Radu, Son

Rahmat Ali, Gulam Rusul

Universiti Putra Malaysia Institutional Repository

Medical and Health Sciences50n Radu and Gulam RU5UIMolecular Characterization of Vibrio vulnificus Isolated from Cockle and ShrimpFaculty of Food Science and BiotechnologyUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaE-mail ofCorrespondingAuthor:son@fsb.upm.edu.myKey words: Vibrio vulnijiclIs, cockle, shrimp, RAPD-PCR.IntroductionVibrio vulnificus has emerged as animportant pathogen responsible forsepticemia and severe wound infec-tions in immunocompromised patients.V. vulnificus is an estuarine bacteriumcommonly found in coastal waters andhas been detected in shellfish and in-testinal contents of fish (Oliver, 1989;Amaro et al., 1995). Molecular typingis being used for epidemiological studyof this potential pathogen in the envi-ronment. Loi et al. (1997) showed thatit is necessary to characterize severalisolates from the same source becauseit could harbor different clones of V.vulnificus. Thus, the aim of this studywas to characterize and compare thestrains of V. vulnificus isolated fromcockles and shrimps. The characteriza-tion was done by randomly amplifiedpolymorphic DNA (RAPD) analysis.Materials and Methods109 of the shrimp or cockles sampleswere stomached in 90 m1 of peptonewater, and the samples were seriallydiluted in lO-fold increments of 100 mlof 0.5% peptone water. One ml of seri-ally diluted sample was transferred into9 ml alkaline peptone broth, incubatedovernight at 37°C and were streakedonto thiosulfate-citrate-bile salt-sucroseagar. Colonies were inoculated intoAPI 20E biochemical strips for identi-fication. Prior to amplification, chro-mosomal DNA of the V. vulnificus wasisolated as described by Ausubel et al.(1987). Reactions mixtures of 25 IIIwere made consisting 2.5 ~l lOx reac-tion buffer, 0.5 III of 10 mM dNTPmix, 2 mM of primer, 2111 of 25 mMMgClz, 0.5 ~I Taq polymerase, 20-30ng DNA and made up to 25 111withsterile distilled water. A thermal cycler(Perkin Elmer model 2400) was usedfor amplification of 45 cycles at 94°Cfor 2 min, 36°C for 1 min and 72°C for2 min. A final elongation step of irefor 5 min was included.Results and DiscussionThe initial experiments were performedwith a subset of Vibrio vulnificusstrains to identify primers that providepolymorphic band patterns. RAPD-PCR using primers GEN1500i andGEN15008 resulted in amplification ofgenomic DNA ranging in sizes from0.25 to 10.0 kilobases pairs (kb). Of the50 strains examined, 46 and 47 differ-ent RAPD patterns were generatedusing primer GENI500i andGENI5OO8, respectively. Four andthree strains were untypable usingthese two primers respectively. Fromthe dendrogram generated, it was ob-served that some strains of Vibrio vul-nificus from cockles and shrimps sam-ples were grouped into the same clus-ters, indicating their possible geneticrelatedness. Interest in the microbio-logical relationship of V. vulnificusfrom seafoods has been driven by therecognition that strains of these speciesare associated with human infections.Despite the increased interest in theVibrio and the rapidly expanding bodyof knowledge concerning the variousassociations of these genera with sea-foods, limited studies have reported thepresence of V. vulnificus in seafoods inMalaysia (Son et al., 1998). Biochemi-cal characterization revealed that someof the Vibrio vulnificus strains isolatedfrom cockles and shrimps in this studywere biotype 1, which is known as anopportunistic human pathogen withinfection resulting from consumptionof contaminated seafood or exposure tomarine environment in the case ofwound infections. Thus it would be ofgreat benefit to apply rapid screeningmethod such as RAPD-PCR to screenfor presence of clonal strains of V.vulnificus that are potentially patho-genic in humans from seafood samples.This PCR sreening method is simpleand rapid and may be useful in epide-miological analysis, particularly inlarge studies and in urgent situations.This report suggested that V. vulnificusbiotype 1 is present in seafood samplesmarketed in Malaysia and thus empha-size the need for caution when dealingwith seafoods contaminated with thisorganism.ConclusionsThe presence of V. vulnificus biotype 1may be a health hazard to consumers ofraw shellfish in the study area as cock-les and shrimps are popular ingredientin many types of local foods.Benefits from the studyThe information on the isolation of V.vulnificus biotype 1 from cockles andshrimps marketed locally would beuseful for the public health agency totake the necessary measures to reducethe V. vulnificus contamination of theseproducts, thereby decreasing the risk ofhuman infection. This research hasprovided training for postgraduatestudent on molecular tools used inmolecular fingerprinting of humanpathogen isolated from food sources.Literature cited in the textAmaro, C., Biosca, E.G., Fonz, B., Alcaide,E. and Esteve, C. 1995. Evidence thatwater transmit Vibrio vulnificus biotype 2infections to eels. Applied andEnvironmental Microbiology. 61: 1133-1137.Ausubel, F.M., Brent, R. and Kingston, R.E.1987. Current protocols in molecularbiology. John Wiley, New York.Loi, L., Dalsgaard, A., Larsen, J.L., Warner,I.M. and Oliver, J.D. 1997. Comparisonof ribotyping and randomly amplifiedpolymorphic DNA CR fr characterizationof Vibrio vulnificus. Applied andEnvironmental Microbiology. 63: 1674-1678.315UPM Research Report 1997-2000, Section 2-Extended AbstractsSon R. Nasreldin EH, Zaiton H. Samuel L.Rusul G. Nimita F. Yuherman andEndang P. 1998. Characterization ofVibrio vulnificus isolated from cockles(Anadara granosay: antimicrobialresistance, plasmid profiles and randomamplification of polymorphic DNAanalysis. FEMS Microbiology Letters.165: 139-143.Project Publications inRefereed JournalsAhbrizal T. Son R. Reezal A. Mutalib AR.Rusul G. Nasreldin EH and Yuherman.2000. Random amplified polymorphicDNA analysis 10 differentiate strains ofVibrio vulnificus isolated from cocklesand shrimps. Malaysian Joul7IIJlofMedical Science. 7: 41-46.Son R. Nasreldin EH. Zaiton H. Samuel L.Rusul G, Nimita F, Yuherman andEndang P. 1998. Characterization ofVibrio vulnificus isolated from cockles(Anadara granosa): antimicrobial316resistance, plasmid profiles and randomamplification of polymorphic DNAanalysis. FEMS Microbiology Letters.165: 139-143.Son R. Yuherman, Rusul, G. Lum KY andNishibuchi M. 2000. Detection andmolecular characterization of Vibriovulnificus from coastal waters ofMalaysia. Southeast Asian Journal ofTropical Medicine and Public Health. 31:668-673.Project Publications inConference ProceedingsNasreldin EH. Son R. Rusul G, Khair J,Hassan Z. Reezal A. Nimita F and SahilahAM. 2000. Prevalence of Vibrio spp. inshrimp (Peneus indicus) marketed inMalaysia. 3'd National Symposium onHealth Sciences. 29-30 April 2000. PutraWorld Trade Center. Kuala Lumpur.Malaysia.Nasreldin EH. Zaiton H. Son R. Rusul Gand Nimita F. 1998. Prevalence studies ofVibrio vulnificus isolated from cockles(Anadara granosa) in Malaysia. FithSymposium of Applied Biology, 5-6 May1998. Universiti Putra Malaysia.Yuherrnan, Son R and Rusul, G. 1999.Characterization of Vibrio vulnificusisolated from sea water by plasmidprofiling. antibiotic resistance pattern andrandom amplified polymorphic DNA(RAPD). 35" Annual Scientific Seminar,Malaysian Society of Parasitology andTropical Medicine. 17-18 March 1999.Institute for Medical Research. Malaysia.Graduate ResearchTg Ahbrizal. 2000. Molecular Biology[MS]. Universiti Putra Malaysia.Yuhennan. 2000. Molecular Biology[Ph.DJ. Universiti Putra Malaysia.Nasreldin Elhadi Hussein. 1999. MolecularBiology [MS]. Universiti Putra Malaysia.

Molecular characterization of vibrio vulnificus isolated from cockle and shrimp

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Molecular characterization of vibrio vulnificus isolated from cockle and shrimp

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