Laboratory serial passage of Yersinia ruckeri: phenotypic and genomic virulence dynamics

Introduction: The routine serial passage of bacterial pathogens on artificial media is common in diagnostic and research laboratories, but such “laboratory domestication” may alter virulence traits and decouple reference strains from the field populations they are meant to represent. Materials and methods: To examine this in Y. ruckeri, isolates were revived from frozen primary stocks, confirmed by species-specific PCR and MALDI-TOF MS, and baseline biochemical and virulence-associated traits (hemolysis, gelatinase, Tween 20/80 hydrolysis, motility, temperature and NaCl tolerance) were recorded. Isolates then underwent 28 days of daily passage on TSA at 28 °C, with virulence phenotypes re-assessed at predefined time points. In parallel, one representative isolate was sequenced on days 0 and 28 using Oxford Nanopore technology, and assemblies were compared by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), followed by virulence factor database (VFDB)-based virulence profiling, OrthoVenn orthologous clustering and antibiotics and secondary metabolite analysis shell (antiSMASH) analysis of secondary metabolite biosynthetic gene clusters. Results: Core biochemical traits (gelatin hydrolysis, γ-hemolysis, growth at 4 °C and 37 °C, and tolerance to high NaCl) remained stable, whereas motility and Tween 20/80 hydrolysis shifted in four of ten isolates, with fewer motile or Tween-positive strains on day-28. Genomically, day-0 and day-28 assemblies were nearly indistinguishable from each other and from Y. ruckeri ATCC 29473, yet showed modest changes in relation to their virulence gene content; secondary metabolite biosynthetic gene cluster (BGC) types were conserved, and OrthoVenn identified 3629 shared orthologous clusters plus only seven small mobile element-associated clusters unique to day-0. Conclusions: These findings indicate that even short-term, routine passage can remodel phenotypic virulence markers and fine-scale virulence repertoires in Y. ruckeri, while core metabolism, stress tolerance and secondary metabolite capacity remain stable. Phenotypic drift toward non-motile, Tween-negative profiles—already known from field biotypes—shows that domestication does not equal simple attenuation and can complicate the interpretation of in vitro markers as surrogates for in vivo virulence, underscoring the importance of passage history and culture conditions as critical metadata for virulence studies, challenge trials and vaccine development.