Molecular characterisation of Flavobacterium spp. and investigation of their biofilm-forming capacity in the tilapia aquaculture system

[摘要] ENGLISH ABSTRACT: Fish infections caused by pathogenic Flavobacterium spp. are a majorproblem in the aquaculture industry worldwide, often leading to largeeconomic losses. Thirty-two Flavobacterium spp. isolates, obtained fromvarious diseased fish species and biofilm growth, were characterisedgenetically using 16S rRNA gene sequencing, 16S rRNA gene PCRrestriction fragment length polymorphism (RFLP), randomly amplifiedpolymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP)element PCR, plasmid profiling, whole cell protein (WCP) and outermembrane protein (OMP) analyses. The biofilm-forming capability of fivegenetically heterogeneous Flavobacterium spp. study isolates wasinvestigated using a modified microtiter-plate adherence assay, as well asflow cell studies. Experimental infection studies with Mozambique tilapia(Oreochromis mossambicus) were carried out in order to determine thevirulence of the Flavobacterium spp. study isolates. 16S rRNA genesequence analysis showed the Flavobacterium spp. study isolates wereclosely related, and 97% sequence similarity was shared with published F.johnsoniae sequences. A high degree of genetic heterogeneity wasdisplayed by the Flavobacterium spp. study isolates following RAPD-PCR,REP-PCR and OMP analysis, however, based on the results obtained byplasmid profiling and WCP analysis, the isolates appeared genetically veryhomogeneous. The biofilm phenotype was displayed by all fiveFlavobacterium spp. isolates tested and varied from weakly to stronglyadherent. No specific correlation was observed between the RAPD, REPand/or OMP profiles and degree of adherence displayed by Flavobacteriumspp. isolates. However, a specific WCP profile (profile B), exhibited by 48% ofthe Flavobacterium spp. isolates, was linked to strong adherence.Experimental infection studies showed that Flavobacterium spp. isolatesdisplayed variable levels of virulence, which could not be linked to biofilmformation, nor specific genotypes. This is the first reported isolation andcharacterisation of Flavobacterium spp. isolated from diseased fish inSouthern Africa, and there appears to be significant diversity amongst theisolates which is not geographically linked nor host related.

[发布机构] Stellenbosch University