Cohabitation of Piscirickettsia salmonis genogroups (LF-89 and EM-90): synergistic effect on growth dynamics.

Piscirickettsia salmonis, the biological agent of Salmonid Rickettsial Septicemia (SRS), is a facultative intracellular bacterium that can be divided into two genogroups (LF-89 and EM-90) with different virulence levels and patterns. Studies have found co-infection of these genogroups in salmonid farms in Chile, but it is essential to assess whether this interaction within the host is related to virulence and changes in pathogen dynamics. In this study, we studied four isolates from EM-90 and one LF-89 isolate chosen based on their genomic differences. The aim was to evaluate how co-cultivation affects bacterial growth performance and virulence factor expression in Atlantic salmon (Salmo salar) in vitro and in vivo. In vitro results using FN2 medium, showed a similar growth curve between co-cultures of LF-89 and EM-90 compared to EM-90 monocultures. This was explained by the higher ratio of EM-90 to LF-89 in all co-cultures. When evaluating the expression of virulence factors, it was discovered that the luxR gene was expressed only in EM-90-like isolates and that there were significant differences between mono- and co-cultures for flaA and cheA, suggesting a response to cohabitation. Moreover, during in vivo co-cultures, transcriptomic analysis revealed an upregulation of transposases, flagellum-related genes (fliI and flgK), transporters, and permeases that could unveil novel virulence effectors used in the early infection process of P. salmonis. Thus, our work has shown that cohabitation of P. salmonis genogroups can modulate their behavior and virulence effector expression. These data can contribute to new strategies and approaches to improve the current health treatments against this salmonid pathogen.

Ongoing diversification of the global fish pathogen Piscirickettsia salmonis through genetic isolation and transposition bursts.

The management of bacterial pathogens remains a key challenge of aquaculture. The marine gammaproteobacterium Piscirickettsia salmonis is the etiological agent of piscirickettsiosis and causes multi-systemic infections in different salmon species, resulting in considerable mortality and substantial commercial losses. Here, we elucidate its global diversity, evolution, and selection during human interventions. Our comprehensive analysis of 73 closed, high quality genome sequences covered strains from major outbreaks and was supplemented by an analysis of all P. salmonis 16S rRNA gene sequences and metagenomic reads available in public databases. Genome comparison showed that Piscirickettsia comprises at least three distinct, genetically isolated species of which two showed evidence for continuing speciation. However, at least twice the number of species exist in marine fish or seawater. A hallmark of Piscirickettsia diversification is the unprecedented amount and diversity of transposases which are particularly active in subgroups undergoing rapid speciation and are key to the acquisition of novel genes and to pseudogenization. Several group-specific genes are involved in surface antigen synthesis and may explain the differences in virulence between strains. However, the frequent failure of antibiotic treatment of piscirickettsiosis outbreaks cannot be explained by horizontal acquisition of resistance genes which so far occurred only very rarely. Besides revealing a dynamic diversification of an important pathogen, our study also provides the data for improving its surveillance, predicting the emergence of novel lineages, and adapting aquaculture management, and thereby contributes towards the sustainability of salmon farming.

Expression of flagellin and key regulatory flagellar genes in the non-motile bacterium Piscirickettsia salmonis.

The Piscirickettsia salmonis genome was screened to evaluate potential flagella-related open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.