Improved understanding of biofilm development by Piscirickettsia salmonis reveals potential risks for the persistence and dissemination of piscirickettsiosis

Abstract

Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socioeconomic impacts for Chilean salmonid aquaculture. The identification of major environmental reservoirs for P. salmonis has long been ignored. Most microbial life occurs in biofilms, with possible implications in disease outbreaks as pathogen seed banks. Herein, we report on an in vitro analysis of biofilm formation by P. salmonis Psal-103 (LF-89-like genotype) and Psal-104 (EM-90-like genotype), the aim of which was to gain new insights into the ecological role of biofilms using multiple approaches. The cytotoxic response of the salmon head kidney cell line to P. salmonis showed interisolate differences, depending on the source of the bacterial inoculum (biofilm or planktonic). Biofilm formation showed a variable-length lag-phase, which was associated with wider fluctuations in biofilm viability. Interisolate differences in the lag phase emerged regardless of the nutritional content of the medium, but both isolates formed mature biofilms from 288 h onwards. Psal-103 biofilms were sensitive to Atlantic salmon skin mucus during early formation, whereas Psal-104 biofilms were more tolerant. The ability of P. salmonis to form viable and mucus-tolerant biofilms on plastic surfaces in seawater represents a potentially important environmental risk for the persistence and dissemination of piscirickettsiosis. Piscirickettsiosis, or salmon rickettsial septicemia, is a fish disease caused by the facultative intracellular bacterium Piscirickettsia salmonis. First described in relation to the Chilean salmon farming industry 1 , this pathogen has since been reported in North America and Europe 2. A decade ago, P. salmonis was considered an obligate intracellular pathogen 3 , but later research achieved growth on artificial cell-free media 4,5. P. salmonis can survive outside of hosts for a long time as free-living cells, having been detected around salmon farms by qPCR up to 30 days after cage emptying 6 or as a biofilm mode-of-growth in a marine broth medium for 15-30 days 7. Biofilm formation is a multi-step process that involves bacterial attachment to surfaces, microcolony formation, growthdependent maturation, and cell detachment from mature biofilms to colonize new habitats 8. One persisting question is if the survival behavior of P. salmonis as a free-living bacterium is linked with a biofilm lifestyle in marine habitats. Bacterial biofilms are nearly ubiquitous in all major habitats 9 , including some synthetic habitats, such as the "plastisphere" of marine environments 10-12 .

Piscirickettsia salmonis does not evidence quorum sensing based on acyl‐homoserine lactones

“…Significant investigative advancements have been made as related to P. salmonis , including in vitro culturing (e.g. Fuentealba et al., 2020), virulence factors (Almarza et al., 2016; Calquín et al, 2018; Oliver et al., 2017), life strategies (Levipan et al., 2020), gene expression during cell infection (Machuca & Martínez, 2016; Zúñiga et al., 2020) and epidemiology (Meyer et al., 2021). However, clarity is almost completely lacking on the molecular signalling pathways underlying the lifestyle of the bacterium.…”

Piscirickettsia salmonis Produces a N-Acetyl-L-Homoserine Lactone as a Bacterial Quorum Sensing System-Related Molecule

“…Additionally, In addition, antibiotics treatments used in aquaculture have not been completely effective in reducing the development of salmonid rickettsial septicemia (SRS) ( Miranda et al., 2018 ), probably due to antibiotic resistance acquired by the pathogen ( Henríquez et al., 2016 ; Sandoval et al., 2016 ; Cartes et al., 2017 ) and SRS outbreaks are recurrent in Chile ( Sernapesca, 2021 ). Several studies have described the intracellular life cycle of P. salmonis ( McCarthy et al., 2008 ; Rojas et al., 2009 ; Pérez-Stuardo et al., 2019 ; Zúñiga et al., 2020 ) and even the biofilm formation as a survival strategy of P. salmonis in the seawater ( Marshall et al., 2012 ; Levipan et al., 2020 ). However, the molecular mechanisms of P. salmonis communication its responses to specific external stimuli are largely unknown.…”

Assessing the impacts of skin mucus from Salmo salar and Oncorhynchus mykiss on the growth and in vitro infectivity of the fish pathogen Piscirickettsia salmonis

“…Furthermore, biofilm formation by P . salmonis occurs independently of the presence of skin mucus of Atlantic salmon, suggesting mucus tolerance (Levipan et al., 2020). This latter finding was consistent with results from the free‐living mode of growth of P .…”

“…Indeed, mucus‐unexposed Psal‐103 and Psal‐104 strains displayed higher cytotoxicity levels within the first 48 hpi without significant differences between planktonic and biofilm‐derived P . salmonis (Levipan et al., 2020). The same has been observed for the strain LF‐89 T (results from another yet‐unpublished study by our team).…”

“…The type strain of Piscirickettsia salmonis , LF‐89 T (ATCC VR‐1361), was studied along with two isolates of this bacterium, Psal‐103 (LF‐89‐like genotype) and Psal‐104 (EM‐90‐like genotype) (Levipan et al., 2020). Both isolates were obtained in 2012 from Atlantic salmon farms in the inner sea of Chiloé Island in the Los Lagos Region (southern Chile) during a field outbreak.…”