Biofilm formation by the fish pathogen Flavobacterium columnare: a quantitative and qualitative study

Abstract

The objective of this study was to evaluate the effect that temperature, pH, salinity, hardness and carbohydrates have on biofilm formation by the fish pathogen Flavobacterium columnare. Nineteen F. columnare strains, including representatives from all genomovars, were compared in this study. Biofilm formation was quantified spectrophotometrically by allowing cells to attach and colonize polystyrene wells that were then stained with crystal violet. Although significant strain-to-strain variation was observed, differences in biofilm formation between genomovars were not significant. The two main factors influencing biofilm formation were salinity and hardness. Low salinity (5 ppt) and high hardness (360 ppm) favored biofilm proliferation. In addition, the carbohydrate mannose also promoted biofilm. On the contrary, warmer temperatures (30°C) inhibited biofilm formation. The ultrastructure of biofilm developed on glass slides was characterized by light, confocal laser and scanning electron microscopy. Surface colonization started within 6 h post-inoculation and microcolonies were observed within 24 h. Extracellular polysaccharide substances (EPS) and water channels were observed in mature biofilms (24-48 h). The virulence potential of biofilm was confirmed by cutaneous inoculation of channel catfish fingerlings with mature biofilm. My results showed that Flavobacterium columnare can attach to inert surfaces and colonize them by producing biofilm. My data showed that some physicochemical parameters modulated biofilm formation by F. columnare. The values identified in this study could be used as reference values to prevent biofilm formation in aquaculture settings. I have experimentally proved that it is possible to induce columnaris disease in channel catfish when compromised skin is exposed to mature biofilm of F. columnare. This suggests that F. columnare could use biofilm as reservoir in the natural environment but also in aquaculture facilities.