Diversity of biofilm architectures within the genus Tenacibaculum

Marine aquaculture sustainability is increasingly threatened by tenacibaculosis, a disease caused by Tenacibaculum species that are thought to persist through biofilm formation. However, the architectural diversity of biofilms across the genus and its contribution to persistence remain unclear. Here, we conducted a large-scale phenotypic analysis of 40 Tenacibaculum isolates including 19 T. maritimum strains, an aquaculture-relevant fish pathogen, and 21 type strains representing different species. Using three complementary growth models: planktonic free-cell cultures, macro-colony biofilms, and submerged biofilms, 17 quantitative parameters were measured. We observed substantial inter- and intra-species heterogeneity. Strains adopted distinct developmental phenotypes, ranging from a high-yield, slow-growth profile associated with thick, structured three-dimensional biofilms to fast-growing communities with limited architectural complexity. Confocal imaging revealed high structural variation across the genus, with Congo red-labeled extracellular materials exhibiting strain-specific differences in porosity and spatial distribution within the biofilm. Multivariate analysis partitioned the collection into four phenotypic clusters defined by distinct growth and structural signatures. Antimicrobial susceptibility assays using oxytetracycline hydrochloride in four representative strains showed that biofilm-associated tolerance was strain-dependent. Together, this work identifies biofilm architecture as a measurable and informative dimension of phenotypic diversification in Tenacibaculum and provides candidate structural markers to support future mechanistic studies and the development of strategies aimed at limiting the persistence of problematic Tenacibaculum species in aquaculture systems.