Additional evidence of the facultative intracellular nature of the fish bacterial pathogen Piscirickettsia salmonis

Abstract

RESuMENPiscirickettsia 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.Palabras clave: Piscirickettsia salmonis, intracelular facultativo, genómica.

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

“…salmonis is a non-encapsulated, pleomorphic, generally coccoid bacterium with a diameter ranging from 0.5 to 1.5 µm (Fryer et al 1992). This bacterium is classified as a facultative intracellular member of the Gammaproteobacteria that is able to grow in cellfree media (Mauel et al 2008, Mikalsen et al 2008, Gómez et al 2009), belongs to the Piscirickettsiaceae family, and is closely related to the genera Coxiella, Francisella, and Legionella (Fryer & Hedrick 2003). Indeed, a recent report described the presence of a fully functional Dot/Icmlike type IV secretion system in P. salmonis that is highly similar to that of L. pneumophila, a species in which this system is directly associated with pathogenicity (Zusman et al 2008).…”

A Novel Liquid Medium for the Efficient Growth of the Salmonid Pathogen Piscirickettsia salmonis and Optimization of Culture Conditions

“…P. salmonis is a Gram-negative bacterium described as non-motile, not encapsulated, pleomorphic but generally coccoid, with a diameter ranging from 0.1 to 1.5 µm [4], [5] and is considered as facultative intracellular organism [6], [7], [8]. Nowadays, the prophylactic treatments available for controlling the disease are vaccines based on either recombinant antigens or bacterins (attenuated bacteria), which are administered through injection or oral immunization [9].…”

“…Nowadays is possible obtain the growth of the bacterium on solid and liquid media free from eukaryotic host cells [6], [7], [8], [13]. The range of temperature for the bacterium cultivation has been reported between 16° and 23°C, but the optimum culture temperature only has been reported as 22°C for agar cultures [7].…”

“…The range of temperature for the bacterium cultivation has been reported between 16° and 23°C, but the optimum culture temperature only has been reported as 22°C for agar cultures [7]. Currently there are three studies that describe growth of the bacterium in liquid media and the growth only have been performed at 17° and 23°C [8], [13], [14], but no studies exist regarding the optimum growth temperature in liquid media. Considering that culture conditions affect cell growth [15] and intra- and extracellular metabolites production [16], media composition and culture temperatures were evaluated in the present work in order to develop a process to produce P. salmonis biomass.…”

Isolation of Tasmanian Rickettsia-like organism (RLO) from farmed salmonids: identification of multiple serotypes and confirmation of pathogenicity

“…It is also culturable in Xenopus laevis (Daudin) XTC-2 and fall army worm Spodoptera frugiperda Sf21 cells (Birkbeck et al 2004a). For many years, P. salmonis was thought to be obligately intracellular (Fryer et al 1992, Almendras & Fuentealba 1997, Mauel & Miller 2002, Fryer & Hedrick 2003, but using specialised enriched media (Mauel et al 2008, Mikalsen et al 2008, Gómez et al 2009, Otterlei et al 2016), the bacterium is now considered fastidious and facultatively intracellular. The development of these new media provided an ideal opportunity to attempt to isolate TRLO in a cell culture-independent fashion.…”