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.
Highlights
Edwardsiellosis is a serious disease affecting a wide range of cultured fish species both in marine and freshwater environments
The isolate contained a spontaneously inducible intact prophage that is genetically similar to both a prophage found in the bacterial genome of an E. piscicida-like strain EA181011 (Reichley et al, 2015) and the free bacteriophage GF-2 (Yasuike et al, 2015) that has been isolated against E. tarda
The E. anguillarum strain EA011113 was isolated from diseased sharpsnout seabream, Diplodus puntazzo, in Greece (Katharios et al, 2015), while E. anguillarum type strain ET080813 was isolated from diseased eel, Anguilla anguilla, in China (Wang Q. et al, 2009)
Summary
Edwardsiellosis is a serious disease affecting a wide range of cultured fish species both in marine and freshwater environments It is increasingly becoming a threat for the viability of aquaculture industries worldwide (Xu and Zhang, 2014) and its main causative agents are Edwardsiella tarda, E. ictaluri, E. piscicida, and E. anguillarum (Shao et al, 2015). We described the first incidence of edwardsiellosis in the Mediterranean Sea, in cultured sharpsnout seabream, Diplodus puntazzo (Katharios et al, 2015) At this incidence we had identified the isolates as E. tarda, based on their phenotypic characteristics and 16s rRNA sequences. The isolate contained a spontaneously inducible intact prophage that is genetically similar to both a prophage found in the bacterial genome of an E. piscicida-like strain EA181011 (Reichley et al, 2015) and the free bacteriophage GF-2 (Yasuike et al, 2015) that has been isolated against E. tarda
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