Frontiers in Marine Science (Jun 2014)
Insights on the virulence mechanisms of European <i>Edwardsiella tarda</i> strains isolated from turbot
Abstract
Edwardsiella tarda is a common inhabitant of diverse ecological niches as well as a common guest of a high variety of animals including fish, reptiles, amphibians, chickens and other warm-blooded animals as humans. With regard to the aquatic environments, E. tarda has been described as the causative agent of infections in more than 20 fish species including some important fish species in aquaculture industry. Several potential pathogenic properties have been suggested to contribute to the infection process of E. tarda, which include adhesins, Type III and Type VI secretion system, and ability to survive and replicate in phagocytes, among others. Identification of these virulence-related genes is essential for understanding the pathogenesis of the species. Since E. tarda causes great losses in the Chinese aquaculture, great efforts have been recently devoted to study the pathogenicity mechanism of E. tarda in Asian countries. However, all these studies were conducted employing Asian isolates. Being E. tarda a pathogen of great economical concern in European turbot aquaculture and since the high intraspecific variability of E. tarda is well known, it becomes evident that additional pathogenicity studies conducted with non-Asiatic strains are needed. Enzymes such as chondroitinase are believed to play an important role in the pathogenicity of bacteria that cause infections (Tam et al., 1982). Chondroitinase activity was proposed to be one virulence contributor in Edwardsiella spp. and mediates the cartilage degradation in the chronic “hole-in-the-head” lesion. In Gram-negative bacteria, the most intensively studied quorum sensing systems rely on the use of N-acylhomoserine lactones (AHLs), which production is common among marine and fish pathogenic Proteobacteria, controlling the expression of key virulence factors. In the case of E. tarda, strain NUF251 from diseased flounder had the ability to produce two kinds of AHL molecules. It is likely that the QS system of E. tarda controls the production of various virulent factors or the infection of host cells. Recently, it had been reported that the European turbot isolate ACC35.1 of E. tarda was able to produce N-hexanoyl-L-homoserine lactone (C6-HSL) and N-oxohexanoyl-L-homoserine lactone (OC6-HSL) in vitro. Iron is an essential element for most bacteria but its bioavailability is limited due to the low solubility of Fe(III) at physiological pH. Due to this shortage, pathogens have developed mechanism to obtain iron from the host such as siderophores and be able to develop the infection. Several authors suggested that siderophores are necessary for pathogenicity of E. tarda, but they are not sufficient, being needed other virulence factors. Moreover, it had been described the presence of genes that encode proteins related with vibrioferrin siderophore biosynthesis and transport in the E. tarda genome. In this report, we present the first study carried out with European E. tarda strains isolated from turbot. To the best of our knowledge, none of the previously published studies placed attention on European strains of this pathogen. A collection of E. tarda strains which comprises isolates from turbot from 5 distinct rearing facilities in 2 areas of Europe (one in northern Europe and 4 in southern Europe) belonging all of them to the same serotype were employed. We proposed to establish the correlation between the presence of virulence-related genes in the genome of European E. tarda isolates from turbot and their phenotypic traits. The selected genes are related with three aspects typically involved in bacterial pathogenesis: chondroitinase activity, quorum sensing and siderophore-mediated ferric uptake systems. The results obtained in this work demonstrated that a candidate gene encoding a chondroitinase was present in all the European turbot isolates of E. tarda, which would be homologous to the gene present in the EIB202 strain with Asian origin. This might indicate that genes of chondroitinase synthesis are highly conserved within the species, and that no variations would be found depending of the geographical origin. In addition, all of our E. tarda isolates showed similar ratios of enzyme production, with no significant differences depending on the strain or its area of isolation. In addition, all the strains showed positive amplification for genes involved in AHL production and our results showed the production in vivo of C6-HSL and OC6-HSL during the infection caused by the European turbot isolate, which is in accordance with in vitro results previously. To our knowledge, our study represents the first report that describes the detection of AHL produced by E. tarda during fish infection. Interestingly, our infection assays showed a strong decrease in the AHL production when fish were inoculated with high doses of bacteria. This could confirm the theory that complex cell–cell communication mechanisms are cell-density-dependent, being the AHL production in E. tarda controlled by the density of cells in the fish. In this study, we isolated and characterized for the first time the siderophore responsible for iron uptake in E. tarda. In contrast to other Enterobacteriaceae that produce phenolate-type siderophores like enterobactin or the hydroxamate-type siderophore aerobactin which is also secreted by Vibrio mimicus and V. hollisa, our results demonstrated that E. tarda produces the hydroxamate-type siderophore vibrioferrin which was originally isolated from Vibrio parahaemolyticus. The information generated from this study will contribute to a better understanding of E. tarda pathogenesis and may prove very useful in the development of new antibacterials or preventive treatments against edwardsiellosis infections.
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