Mechanism of action of an Enterobacter probiotic on Flavobacterium psychrophilum
Schubiger, Carla B.
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Flavobacterium psychrophilum is a Gram-negative, filamentous bacterium that causes systemic Coldwater Disease (CWD) in multiple fish species including salmonids. CWD is one of the leading problems in the rainbow trout (Oncorhynchus mykiss Walbaum) and salmon industries and induces mortality rates up to 70%, thereby causing severe economic losses. To date no vaccine is available for commercial use and the disease is treated with antibiotics. Alternative management strategies, including the use of probiotics, may prove to be a more cost effective and environmentally sustainable approach to controlling CWD. Probiotics are live microbes that are beneficial to the health of the host and can be administered as a dietary supplement. Recently, an autochthonous Enterobacter strain (C6-6) was shown to inhibit the growth of F. psychrophilum in vitro. Using two different assays (exposure to C6-6 supernatant and a "diffusion assay"), F. psychrophilum growth was inhibited in a manner consistent with exposure to a soluble inhibitor produced by C6-6. Furthermore, when trout were fed Enterobacter C6-6 as a probiotic supplement, mortality from a F. psychrophilum challenge was significantly reduced. For this study, protease digest of the supernatant resulted in a loss of inhibiting activity and size fractionation revealed that the inhibiting protein is approximately 3 kDa or less. SDS-PAGE of concentrated supernatant yielded in a protein band at ≤3 kDa and subsequent LC-MS/MS analysis indicated the presence of a putative pore-forming entericidin. Using agar-plate diffusion assays, entericidin gene knockouts and their respective complements were used against F. psychrophilum to confirm entericidin as the inhibiting agent; the entericidin knockout lost its inhibiting phenotype against F. psychrophilum and the complemented knockout showed restored inhibiting activity. Enterobacter C6-6, its entericidin gene knockout and its complement were also mixed into a commercial salmonid diet and administered to rainbow trout fry that were subsequently challenged with F. psychrophilum by subcutaneous injection on the eleventh day of the feeding trial. Fish receiving feed containing entericidin showed a higher relative survival rate (58.1%) than fish receiving the knockout (19.4%) or were not fed any probiotics (reference value). These results provide new insight into the mechanism of action of this probiotic and yields new findings relevant to control CWD and other pathogens. Besides application of the probiotic itself, by identifying the inhibitory compound it might be possible to produce the inhibitor directly or as part of transgenic feed components thereby decreasing cost and increasing efficiency of administration.