DataSheet_1_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.

鱼类立克次体病（Piscirickettsiosis）是由革兰氏阴性菌Piscirickettsia salmonis引起的一种鱼类疾病。该疾病对智利鲑鱼养殖业具有显著的社会经济影响。该细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多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制的风险因素，但其在水生动物疾病，包括鱼类立克次体病中的微生物学意义却鲜有评估。本研究采用多方法手段分析了P. salmonis 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P. salmonis LF-89T优势菌株在其生物膜状态下的潜在致病力。P. salmonis表现出快速的生物膜形成动力学，遵循多步且菌株依赖性极高的过程。在酶谱方面没有显著差异，在细胞毒性方面（如在王鲑胚胎细胞系中测试）与生物膜来源的细菌和浮游等量细菌之间也未发现显著差异。通过全转录组测序和差异基因表达分析确定的P. salmonis LF-89T在生物膜中的潜在致病力武器库包括细胞粘附、多糖生物合成、转录调控和基因移动等基因。值得注意的是，P. salmonis LF-89T的全局基因表达谱在生物膜发育阶段的24小时和48小时内并未富集与致病性相关的基因，且在生物膜中特异表达的致病性相关基因的富集也未被发现。这些结果表明，P. salmonis 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对应物更具致病性，这一点由细胞毒性试验得到证实，该试验揭示了两种生长模式都对鲑鱼细胞系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对应物相比，并不构成致病性热点。本研究首次提供了转录组目录，以选择可用于预防或控制P. salmonis（体外和/或体内）粘附和/或生物膜形成的特定基因，并为进一步深入了解鱼类立克次体病的发病机制提供了新的见解。