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.IMPORTANCEThe genus Tenacibaculum includes several fish-pathogenic species that pose major concerns in marine aquaculture, yet their biofilm biology remains poorly characterized. By analyzing 40 isolates across complementary growth models, we show that biofilm architecture represents a quantifiable source of phenotypic variation within the genus. Strains adopted various developmental profiles that uncouple the growth rate, biomass yield, and three-dimensional structural complexity. Importantly, experiments performed on four representative strains indicate that biofilm-associated antimicrobial survival is strain-dependent and may be influenced by biofilm structural organization. These findings emphasize the need to account for structure-based functional profiling in addition to species-based classification. By identifying candidate architectural markers and representative phenotypic clusters, this work provides a rational framework for future mechanistic studies and for evaluating whether specific biofilm traits are associated with persistence or risk in aquaculture systems.