Entericidin is required for a probiotic treatment (Enterobacter sp. strain C6-6) to protect trout from cold-water disease challenge.
Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB<pET101::ecnAB>) and the wild-type strain (C6-6) were added to the fish diet every day 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 longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens.
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USA Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA.
Carla B
CB
Schubiger
Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA.
Lisa H
LH
Orfe
Department of Fish and Wildlife Sciences and the Aquaculture Research Institute, University of Idaho, Moscow, Idaho, USA.
Ponnerassery S
PS
Sudheesh
Department of Fish and Wildlife Sciences and the Aquaculture Research Institute, University of Idaho, Moscow, Idaho, USA.
Kenneth D
KD
Cain
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USA Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA.
Devendra H
DH
Shah
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, USA Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, USA drcall@wsu.edu.
Douglas R
DR
Call
D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000898:Antibiosis; D001424:Bacterial Infections / Q000517:prevention & control / Q000662:veterinary*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004754:Enterobacter / Q000254:growth & development / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000378:metabolism; D005393:Fish Diseases / Q000517:prevention & control*; D005417:Flavobacterium / Q000187:drug effects / Q000254:growth & development*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000382:microbiology*; D019936:Probiotics / Q000008:administration & dosage*; D017422:Sequence Analysis, DNA
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial