Molecular Characterisation of Clinical and Environmental Isolates of Vibrio parahaemolyticus
Creators: name: Kadhim, Hadaf Mahdi
Publisher: name: University of Plymouth
Year: 2013
Subjects: Vibrio parahaemolyticus, molecular characterisation

description: The halophilic bacterium Vibrio parahaemolyticus is widely distributed as a natural inhabitant of marine and estuarine environments. Some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Studies were undertaken to differentiate clinical (virulent) and environmental (mainly avirulent) forms of Vibrio parahaemolyticus. Initially, identification and confirmation of a total of 55 V. parahaemolyticus isolates (23 clinical and 32 environmental) was carried out by using selective media, biochemical and nutritional tests. Identity was confirmed by specific polymerase chain reaction (PCR) targeting the toxR gene. In an attempt to differentiate between virulent and avirulent forms V. parahaemolyticus, potential virulence factors (enzyme activities), presence of plasmids and analyses of whole cell protein profiles and extracellular products (ECPs) by SDS-PAGE were performed. The results suggested that the presence of plasmids in isolates was not linked to virulence and SDS-PAGE profiles did not differentiate between virulent and avirulent forms, but a combination of enzyme activities may contribute to virulence. The ECPs of all 55 V. parahaemolyticus isolates were tested for their cytotoxicity towards two types of cell lines, the clinical isolates showed that 21 out of 23 (91%) and 2 out of 23 (8.69%) showed high and medium cytotoxicity, respectively. Amongst the environmental isolates 2 out of 32 (6.25%), 2 out of 32 (6.25%) and 28 out of 32 (87.5%) showed high, medium and low cytotoxicity, respectively. Randomly amplified polymorphic DNA (RAPD)-PCR was used to analyse the two groups of isolates. Firstly a 600 bp band was recognised in mainly clinical isolates. This DNA fragment was cloned and sequenced and found to code for an outer membrane protein (OMP). Two PCR primers were designed to specifically amplify a 200bp unique sequence from presumptive virulent strains (PCR-OMP); however, not all clinical isolates were positive (21 out of 23, 91%). A second RAPD-PCR identified a further unique band of approximately 310 bp in mostly clinical isolates. After cloning this band’s DNA, the DNA sequence revealed a hypothetical gene, htp, whose function is not known. Specific primers, VPHTP1 and VPHTP2 were developed for the detection of the htp sequence, but again not all clinical isolates were positive (19 out of 23, 82.6%). This led to the development of a multiplex M-PCR which detects all isolates of V. parahaemolyticus and differentiates them into potentially virulent and avirulent forms. The M-PCR works by targeting the toxR gene, and sequences for omp and htp. The M-PCR was performed on all V. parahaemolyticus isolates used in this study, as well as other Vibrio species and a selection of non-Vibrio species. The amplification of toxR gene 367 bp fragment was found in all V. parahaemolyticus tested; all clinical isolates (100%) showed amplification of omp and/or htp. This multiplex PCR detected 3 (9.37%) environment isolates, which may potentially be able to cause disease. No amplification was seen for the other species tested. Thus, the M-PCR could be used for identifying V. parahaemolyticus and detecting / differentiating potentially virulent and avirulent forms. This method should be