Identification of Putative Vibrio Species Isolated from Processed Marine Fish Using Thiosulphate-Citrate-Bile-Sucrose (TCBS) Agar

Abstract

Aims: To identify putative Vibrio isolates obtained from processed hake, pilchards and horse mackerel using Thiosulphate-Citrate-Bile-Sucrose (TCBS) agar. Place and Duration of Study: Sampling during April – June 2005 in Walvis Bay Namibia. Further analyses performed at the Department of Microbiology and Plant Pathology, University of Pretoria, South Africa between 2005 and 2007. Methodology: The 247 putative Vibrio isolates obtained from Thiosulphate-Citrate-Bile-Sucrose (TCBS) agar were initially grouped according to their Gram, oxidase and oxidation-fermentation reactions. Thereafter PCR was used to screen the isolates for genes specific to human pathogenic Vibrio species such as Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. This was followed by sequencing the 16S rRNA gene for isolates of interest and drawing Neighbour Joining phylogenetic trees based on the data.  All atypical isolates were further characterised by a combination of selected phenotypic tests and the V. alginolyticus specific collagenase gene PCR.  Results: Of the 247 isolates obtained from TCBS, four were Gram positive. Many of the Gram negative isolates belonged to the family Enterobacteriaceae and others were members of the Pseudomonadaceae. Of the 91 isolates identified as belonging to the Vibrionaceae and related families, Vibrio alginolyticus was the dominant Vibrio species. None of the bacteria isolated from the processed fish belonged to the human pathogenic Vibrio species. Conclusion: This study again demonstrated that TCBS agar is not selective for the isolation of Vibrio species and that a wide range of bacteria could be isolated on this medium when analysing marine fish. The identity of putative Vibrio isolates obtained from this medium should therefore be confirmed employing a number of phenotypic and genetic techniques to accurately identify the atypical isolates.