Multi-country and intersectoral assessment of cluster congruence between different bioinformatics pipelines for genomics surveillance of foodborne bacterial pathogens

Food and waterborne disease (FWD) surveillance requires Whole-Genome Sequencing (WGS)-based systems following a One Health approach. However, different laboratories employ different WGS pipelines in their routine surveillance activities, casting doubt on the comparability of their results and hindering optimal communication at intersectoral and international levels. Through a collaborative effort involving eleven European institutes across seven countries and spanning the food, animal and human health sectors, we aimed to assess the inter-laboratory comparability of WGS clustering results for four important foodborne pathogens: Listeria monocytogenes, Salmonella enterica, Escherichia coli and Campylobacter jejuni. Each participating institute (n=9) applied its surveillance pipeline over the same WGS datasets (>2000 isolates per species), and, for each pipeline, genetic clusters were identified at each possible allele/SNP distance threshold. Inter-pipeline clustering congruence was assessed by calculating a Congruence Score (relying on Adjusted Wallace and Adjusted Rand coefficients) across all resolution levels, followed by an in-depth comparative analysis of cluster composition at outbreak level. An additional cluster congruence assessment was performed between WGS and traditional typing, which, depending on the species, included Sequence Type (ST), Clonal Complex (CC) and/or serotype. Our results revealed a general high concordance between allele-based pipelines at all resolution levels for all species, except for C. jejuni, where the different resolution power of available allele-based schemas led to marked discrepancies. Still, this study identified non-negligible differences in allele-based pipeline performance for outbreak cluster detection, suggesting that a threshold flexibilization is important for the detection of similar outbreak signals by different laboratories. These results, together with the observation that different STs, CCs and serotypes exhibit remarkably different genetic diversity, should inform future threshold selections for outbreak case definitions. In conclusion, this study provides valuable insights into the comparability of pipelines commonly used for routine genomics surveillance, and reinforces the need, while demonstrating the feasibility, of conducting continuous and comprehensive WGS pipeline comparability assessments. Ultimately, it opens good perspectives for a smoother international and intersectoral cooperation and communication towards a sustainable and efficient One Health FWD surveillance. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by co-funding from the European Union's Horizon 2020 Research and Innovation program under grant agreement No 773830: One Health European Joint Programme (2020 to 2022) (https://onehealthejp.eu/projects/foodborne-zoonoses/jrp-beone) and by the ISIDORe project (funding from the European Union's Horizon Europe Research & Innovation Programme, Grant Agreement no. 101046133). VM contribution was funded by national funds through FCT - Foundation for Science and Technology, I.P., in the frame of Individual CEEC 2022.00851.CEECIND/CP1748/CT0001 (2023 onwards). JDS contribution was supported by the project "Sustainable use and integration of enhanced infrastructure into routine genome-based surveillance and outbreak investigation activities in Portugal" (GENEO, https://www.insa.min-saude.pt/category/projectos/geneo/) on behalf of the EU4H programme (EU4H-2022-DGA-MS-IBA-1). Research at the National Veterinary Research Institute (PIWet) Poland was supported by the Polish Ministry of Education and Science from the funds for science in the years 2018-2022 allocated for the implementation of a co-financed international project. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Anonymized sequencing reads of the BeONE dataset are deposited in the European Nucleotide Archive (ENA) database under the BioProjects PRJEB57166, PRJEB57179, PRJEB57098 and PRJEB57119. Genome assemblies are deposited in the Zenodo repository (L. monocytogenes: 10.5281/ZENODO.7267486; S. enterica: 10.5281/ZENODO.7267785; E. coli: 10.5281/ZENODO.7267844; C. jejuni: 10.5281/ZENODO.7267879). The public dataset data was retrieved from Zenodo (L. monocytogenes: 10.5281/ZENODO.7116878; S. enterica: 10.5281/ZENODO.7119735; E. coli: 10.5281/ZENODO.7120057; C. jejuni: 10.5281/ZENODO.7120166). The collection of scripts used to conduct these analyses are available at the github repository https://github.com/insapathogenomics/WGS\_cluster\_congruence. Supplementary data are available in the Zenodo repository (https://doi.org/10.5281/zenodo.12805750).