Biofilm formation by the fish pathogen Flavobacterium columnare: development and parameters affecting surface attachment

Abstract

Flavobacterium columnare is a bacterial fish pathogen that affects many freshwater species worldwide. The natural reservoir of this pathogen is unknown, but its resilience in closed aquaculture systems posits biofilm as the source of contagion for farmed fish. The objectives of this study were (i) to characterize the dynamics of biofilm formation and morphology under static and flow conditions and (ii) to evaluate the effects of temperature, pH, salinity, hardness, and carbohydrates on biofilm formation. Nineteen F. columnare strains, including representatives of all of the defined genetic groups (genomovars), were compared in this study. The structure of biofilm was characterized by light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. F. columnare was able to attach to and colonize inert surfaces by producing biofilm. Surface colonization started within 6 h postinoculation, and microcolonies were observed within 24 h. Extracellular polysaccharide substances and water channels were observed in mature biofilms (24 to 48 h). A similar time course was observed when F. columnare formed biofilm in microfluidic chambers under flow conditions. The virulence potential of biofilm was confirmed by cutaneous inoculation of channel catfish fingerlings with mature biofilm. Several physicochemical parameters modulate attachment to surfaces, with the largest influence being exerted by hardness, salinity, and the presence of mannose. Maintenance of hardness and salinity values within certain ranges could prevent biofilm formation by F. columnare in aquaculture systems.

Fig 1

Colonization and biofilm development by F. columnare strain ALG-00-530 on glass slides. The bright-field light microscopy images in panels A to F display cell attachment and biofilm formation at 6, 12, 18, 24, 36, and 48 h postinoculation. Scale bars, 20 μm in panels A, C, D, E, and F and 10 μm in panel B.

Fig 2

Colonization and biofilm development by F. columnare strain ALG-00-530 on glass slides analyzed by SEM. (A) Cells attached at 12 h postinoculation (scale bar, 30 μm). (B and C) Details of cell aggregation at 48 h (scale bars, 20 μm). (D) Closeup of F. columnare cells in the early stages of EPS secretion (scale bar, 1 μm). The black arrows indicate bacterial surface blebbing (SB), and the white arrow indicates extracellular polysaccharide.

Fig 3

SEM of F. columnare cells attached to glass slides and fixed with HMDS (A) and osmium tetroxide (B). Arrows indicate EPS and bacterial cells (BC). Scale bars, 2 μm.

Fig 4

Viability of F. columnare cells in biofilm determined with the Live/Dead cell viability kit and examination by CLSM. Biofilm was grown on glass slides for 48 h postinoculation. Live cells are stained green, dead cells are red, and EPS is stained blue with calcofluor white. Scale bar, 10 μm.

Fig 5

Biofilm formation by F. columnare inside microfluidic chambers. F. columnare strains ALG-00-530 and ARS-1 were introduced into microfluidic chambers kept under constant flow of MS broth, and growth and biofilm formation were monitored microscopically. Time zero indicates that the images were acquired 24 h after the introduction of bacterial cells into the chamber. The following columns show images captured 1 and 3 h after the cells were introduced into the chamber. Note that strain ALG-00-530 formed a biofilm that occupied most of the channels, while ARS-1 formed cell aggregates only at the bottom of the channel under reduced shear stress. Scale bars, 20 μm.

Fig 6

Adhesion (mean absorbance ± standard error) of F. columnare strains to microtiter plates. Light gray bars show cell growth, and dark gray bars represent attached cells at 48 h postinoculation. Panels A to E show the effects that temperature, pH, salinity, hardness, and the addition of carbohydrates had on cell attachment and proliferation. Different letters a to e indicate significant differences (P < 0.05) in cell growth. Different letters w to z indicate significant differences (P < 0.05) in cell attachment.

Fig 7

Clustering analysis of cell attachment using a composite data set with all of the parameters assayed. A similarity matrix was obtained by using the Pearson product moment correlation coefficient, and the dendrogram was generated by UPGMA. The scale bar shows the percent similarity between strains. The two main groups were delineated at 50% similarity.