Phase variation in Flavobacterium psychrophilum: characterization of two distinct colony phenotypes

Abstract

Four 'smooth' and 4 'rough' colony phenotypes of the Gram-negative fish pathogen Flavobacterium psychrophilum isolated from rainbow trout Oncorhynchus mykiss were characterized using biochemical, physiological, molecular and virulence tests to better understand the pathogenesis of the bacterium. Biochemically, the 2 cell types did not react significantly differently. Physiologically, the 2 phenotypes had distinct characteristics, and, when grown in broth, the smooth cells were found to be autoagglutinating and able to switch into the non-agglutinating rough phenotype. The rough cells did not switch into the smooth phenotype under any growth conditions tested, indicating that the phase variation from the smooth to rough phenotype is irreversible or that the conditions for the reversible switch are still to be found. Smooth cells were hydrophobic and more adhesive compared to the hydrophilic rough cells, suggesting that the phase variation most probably involves one or several surface structures other than outer membrane proteins and lipopolysaccharides that were found to be similar in both types. Analysis of extracellular products produced by the 2 cell types indicated furthermore that a difference in enzymatic activities could exist. Both cell types were virulent for rainbow trout in an intramuscular challenge; thus, the distinct physiological characteristics of the phenotypes do not seem to be directly associated with virulence, when the body surface of the fish is disregarded. The results suggest that phase variation occurs in F. psychrophilum, but that the importance of the 2 phenotypes for the pathogenesis of the bacterium has still to be investigated.

In vitro opsonin-independent interactions between cells of smooth and rough phenotypes of Flavobacterium psychrophilum and rainbow trout (Oncorhynchus mykiss) head kidney macrophages

“…The rough surface, a consequence of the reduction of a glycopeptidolipid in the cell wall associated with motility and biofilm formation in the smooth variant, was suggested to be the reason for this. The rough variant was also reported to be more virulent than the smooth variant in mice infection models [19] which does not, however, seem to be the case for the smooth and rough variants of F. psychrophilum as both are virulent for rainbow trout when injected [8].…”

“…The phenotype of these F. psychrophilum cells was however not noted [7]. Both smooth and rough cells have been reported to cause disease in rainbow trout [8]. In the future it needs to be investigated whether the less cytotoxic smooth F. psychrophilum cells are able to survive and multiply better in macrophages compared with the Fig.…”

“…All isolates contained cells of smooth (S) and rough (R) colony phenotypic variants that were separated and named after the corresponding colony type as P13-4S/96, P13-4R/96, P6-1S/07, P6-1R/07, P6-3S/07 and P6-3R/07 [8]. Stock suspensions were maintained in trypton yeast extract salts (TYES) broth [20] with 20% (v/v) glycerol at À70 C. For all assays, bacteria were grown on TYES agar [20] for five days at 15 C and suspended in L-15 medium (Sigma) to an optical density (OD) of 1.5 AE 0.05 at 520.…”

“…F. psychrophilum has previously been reported to stimulate [4], survive and multiply in [5], adhere to [6] and be cytotoxic to [7] rainbow trout (Oncorhynchus mykiss) phagocytes. In these studies however, the phase varying nature of F. psychrophilum, expressed as smooth and rough phenotypic variants [8], was not taken into consideration. In our previous study, we demonstrated different hemolytic activity in smooth and rough phenotypes of F. psychrophilum isolated from rainbow trout.…”

Multilocus Sequence Typing Identifies Epidemic Clones of Flavobacterium psychrophilum in Nordic Countries

“…F. psychrophilum from rainbow trout included isolates from eggs (n ϭ 4), ovarian fluid (n ϭ 10), and milt (n ϭ 12). Environmental isolates from water samples (n ϭ 11) from fish farm environments, as well as three rough laboratory mutant strains prepared from isolated smooth colony types by repeated passages in TYES broth (34), were included in the collection.…”