorities under the European Green Deal and “An Economy that Works for People” by improving understanding of antimicrobial-resistant pathogens and supporting more sustainable and effective responses to infection control.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The EXCELLGEN project combined comparative genomics, large-scale screening, and quantitative phenotyping to uncover the genomic and cellular basis of virulence in extraintestinal pathogenic Escherichia coli (ExPEC). A comprehensive collection of 101 ExPEC isolates was sequenced using long-read technology. Comparative analysis with a laboratory non-pathogenic strain revealed over 3,000 genomic regions of difference encompassing nearly 12,000 unique genes. Importantly, many of those were highly associated with ExPEC, including several linked to carbohydrate metabolism and others of unknown function. These associations could be observed thanks to assembling a large collection of over 131000 genomes annotated with detailed metadata, which in many cases helped us to link them to their clinical status, including ExPEC (20523 genomes) or IPEC (42791 genomes) labels. Notably, the we identified distinct patterns of Type VI secretion systems (T6SS), a bacterial nanoweapon, between ExPEC and IPEC and we trained a model to distinguish these pathogroups based on these and other virulence markers. These results were published in mSystems (Nesporova et al., 2025; https://pubmed.ncbi.nlm.nih.gov/40530882/).

In parallel, high-throughput single-cell fluorescence microscopy was employed to quantify morphological and behavioural traits across ExPEC strains under different environmental conditions. The analyses showed that even closely related strains can exhibit striking phenotypic variability, with major changes in cell morphology linked to the loss of a small genomic region containing nine genes. In a complementary study, we provided functional annotations of multiple genes with unknown functions and we showed their conservation across E. coli and other bacterial species. This work was published in mSystems (Sondervorst et al., 2025; https://pubmed.ncbi.nlm.nih.gov/40492708/).

Together, these achievements have generated one of the largest curated genomic resources for E. coli, provided new insights into the molecular determinants of ExPEC virulence, and identified promising genetic and phenotypic targets for future translational research and infection control strategies.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

EXCELLGEN advances current knowledge by combining two large-scale approaches rarely integrated in microbiology: comparative genomics of over 130,000 genomes and quantitative single-cell phenotyping. This unique combination enables linkage between genetic variation and cellular function.
The project uncovered several ExPEC-associated genes, including metabolic and secretion system components, that likely play a key role in pathogenic adaptation. By identifying these specific molecular signatures, EXCELLGEN lays the foundation for developing new diagnostic markers and therapeutic targets against ExPEC infections.
All computational pipelines, genomic data, and preprints were shared openly via GitHub and public repositories, reinforcing FAIR and Open Science principles and facilitating follow-up research. Future work will expand functional validation of ExPEC-associated genes and explore their mechanistic roles, potentially leading to translational applications in human and veterinary medicine.

Clusters of virulence-associated genes linked to ExPEC and IPEC, © 2025 Nesporova et al. under CC 4

PCA based on VAGs for different types of metadata. © 2025 Nesporova et al. under CC 4