The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Biomedical Center Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

CEITEC VFU University of Veterinary and Pharmaceutical Sciences Brno Brno Czech Republic

Center for Infectious Disease Control National Institute for Public Health and the Environment Bilthoven The Netherlands

Department of Bacteria Parasites and Fungi Statens Serum Institut Copenhagen Denmark

Department of Bacteriology and Epidemiology Wageningen Bioveterinary Research Lelystad The Netherlands

Department of Biology and Wildlife Diseases Faculty of Veterinary Hygiene University of Veterinary and Pharmaceutical Sciences Brno Brno Czech Republic

Department of Infectious Diseases and Immunology Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands

Department of Microbiology Faculty of Medicine and University Hospital in Pilsen Charles University Pilsen Czech Republic

Department of Veterinary and Animal Sciences Faculty of Health and Medical Sciences University of Copenhagen Frederiksberg Denmark

Institute of Hygiene and Infectious Diseases of Animals Justus Liebig University Giessen 35392 Giessen Germany

Université de Lyon ANSES Unité Antibiorésistance et Virulence Bactériennes Lyon France

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