Laboratory investigation for clonality of a foodborne outbreak due to Vibrio parahaemolyticus in Singapore, 2009 My-Van La1, Siti Zulaina1, Lin Cui1, Roland Jureen2, Raymond T.P Lin1,2 ______________________________________________________________________________________________ 1National Public Health Laboratory, Ministry of Health, Singapore 2Department of Laboratory Medicine, National University Hospital, Singapore INTRODUCTION Vibrio parahaemolyticus is a halophilic gram-negative bacterium that causes acute gastroenteritis in human after their consumption of contaminated foods, most often raw or partially cooked seafood. There was a suspected point source of gastroenteritis outbreak with V. parahaemolyticus following the consumption of the local salad dish “Indian rojak” from a popular hawker stall (Figure 1) in Singapore in early April, 2009. The total number of involved cases was 154, with 48 cases hospitalized and 2 dead. The National Public Health Laboratory (NPHL) collaborated with the MOH investigation team on the determination of genetic relatedness of V. parahaemolyticus isolates collected. Figure 3. Gel image generated by Bioanalyzer. REP-PCR profiles of outbreak isolates (lane 1-9) and unrelated control strains (lane 10-12). Figure 1A. Indian rojak stall (Source: Channel NewsAsia) Figure 1B. Indian rojak (Source: http://www.foodliabilitylaw.com) Figure 4. Dendrogram showed that 15 of 16 investigated isolates were in the same cluster. METHOD Sixteen V.parahaemolyticus isolates obtained from specimens of the outbreak patients and food handlers were sent to NPHL for investigation. Repetitive extragenic palindromic PCR (REP-PCR)1, PCR for the thermostable direct hemolysin gene (tdh) and the tdh-related hemolysin gene (trh)2 as well as serotyping were performed on all isolates from suspected outbreak cases and some unrelated control strains. The REP-PCR fingerprint was generated with the Agilent® Bioanalyzer using DNA 1000 LabChip® kit, and then analyzed with Bionumerics software. All 15 outbreak isolates had serotype O4:K55. The isolate No22, which was not present in the outbreak cluster based upon REP-PCR typing, had serotype O3:K6. Tdh and Trh are considered as major virulence factors for this organism. PCR carried out on 16 isolates were positive for tdh (Figure 5) and negative for trh (Figure 6). Figure 6. PCR detecting trh were negative for 16 / 16 investigated isolates Outbreak isolates Figure 5. PCR detecting tdh were positive for 16 / 16 investigated isolates 250 bp 251 bp Figure 2. Agilent® Bioanalyzer platform RESULTS AND DISCUSSION In total, REP-PCR was carried out on 26 isolates, i.e. 16 isolates from suspected outbreak cases and 10 control strains. Using the unweighted pair group method with arithmetic mean (UPGMA), the resulting dendrogram showed that REP-PCR profiles obtained from 15 of 16 investigated isolates (93.75%) were identical and different from all control strains (Figure 3 and 4). REP-PCR typing appeared to be as discriminatory as pulse-field gel electrophoresis in this outbreak investigation (personal communication). CONCLUSION REP-PCR in this setting was a rapid and useful molecular typing method for the laboratory evaluation of genetic and epidemiological relationships among V. parahaemolyticus strains. REFERENCES 1. Wong HC & Lin CH (2001) J Clin Microbiol 39: 4233-4240. 2. Tada J et al. (1992) Mol Cell Probes 6: 477-478.