CHARACTERIZATION OF A PUTATIVE PROBIOTIC ENTEROBACTER STRAIN (C6-6) AND POTENTIAL MECHANISMS ASSOCIATED WITH PROTECTION OF RAINBOW TROUT CHALLENGED WITH Flavobacterium psychrophilum

An autochthonous Enterobacter strain (C6-6) provides protection against coldwater disease/rainbow trout fry syndrome (CWD/RTFS) in fish when administered as a microbial feed additive, and directly inhibits the in vitro growth of Flavobacterium psychrophilum. This putative probiotic works equally well with or without microencapsulation when top coated onto the diet, and appears to be a potential alternative strategy for managing CWD in aquaculture. It was originally isolated from healthy rainbow trout and appears to colonize the gastrointestinal (GI) tract and exist as part of the normal flora. Continued investigation of this Enterobacter strain has suggested that growth inhibition of F. psychrophilum and CWD related protection in fish is directly linked to a low-molecular-mass (< 3kDa) antimicrobial fraction found in bacterial culture supernatant. The mechanisms associated with health benefits of probiotics are seldom investigated; however, SDS-PAGE, and subsequent tandem mass spectroscopy of this < 3kDa fraction identified an entericidin (EcnB), a small membrane lipoprotein considered a putative pore-forming toxin. By creating ecnAB knockout strains of Enterobacter C6-6, inhibition of F. psychrophilum was negated, while ecnAB-complemented knockouts showed recovery of the inhibitory phenotype. In fish experiments, the engineered C6-6 strains and the wild-type (C6-6) strain were added to the diet and fish were fed for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly greater survival than fish fed the ecnAB knockout strain (P<0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (Fig 1). This demonstrates that this antimicrobial peptide is the predominant molecule associated with the probiotic activity of Enterobacter C6-6, and may present new therapeutic and prophylactic treatment opportunities for F. psychrophilum or other similar pathogens.