Molecular characterization of Edwardsiella spp. and Flavobacterium columnare

Abstract

Bacterial diseases are responsible for large economic losses in aquaculture around the world. Flavobacterium columnare and Edwardsiella spp. negatively impact the channel catfish industry in southern USA. However, limited biological information is available for F. columnare and Edwardsiella ictaluri, which has hampered the development of accurate detection methods. In addition, the mechanisms of virulence of these pathogens are poorly understood which has prevented us to propose effective control/treatment methods for them. In the present study, new signature sequences for these pathogens were identified and evaluated as target candidates in PCR-based approaches. Unfortunately, multiplex PCR and real-time PCR tests developed in this study failed to provide the required specificity and sensitivity and could not be implemented as diagnostic tools. However, an intervening sequence (IVS) was discovered in E. ictaluri when sequencing the 23S rRNA gene. IVS are seldom found in bacteria and this is the first time an IVS has been described in the genus Edwardsiella. This IVS exhibited 97% similarity to the IVS in Salmonella typhimurium. A 23S rRNA gene-based phylogenetic tree was constructed placing E. ictaluri and E. tarda in context with other enterobacteria. This tree showed that Edwardsiella spp. are phylogenetically closer to the genus Erwinia than to the core members of the Enterobacteriaceae family. A phenotypic and genotypic comparison among F. columnare strains with different degrees of virulence was carried out in order to identify virulence markers. Lipopolysaccharide (LPS) and total protein profiles were characterized to illustrate the phenotypic differences between virulent and avirulent F. columnare strains. A F. columnare avirulent mutant lacked the high molecular weight bands of the LPS but showed two low molecular weight proteins that were absent in virulent strains. Four putative virulence genes were identified (gtf, hemH, norB, trx) by partially sequencing a shotgun genomic library from a virulent F. columnare strain. Nucleotide sequences of these genes divided the F. columnare strains analyzed into two populations that correlated well with previously described genomovars. Expression of these genes differed among strains under the same conditions. Differential gene expression was also observed when cells were grown under iron-restricted conditions and in the presence of catfish skin explants. These results provide new insights into the understanding of genetics and pathogenesis of F. columnare.