BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

CEITEC VFU University of Veterinary Sciences Brno Brno Czech Republic

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees Faculty of Veterinary Hygiene and Ecology University of Veterinary Sciences Brno Brno Czech Republic

Department of Infectious Diseases and Microbiology Faculty of Veterinary Medicine University of Veterinary Sciences Brno Palackeho Trida 1946 1 612 42 Brno Czech Republic

Department of Infectious Diseases and Preventive Medicine Veterinary Research Institute Brno Brno Czech Republic

Department of Molecular and Clinical Pathology and Medical Genetics University Hospital Ostrava Ostrava Czech Republic

Department of Zoology Fish Production Hydrobiology and Apiculture Faculty of Agriculture Mendel University Brno Czech Republic

Faculty of Electrical Engineering and Communication Department of Biomedical Engineering Brno University of Technology 616 00 Brno Czech Republic

Molecular Systems Biology Department of Functional and Evolutionary Ecology Faculty of Life Sciences University of Vienna 1030 Vienna Austria

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