Characterization of a Highly Virulent Edwardsiella anguillarum Strain Isolated From Greek Aquaculture, and a Spontaneously Induced Prophage Therein

Abstract

Edwardsiella-associated outbreaks are increasingly reported on both marine and freshwater aquaculture setups, accounting for severe financial and biomass losses. E. tarda, E. ictaluri, and E. hoshinae have been the traditional causative agents of edwardsiellosis in aquaculture, however, intensive studies due to the significance of the disease have just recently revealed two more species, E. piscicida and E. anguillarum. Whole genome sequencing that was conducted on the strain EA011113, isolated from farmed Diplodus puntazzo after an edwardsiellosis outbreak in Greece, confirmed it as a new clinical strain of E. anguillarum. Extensive phylogenetic analysis showed that this Greek strain is closely related to an Israeli E. piscicida-like clinical strain, isolated from diseased groupers, Epinephelus aeneus and E. marginatus in Red Sea. Bioinformatic analyses of E. anguillarum strain EA011113 unveiled a wide repertoire of potential virulence factors, the effect of which was corroborated by the mortalities that the strain induced in adult zebrafish, Danio rerio, under different levels of infection intensity (LD50 after 48 h: 1.85 × 104 cfu/fish). This strain was non-motile and according to electron microscopy lacked flagella, a fact that is not typical for E. anguillarum. Comparative genomic analysis revealed a deletion of 36 nt found in the flagellar biosynthetic gene (FlhB) that could explain that trait. Further in silico analysis revealed an intact prophage that was integrated in the bacterial genome. Following spontaneous induction, the phage was isolated, purified, characterized and independently sequenced, confirming its viability as a free, inducible virion as well. Separate genomic analysis of the prophage implies a plausible case of lysogenic conversion. Focusing on edwardsiellosis as a rapidly emerging aquaculture disease on a global scale, this work offers some insight into the virulence, fitness, and potential lysogenic conversion of a of a newly described, yet highly pathogenic, strain of E. anguillarum.

Edwardsiella piscicida: A versatile emerging pathogen of fish

“…piscicida now includes the fish pathogens under the old species name of E. tarda that are isolated from diseased fish and contains one type III and one type VI secretion system (T3SS and T6SS) [12,14]. E. anguillarum contains two T3SSs and three T6SSs and is highly virulent to fish although not much work has been done on this organism [13,[15][16][17][18]. It is interesting to note that isolates of this species have gone by different names; E. piscicida-like species, atypical E. piscicida, or just E. tarda strains [15,18].…”

Characterization and Pathological Analysis of a Virulent Edwardsiella anguillarum Strain Isolated From Nile Tilapia (Oreochromis niloticus) in Korea

“…As this is the first study reporting an E. anguillarum infection in a tilapia farm located in Asia, the pathogenicity of the strain was estimated by implementing a challenge trial in tilapia itself, which was different to past studies where such trials were performed in zebrafish (3). Moreover, histopathological analysis was undertaken to investigate the pathogenicity of the strain and compare the results to existing studies.…”

“…The genus Edwardsiella comprise rod-shaped, gram-negative, facultative anaerobic bacteria belonging to the family Enterobacteriaceae and order Enterobacteriales (2). The motility of these bacteria depends on the existence of flagella; while Edwardsiella is largely considered motile, there have been reports of some bacteria being non-motile due to the deletion of the flagella biosynthetic gene (3). The genus Edwardsiella comprises five species: Edwardsiella hoshinae, Edwardsiella ictaluri, Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella anguillarum (4); E. tarda is one of the most common species found under normal environmental conditions (5), and infects a variety of animals including fish, amphibians, and mammals (6,7).…”

“…), seabream species, channel catfish (Ictalurus punctatus), hybrid catfish (Ictalurus furcatus), and tilapia (Oreochromis sp.) (3,(14)(15)(16).…”

“…These bacteria were originally isolated from diseased eels (Anguilla spp.) in China, and studies concerning the virulence of the bacterial isolates from a variety of fish species have been increasing in other countries, including China, Japan, USA, and Greece (3,4,15). In this study, we aim to identify the species of Edwardsiella isolated from infected Nile tilapia cultured in Korea; we additionally attempt to characterize the pathogenicity of the species identified, examine the damage to internal organs by histopathological examination, and determine antimicrobial susceptibility.…”