Table_2_Characterization of a Highly Virulent Edwardsiella anguillarum Strain Isolated From Greek Aquaculture, and a Spontaneously Induced Prophage Therein.XLSX

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.

与爱德华氏菌属（Edwardsiella）相关的暴发疫情在海水及淡水养殖系统中日益频发，造成了严重的经济损失与养殖生物量损失。迟钝爱德华氏菌（E. tarda）、鮰爱德华氏菌（E. ictaluri）与星爱德华氏菌（E. hoshinae）一直是水产养殖中爱德华氏菌病（edwardsiellosis）的传统致病菌；然而，鉴于该病害的重大危害，近期的深入研究又新发现了另外两个致病菌种：杀鱼爱德华氏菌（E. piscicida）与鳗爱德华氏菌（E. anguillarum）。从希腊某养殖场爱德华氏菌病暴发后的养殖平鲷（Diplodus puntazzo）体内分离得到的菌株EA011113，经全基因组测序（Whole genome sequencing）确认其为鳗爱德华氏菌的一株新型临床菌株。全面的系统发育分析显示，这株希腊菌株与以色列一株分离自红海患病石斑鱼（Epinephelus aeneus及Epinephelus marginatus）的类杀鱼爱德华氏菌临床菌株亲缘关系紧密。对鳗爱德华氏菌菌株EA011113的生物信息学分析揭示了大量潜在毒力因子，该菌株在不同感染强度下对成年斑马鱼（Danio rerio）造成的死亡率证实了这些毒力因子的致病作用：48小时半数致死量（LD50）为1.85×10^4菌落形成单位（cfu）/尾鱼。该菌株无运动能力，且经电子显微镜观察未发现鞭毛，这一特性与鳗爱德华氏菌的典型表型不符。比较基因组学分析显示，该菌株的鞭毛生物合成基因FlhB中存在一段36 nt的缺失，这可解释其无鞭毛的表型。进一步的计算机模拟分析（in silico analysis）发现，该细菌基因组中整合有完整的前噬菌体（prophage）。经自发诱导后，该噬菌体被分离、纯化、鉴定并完成独立测序，证实其可作为游离的可诱导病毒粒子存活。对该前噬菌体的单独基因组分析表明，其存在溶原性转换（lysogenic conversion）的潜在可能。鉴于爱德华氏菌病是全球范围内快速新发的水产养殖病害，本研究对一株最新报道但具有高致病性的鳗爱德华氏菌菌株的毒力、适应性及潜在溶原性转换机制提供了新的科学认识。