DataSheet_2_Collective behavior and virulence arsenal of the fish pathogen Piscirickettsia salmonis in the biofilm realm.pdf

Piscirickettsiosis is a fish disease caused by the Gram-negative bacterium Piscirickettsia salmonis. This disease has a high socio-economic impact on the Chilean salmonid aquaculture industry. The bacterium has a cryptic character in the environment and their main reservoirs are yet unknown. Bacterial biofilms represent a ubiquitous mechanism of cell persistence in diverse natural environments and a risk factor for the pathogenesis of several infectious diseases, but their microbiological significance for waterborne veterinary diseases, including piscirickettsiosis, have seldom been evaluated. This study analyzed the in vitro biofilm behavior of P. salmonis LF-89T (genogroup LF-89) and CA5 (genogroup EM-90) using a multi-method approach and elucidated the potential arsenal of virulence of the P. salmonis LF-89T type strain in its biofilm state. P. salmonis exhibited a quick kinetics of biofilm formation that followed a multi-step and highly strain-dependent process. There were no major differences in enzymatic profiles or significant differences in cytotoxicity (as tested on the Chinook salmon embryo cell line) between biofilm-derived bacteria and planktonic equivalents. The potential arsenal of virulence of P. salmonis LF-89T in biofilms, as determined by whole-transcriptome sequencing and differential gene expression analysis, consisted of genes involved in cell adhesion, polysaccharide biosynthesis, transcriptional regulation, and gene mobility, among others. Importantly, the global gene expression profiles of P. salmonis LF-89T were not enriched with virulence-related genes upregulated in biofilm development stages at 24 and 48 h. An enrichment in virulence-related genes exclusively expressed in biofilms was also undetected. These results indicate that early and mature biofilm development stages of P. salmonis LF-89T were transcriptionally no more virulent than their planktonic counterparts, which was supported by cytotoxic trials, which, in turn, revealed that both modes of growth induced important and very similar levels of cytotoxicity on the salmon cell line. Our results suggest that the aforementioned biofilm development stages do not represent hot spots of virulence compared with planktonic counterparts. This study provides the first transcriptomic catalogue to select specific genes that could be useful to prevent or control the (in vitro and/or in vivo) adherence and/or biofilm formation by P. salmonis and gain further insights into piscirickettsiosis pathogenesis.

鲑鱼立克次体病是由革兰氏阴性菌鲑鱼立克次体引起的鱼类疾病。该疾病对智利鲑鱼养殖业产生了显著的社会经济影响。该菌在环境中的隐匿性特征显著，其主要的储存宿主尚不清楚。细菌生物膜是细胞在各种自然环境中的持久性普遍机制，也是多种传染性疾病发病机理的风险因素，然而，对于水生兽医疾病，包括鲑鱼立克次体病，其微生物学意义却鲜有评估。本研究采用多方法手段分析了鲑鱼立克次体LF-89T（基因群LF-89）和CA5（基因群EM-90）的体外生物膜行为，并阐明了鲑鱼立克次体LF-89T型菌株在其生物膜状态下的潜在致病力。鲑鱼立克次体表现出快速的多步骤且菌株依赖的生物膜形成动力学。生物膜来源的细菌与浮游等量菌在酶谱或对大马哈鱼胚胎细胞系的细胞毒性方面没有显著差异。通过全转录组测序和差异基因表达分析确定的鲑鱼立克次体LF-89T在生物膜中的潜在致病力武器库包括参与细胞粘附、多糖生物合成、转录调控和基因移动等基因。值得注意的是，鲑鱼立克次体LF-89T的全局基因表达谱在24小时和48小时的生物膜发育阶段中，并未富集与致病性相关的基因，且仅在生物膜中特异性表达的致病性相关基因也未检测到。这些结果表明，鲑鱼立克次体LF-89T的早期和成熟生物膜发育阶段在转录水平上并不比其浮游同源菌株更具致病性，这一点得到了细胞毒性试验的支持，该试验进一步揭示了两种生长模式对鲑鱼细胞系均诱导了重要且极为相似水平的细胞毒性。我们的研究结果表明，上述生物膜发育阶段与浮游同源菌株相比，并不构成致病性热点。本研究首次提供了转录组目录，以选择可能有助于预防或控制鲑鱼立克次体（体外和/或体内）粘附和/或生物膜形成的特定基因，并进一步深入理解鲑鱼立克次体病的发病机理。