Characterization methods for Shiga toxin-producing E. coli – a part of OH-HARMONY-CAP G.S Johannessen 1 • R. Tozzoli 2 • A. Pista 3 • C. Flink 4 • F. Alves3 • D. Bolton5 • M. Kirchner6 • N. Boisen7 1. Norwegian Veterinary Institute, Ås, Norway • 2. Istituto Superiore di Sanità, Rome, Italy • 3. National Institute of Health Doutor Ricardo Jorge, Lisbon, Portugal • 4. Swedish Food Agency, Uppsala, Sweden • 5. Teagasc Food Research Centre, Ashtown, Dublin, Ireland • 7. Animal and Plant Health Agency, Addlestone, United Kingdom • 7. Statens Serum Institut, Copenhagen, Denmark In summary, 41 laboratories responded that they tested for STEC, including human clinical (17), animal (13), food (24), feed (6) and environmental samples (11) [1]. For the typing of STEC, serotyping is commonly being used, although it is clear that the pathogenic potential of the isolates cannot be determined by serotypes alone and that all STEC are considered as pathogenic to humans [2]. A total of 25 and 15 labs performed O- and H-typing, respectively, using serological, molecular and a combination of these methods (fig 1). A total of 35 respondents tested their isolates for virulence-associated genes, where a majority tested for stx and eae genes. Approximately half of the labs testing for stx genes also carried out subtyping of these, while subtyping of eae was not common. From the responses to the questionnaire it was not possible to discriminate between laboratories using PCR or WGS for the detection of virulence genes, but we assume that both methods are employed. In addition 17 laboratories reported that they performed phylogenetic analyses, i.e. genetic comparisons to determine the relatedness of isolates (fig 2). While the literature search yielded around 130 hits, we also looked into recent publications from organizations such as EFSA and FAO/WHO [2,3]. Several different methods for typing and characterization of STEC have been tested, but serotyping and virulence gene profiling of stx genes and their subtypes seem to be the most used methods. For further characterization molecular methods such as PFGE and MLVA have been commonly used, but with recent developments in sequencing technology, many laboratories have implemented whole genome sequencing (WGS) based methods for further characterization of STEC, such as single nucleotide polymorphisms (SNP) and core and/or whole genome multi locus sequence typing (cg and/or wg MLST). References: [1] D-IA.2.2.OH-Harmony-Cap 3.2. 2021. https://zenodo.org/record/5788415#.Yjm6--fMKUk, [2] EFSA, 2020. DOI: https://doi,org/10.2903/j.efsa.2020.5967, [3] FAO/WHO, 2018. https://www.fao.org/documents/card/en/c/CA0032EN/ This poster is part of the European Joint Programme One Health EJP. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No 773830. RESULTS METHODS The work package (WP) 3 of the OH-HARMONY-CAP project (One Health Harmonization of Protocols for the detection of foodborne pathogens and AMR determinants) concerns laboratory interoperability, i.e. the potential for using harmonized method across the human, veterinary and food sectors. Here we aimed to map characterization methods for Shiga toxin-producing E. coli currently in use, identify gaps and recommend a harmonised strategy for such methods. • A questionnaire was developed in WP3 to examine current practices in public health, veterinary, food testing and National Reference Laboratories and the responses from the questionnaire with respect to typing and characterization of STEC were analysed. • A literature search in PUBMED using the search string (Shiga toxin-producing Escherichia coli) AND (characterization methods) AND (typing methods) with criteria English language and publication period 2000 - 2021, was performed. • Other sources, such as EURL for E. coli, EFSA, ECDC, OIE and FAO/WHO were also examined. CONCLUSION AND PERSPECTIVES Molecular typing methods, including t