Flavobacterium psychrophilum adhesion and biofilm formation

Abstract

Flavobacterium psychrophilum causes serious infections in farmed rainbow trout (Oncorhynchus mykiss) and subsequent substantial economic losses to fish farmers, globally. Over the past years, few studies have conclusively suggested that adhesion to and biofilm formation on surfaces, are of importance for F. psychrophilum, probably conferring a selective advantage by increasing its ability to persist in fish-farming environments. It is known that phase variation occurs in F. psychrophilum with two different morphological colony phenotypes, smooth and rough, however the role of these phenotypes in adhesion and biofilm formation are not well known yet. The aim of this thesis was to provide information of the adherence of different F. psychrophilum phenotypes to host tissues and screen the temporal persistence of bacterial cells on tissues. Another aim of this thesis was to assess the inhibiting effect of different compounds and bacteriophages on adhesion and the biofilm formation and mature biofilm of both phenotypes. The role of environmental conditions and factors on the biofilm formation of both phenotypes of F. psychrophilum in vitro was also evaluated. Cells of both phenotypes initially adhered to mucosal surfaces of rainbow trout, but only the rough cells were present on tissues for a longer time. Both phenotypes showed a tissue tropism with the fin tissue being the most adhered. Skin mucus promoted the growth of both colony phenotypes but none of the isolates were able to form biofilms in mucus. The results of this thesis showed that a treatment with a combination of carbohydrates, D- and L-amino acids, A-type proanthocyanidins, EDTA and proteinase K reduced the adhesion of mainly smooth cells. Smooth cells produced stronger biofilms compared to rough cells in nutrientrich media, and development of these biofilms was Ca2+ dependent. Most of the examined compounds inhibited the biofilm formation of smooth isolates reaching up to 80% inhibition, while only 2-aminoimidazole, emodin, parthenolide and D-leucine reduced the biomass of mature biofilms at a concentration-dependent manner. Lytic bacteriophages inhibited the biofilm formation of F. psychrophium, while they partially reduced the biomass of the mature biofilms. In conclusion, mucosal surfaces of rainbow trout colonized by F. psychrophilum, and the growth of bacterial cells in skin mucus suggest that mucus-binding sites were available for the bacterial adhesins. The inhibition of mainly smooth cell adhesion to surfaces by most of the compounds was probably due to disruption of the protein-protein interactions that hold the cells together and to cell-surface hydrophobicity. In contrast, the examined rough isolates exhibited resistance to most inhibiting compounds during their adhesion to and biofilm formation on inert surfaces. The ability of F. psychrophilum cells to use fish feed for biofilm formation suggests that there are probable enough nutrients in fish-farming environments for the F. psychrophilum to form biofilms. The results of this thesis also showed that the biofilm formation and mature biofilms can be modulated by using certain compounds and a combination of bacteriophages indicating the potential to reduce the F. psychrophilum biofilms in fish farms, if F. psychrophilum produce biofilms on fish-farming surfaces.