Nishibuchi, Mitsuaki

Radu, Son

Rusul, Gulam

Universiti Putra Malaysia Institutional Repository

FOR Category F11000: Medical and Health SciencesMOLECULAR CHARACTERISATION OF VIBRIOVULNIFICUS (AN EMERGING FOODBORNEPATHOGEN)Son Radu, Gulam Rusul and Mitsuaki Nishibuchi 1Faculty of Food Science and BiotechnologyUniversiti Putra Malaysia, 43400 UPM, Serdang, Selangor,Malaysiaand IDivision of Human Environment, Center for SoutheastAsian Studies, Kyoto University, JapanKeywords: Vibrio vulnificus, plasmid, antibiotic resis-tance, RAPD-PCR.IntroductionOf the vibrio pathogens, Vibrio vulnificus is a common bac-terium in estuarine waters in temperate and tropical waters.V. vulnificus has been identified as the causative agents of apotentially fatal septicaemia (mortality >50%), which occursfollowing ingestion of raw shellfish, and nonfatal infectionshave resulted from handling of shellfish or from exposure ofa pre-existing wounds to seawater. However, the relationshipbetween the density of the ingested pathogen and the generalpublic of the human host (with regards to the probability ofdisease) is unclear. To better determine the health risk asso-ciated with exposure to V. vulnificus, epidemiological track-ing of strains is required. This may be achieved by the use ofDNA fingerprinting which allows rapid and sensitive differ-entiation between V. vulnificus strains. In this study, V. vul-nificus strains isolated from cockles (Anadara granosa) andsea water were characterised by antimicrobial resistance,plasmid profiles and random amplified polymorphic DNA(RAPD) analysis.Materials and MethodsCockles from wet markets were shucked with a sterile scal-pel and the muscle and intravalvar fluids were collected insterile stomacher bags for a cluster of individuals (-50g). Seawater samples were collected in sterile bottles from the resortare of Pulau Kapas, Terengganu. The cockles and seawatersamples were enriched in peptone water overnight and werestreaked onto thiosulfate-citrate-bile-salts agar. Identificationof isolates followed the scheme of Tison et al. (1984). Diskdiffusion tests were performed with antibiotic-containingdisks as recommended by the manufacturer. Plasmid DNAwas extracted followed by electrophoresis, essentially as de-scribed by Sambrook et al. (1989). Prior to amplification,chromosomal DNA of the V. vulnificus strains was extractedby the mini-preparation method by Wilson (1989). Reactionmixtures of 25 III were made consisting of 2.5 III lOx reac-tion buffer, 1 mM (final conc.) of each dNTP, 2 pM primer,2.5 mM MgCI2, 20-30 ng genomic DNA and 1 unit Taqpolymerase. Amplifications were performed for 30 cycles(Perkin Elmer 2400) at 94°C for 2 min, 36°C for 1 min andnoc for 2 min. A final elongation step at noc for 5 min wasincluded.Results and DiscussionV. vulnificus was isolated from 25 of the 100 cockles samplesand all the 50 sea water samples analysed. All the 57 V. vul-nificus strains from seawater were of biotype 1, whereas 26and 10 strains from cockles were identified as biotype 1 and2 respectively. Although biotype 2 strains have always beenrecovered from diseased eels and seem to be obligate fishpathogens, evidence of its survival in brackish water havebeen reported (Amaro et al. 1995). In the present study, wereport the first observation on the isolation of V. vulnificusbiotype 2 from cockles, found and harvested from shallowriver estuaries along the coastal area of peninsular Malaysia(Son et al. 1998). Thirty one (86.1%) and 9 (15.8%) ofstrains from cockles and sea water demonstrated resistance toone or more antibiotics tested. About 23 (%) and 10 (17.5%)strains from cockles and sea water contained plasmid bandsranging in sizes from 1.4 to 9.7 MDa, which are in generalagreement with previous reports on the plasmid carriage in V.vulnificus. Two lO-mer primers (GENI5003: 5'-AGGATACGTG-3' and GENI5009: 5'-AGAAGCGATG-3') gener-ated polymorphism within the V. vulnificus strains. For thestrains from cockles, six and five different RAPD types wereapparent for primer GEN15003 and GEN15009, respectively(Son et a1. 1998). Strains isolated from seawater demon-strated 27 and 37 RAPD types with primer GEN15003 andGEN15009. The results of the present study allowed us torecognise a high level of intraspecific diversity among the V.vulnificus strains. These laboratory observations, based onantibiotic susceptibility, plasmid profiles and RAPD types,suggest how complex the epidemiology of V. vulnificus inthe study area, probably as a result of the concurrent actionof several strains as opposed to the widespread transmissionof a single type.ConclusionsThe presence of this pathogen may be a hazard to consumersof raw shellfish in the study area, as the cockle (Anadaragranosa) is a popular ingredient in several types of 10ciIfoods and as in many areas in Asia they are frequently con-sumed in a semi-cooked condition. The heterogeneity mayhave implication for our understanding of the distributionand evolution of this species in this geographic area.ReferencesAmaro, C., Biosca, E.G., Fonz, B., Alcaide, E. and Esteve, C. 1995.~vidence that water transmits Vibrio vulnificus biotype infec-nons to eels. Applied and Environmental Microbiology. 61: 1133-1137.Sambrook, J., Fritsch, E.F. and Maniatis, M. 1989. Molecular clon-ing: a laboratory manual. Cold Spring Harbor Press, Cold SpringHarbor,NY.Son Radu, Nasreldin, E., Hassan, Z., Rusul, G., Lihan, S., Fifadara,N., Yuherman, and Purwati, E. 1998. Characterization of Vibriovulnificus isolated from cockles (Anadara granosa): antimicro-bial resistance, plasmid profiles and random amplification ofpolymorphic DNA analysis. FEMS Microbiology Letters. 165:139-143.Tison, D.L., Greenwood, J., Nishibuchi, M. and Seidler, R.I. 1984.Molecular taxonomy of lactose-fermenting vibrio. In: Vibrios inthe environment (Colwell, R. R. ed.) John Wiley and Sons, NewYork. p. 217-137.Wilson, K. 1989. Preparation of genomic DNA from bacteria. In:curre~t protocols in molecular biology (Ausubel, F. M., Brent, R.E., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A.and Struhl, K. eds), John Wiley and Sons, New York. p. 2.4.1-2.4.5.Supported by [RPA Grant 06-02-04-0018317

Molecular characterisation of Vibrio vulnificus (an emerging foodborne pathogen)

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Molecular characterisation of Vibrio vulnificus (an emerging foodborne pathogen)

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