Biofilm Produced In Vitro by Piscirickettsia salmonis Generates Differential Cytotoxicity Levels and Expression Patterns of Immune Genes in the Atlantic Salmon Cell Line SHK-1 (original) (raw)
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Scientific Reports, 2020
Piscirickettsia salmonis is the causative agent of piscirickettsiosis, a disease with high socio-economic 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 ...
Piscirickettsia salmonis is a bacterial fish pathogen seriously threatening the sustainability of the Chilean salmon industry. The biology and life cycle of this bacterium is not completely understood and there are no reports explaining how it survives or persists in marine environments. This work provides descriptive data of P. salmonis behavior when it is exposed to stress conditions, producing large cell aggregates closely resembling typical biofilm structures. In order to track this putative biofilm, we used indirect fluorescence and scanning electron microscopy. Complex masses were observed over time; the bacteria appear to be embedded within a matrix which disappears when it is exposed to cellulase, suggesting a polysaccharide nature typical of biofilm formation. Two lectins (ConA and WGA) were used to characterize the matrix. Both lectins showed a strong reaction with the structure, validating the exopolysaccharide nature of the matrix. Recently, several studies have demonstrated a correlation between toxin/anti-toxin system expression at initial stages of biofilm formation. In this report, QRT-PCR analysis was used with the P. salmonis toxin/anti-toxin mazEF operon, showing induction of these genes at early stages of biofilm formation, suggesting that said formation may be an adaptive strategy for survival and persistence under stress conditions in marine environments.
Frontiers in Cellular and Infection Microbiology, 2022
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.
Archivos de medicina veterinaria, 2009
Piscirickettsia salmonis es un microorganismo altamente contagioso y virulento que afecta a la salmonicultura mundial desde el último tercio del siglo pasado y del cual sus mecanismos de sobrevivencia intracelular son completamente desconocidos. Después de algunos reportes recientes en donde se cuestiona su condición de intracelular obligado, hemos decidido mostrar evidencia adicional para cambiar este paradigma, llevando a cabo experimentos tanto clásicos como moleculares que confirman su naturaleza de intracelular facultativo. En este reporte se demuestra inequívocamente que la bacteria recuperada desde cultivos celulares infectados, de placas de agar o de medio líquido, es el mismo organismo, el cual cumple con los postulados de Koch. Además, análisis genéticos y proteómicos confirman que la bacteria obtenida de diferentes fuentes de crecimiento corresponde a la misma cepa tipo LF-89, la que fue originalmente descrita por Fryer en 1992. Sin embargo, el crecimiento de la bacteria, tanto en medios libres de células como en cultivo celular, es subóptimo, por lo cual se requieren más análisis para entender completamente la biología del patógeno. Interesantemente, en este trabajo se logró la mantención de la bacteria en medio líquido, pero a una tasa muy baja de crecimiento. En conclusión, y sumado a reportes anteriores, hemos confirmado la naturaleza intracelular facultativa de este patógeno de peces de cultivo.
Aquaculture, 2020
The propagation of intracellular bacteria in culture mediums implies physiological adaptation to an artificial environment. If these conditions are persistent enough time, a permanent adaptation to the new environment can be expected. This study aimed to explore the genomic and transcriptomic rearrangements that the intracellular bacterium Piscirickettsia salmonis undergo during a long-term serial culture experiment and the potential impact on virulence. Here, PacBio and Illumina sequencing were conducted after 200 passages (~2 years) of P. salmonis in a cell-free culture medium. The results evidenced genomic rearrangements of the P. salmonis genome during the continuous culture period, where a chromosome segment of 35 Kb was translocated from the P0 bacterial chromosome to a P200 plasmid. Notably, the genomic structure of this segment revealed the presence of the Dot/Icm secretion system, which has been previously associated with P. salmonis pathogenesis. On the other hand, a reduced transcriptomic response was evidenced in P. salmonis after 200 passages affecting the expression of different pathways including iron acquisition and metabolism. Furthermore, in vitro infections revealed that after 200 passages P. salmonis is less capable of generating cytopathic effects than the original P0 form. Overall, our results evidence that the continuous propagation of P. salmonis leads to genomic and transcriptomic rearrangement that impact on bacterial virulence. These results open new perspectives about the adaptation of intracellular bacteria to artificial culture conditions, providing useful information to develop live attenuated vaccines against P. salmonis in salmon aquaculture.
2016
P. salmonis ist ein gram-negatives, fakultativ intrazelluläres Bakterium, der Erreger des Salmon Rickettsial Syndroms (SRS), das in den letzten dreißig Jahren große Verluste in der chilenischen Lachsindustrie verursacht hat. Dieses Bakterium wurde erstmals im Jahr 1989 von Bravo und Campos im Kisutsch-Lachs im Süden Chiles identifiziert und zunächst als obligat intrazellulär beschrieben. Erst vor ein paar Jahren gelang es, das Bakterium auf einem soliden künstlichen Medium frei von Zellen wachsen zu lassen. Allerdings wurden in vivo aufgrund der intrazellulären Eigenschaft dieses Pathogens mit den bisherigen auf Antibiotika basierenden Kontrollprogrammen keine guten Ergebnisse erzielt, weshalb es unbedingt von Nöten ist, die Informationen zu diesem Krankheitserreger auszuweiten und zu vertiefen. In dieser Arbeit wurde als Gesamtziel angestrebt, das globale Expressionsmuster von P. salmonis auf Transkriptions- als auch Proteomebene zu analysieren, um Gene und Proteine zu identifizier...
BMC Veterinary Research, 2021
Background Vertebrate hosts limit the availability of iron to microbial pathogens in order to nutritionally starve the invaders. The impact of iron deficiency induced by the iron chelator deferoxamine mesylate (DFO) was investigated in Atlantic salmon SHK-1 cells infected with the facultative intracellular bacterium Piscirickettsia salmonis. Results Effects of the DFO treatment and P. salmonis on SHK-1 cells were gaged by assessing cytopathic effects, bacterial load and activity, and gene expression profiles of eight immune biomarkers at 4- and 7-days post infection (dpi) in the control group, groups receiving single treatments (DFO or P. salmonis) and their combination. The chelator appears to be well-tolerated by host cells, while it had a negative impact on the number of bacterial cells and associated cytotoxicity. DFO alone had minor effects on gene expression of SHK-1 cells, including an early activation of IL-1β at 4 dpi. In contrast to few moderate changes induced by single t...
Journal of Fish Diseases, 2019
Piscirickettsiosis, caused by the intracellular Gram‐negative bacteria Piscirickettsia salmonis, is at present the most devastating disease in the Chilean salmon industry. The aim of this study was to analyse disease development after challenge with a P. salmonis strain (EM90‐like) under a controlled environment by comparing intraperitoneal challenge with cohabitation challenge. The P. salmonis EM90‐like isolate was cultured in a liquid medium for the challenge of 400 Atlantic salmon (Salmo salar) smolts. Cumulative mortality was registered, necropsy was performed, and bacterial distribution in the tissues and histopathological changes were analysed. The results revealed a similar progression of the disease for the two different challenge models. Pathological and histopathological changes became more visible during the development of the clinical phase of the disease. Bacterial DNA was identified in all the analysed tissues indicating a systemic infection. Bacterial tropism to visce...
Microbial pathogenesis, 2018
Disease outbreaks related to waterborne pathogen contamination throughout the world as well as challenges that lie ahead for addressing persistent infection are of renewed interest. In this research, we studied the effects of prolonged exposure of Salmonella enterica serovar Typhimurium to the cues encountered in the extracellular environment particularly in seawater microcosm on bacterial virulence and subsequent infection in Caco-2 cells. Our data show a significant difference in biofilm formation, swimming and swarming motilities between normal and stressed cells of S. Typhimurium under differing NaCl conditions (P < 0.05). Interestingly, adhesion, invasion and apoptotic activity to Caco-2 epithelial cells were determined during infection with normal and stressed Salmonella. Furthermore, we compared the expression of SPI-1 virulence genes (sopA, sopB, sopD, sopE2 and hilA) of normal and stressed S. Typhimurium in response to salt conditions encountered in the extracellular env...