Abstract: Salmonid rickettsial septicemia (SRS) or Piscirickettsiosis is the main, and the most prevalent, systemic bacterial disease that causes high mortality rates in the three most important farmed salmonid species for the Chilean aquaculture industry, resulting in significant economic losses. The etiologic agent is Piscirickettsia salmonis, a facultative intracellular Gram-negative bacterium, which makes its prevention and treatment extremely complicated when an infectious outbreak of Piscirickettsiosis is established. Nowadays, several commercial vaccine formulations have been used in fish to prevent bacterial infections. However, these preparations do not provide the expected protection against Piscirickettsiosis. On the other hand, antibiotic-based treatments, mainly florfenicol (FLO) and oxytetracycline (OTC), bacteriostatic agents commonly used in the salmon industry, have not been effective in controlling SRS. It has been reported that the consumption of antibiotics in the salmon industry in Chile has increased by 56% from 2005 to 2015. The highest consumption was recorded in 2014, with a total use of 563.2 tons of antibiotics. However, based on frequency of outbreaks and mortalities registered Chilean fish farms, these antibiotics have not been effective in controlling Piscirickettsiosis, which remains largely as an unresolved problem for the Chilean aquaculture. In this way, the indiscriminate use of antimicrobials has led to a selective pressure on the P. salmonis population, with the subsequent development of resistant strains of P. salmonis. It is known that P. salmonis is capable of surviving up to 40 days in seawater, which could be related to the development of Biofilm where the bacteria would be embedded, therefore suggesting survival strategies under stressful marine conditions. Many studies in other bacterial pathogens have demonstrated a strong relationship between the biofilm formation and the antibiotic resistance. In addition, the bacterial Biofilm matrix contains outer membrane vesicles (OMVs), and nucleic acids among other components. However, until now little is known regarding the role of oligonucleotides and proteins such as chemotaxis components involved in biofilm formation and components of efflux pumps and other proteins related to antimicrobial resistance contained in P. salmonis OMVs, which could effectively modulate the biofilm production, increasing its antibiotic resistance and affecting its bacterial virulence. Thus, these vesicles could also serve as vehicle for the transport of antibiotic-inactivating enzymes or proteins involved in the activation of antibiotic resistance mechanisms.
Similar to other bacteria, P. salmonis is able to produce biofilm and also release OMVs during infection in CHSE-214 cells and during normal growth in liquid media as response to stress conditions. SDS-PAGE analysis demonstrated that the protein profile of the OMVs was similar to the outer membrane protein profile of P. salmonis. Importantly, in vitro infection assays showed that purified OMVs generated a cytopathic effect on fish cell lines, an also in an in vivo assay using adult zebrafish model as a model suggesting a role in pathogenesis. Thus, OMVs from P. salmonis have been evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with OMVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis. This research will benefit the understanding of resistance mechanisms mediated by Biofilm matrix-derived OMVs of P. salmonis, which will allow the design of alternative therapies of antibiotics for the prevention and/or control of Piscirickettsiosis.