Entericidin Is Required for a Probiotic Treatment (Enterobacter sp. Strain C6-6) To Protect Trout from Cold-Water Disease Challenge

Abstract

c 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 ⌬ecnAB and C6-6 ⌬ecnAB<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. P robiotics are defined as live or dead microbial feed supplements that provide health benefits to the host animal (1). Probiotic strains of Bifidobacterium, Lactobacillus, and Streptococcus (2) have been used successfully in people and terrestrial animals, including poultry (3) and cattle (4), for the past century. Probiotic supplementation has gained attention from the aquaculture industry during the last 2 decades, especially for shrimp production, where antibiotic treatment has been reduced dramatically in favor of supplementing feed with commercially available probiotics composed of Bacillus subtilis and Vibrio alginolyticus (5, 6). In aquaculture, a probiotic is further defined as a live microbial cell that, when administered via the feed or to the rearing water, benefits the host by improving disease resistance, health and growth performance, feed utilization, stress response, and meat quality (7,8). The mechanism of action of probiotics has been defined vaguely as improving the host's "microbial balance" (9) or the balance of the ambient environment, but molecular mechanisms are rarely investigated further, even though a number of applied studies have demonstrated probiotic efficacy against a variety of Gram-negative and Gram-positive pathogens in fish and shellfish (7).In the case of salmonid species, such as Atlantic salmon (Salmo salar Linnaeus) and rainbow trout (Oncorhynchus ...

Nisin Z Production by Lactococcus lactis subsp. cremoris WA2-67 of Aquatic Origin as a Defense Mechanism to Protect Rainbow Trout (Oncorhynchus mykiss, Walbaum) Against Lactococcus garvieae

“…Moreover, Millette et al (2008) showed the anti-infective properties of two bacteriocinogenic LAB strains, the NisZ producer L. lactis MM19 and the pediocin PA-1/AcH producer P. acidilactici MM33, which were capable of reducing the intestinal colonization by vancomycin-resistant enterococci in murine models. Recently, Schubiger et al (2015) demonstrated that entericidin produced by the Gram-negative strain Enterobacter sp. C6-6 is required to protect rainbow trout against cold water disease caused by Flavobacterium psychrophilum.…”

The ecnA antitoxin is not only important for human pathogens: Evidences of its role in the plant pathogen Xathomonas citri subsp. citri

“…The E. coli ecnAB locus encodes the synthesis of two small lipoproteins that are part the entericidin locus TA system, because of hypothesized similarities to a plasmid addiction module, mazEF , that has been studied in E. coli (24). EcnA was designated as the antitoxin that counteracts the EcnB toxin, whose overproduction results in a more pronounced bacterial lysis than that observed in the co-expression of both ecnA and ecnB (25). Different functions have been proposed for the ecnAB TA system, including programmed cell death (24), adherence in the respiratory tract of humans (26), and its requirement for the probiotic functions of Enterobacter sp.…”

“…Different functions have been proposed for the ecnAB TA system, including programmed cell death (24), adherence in the respiratory tract of humans (26), and its requirement for the probiotic functions of Enterobacter sp. strain C6-6 (25).…”

The ecnA Antitoxin Is Important Not Only for Human Pathogens: Evidence of Its Role in the Plant Pathogen Xanthomonas citri subsp. citri

“…Many of these genes have been extensively studied (18,22,23); however, there are genes whose functions in X. citri are still unknown. These genes include the components of the ecnAB toxin/antitoxin (TA) system (XAC_RS20190/ XAC_RS20185 [GenBank database; genome coordinates 4700807 to 4700959/4700598 to 4700732]), which is known to play an important role in Escherichia coli (24,25). The E. coli ecnAB locus encodes two small lipoproteins that are considered to be components of the entericidin locus TA system because of hypothesized similarities to a plasmid addiction module, mazEF, that has been studied in E. coli (24).…”

“…The E. coli ecnAB locus encodes two small lipoproteins that are considered to be components of the entericidin locus TA system because of hypothesized similarities to a plasmid addiction module, mazEF, that has been studied in E. coli (24). EcnA was identified as the antitoxin that counteracts the EcnB toxin, whose overproduction results in more pronounced bacterial lysis than that observed under coexpression of ecnA and ecnB (25). Different functions have been proposed for the ecnAB TA system, including roles in programmed cell death (24) and adherence to the respiratory tract of humans (26) and a requirement of this system for the probiotic functions of Enterobacter sp.…”