Radu, Son

Universiti Putra Malaysia Institutional Repository

Agricultural SciencesRecovery of pathogenic bacteria from aquatic environment, fish and cockles50n RaduDepartment of BiotechnologyFacultyof Food Science and BiotechnologyUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaTelephone Number a/Corresponding Author: 03-8946836/E-mail olCorrespondingAuth(}rs(Jn@f~h.upm.edu.myKey words:Prevalence, .Aeromonas, Listeria, Vibrio, SalmonellaIntroductionThe true incidences of diseases transmitted by seafood (or from aquatic environment) is not known, due to lack of awarenessof the etiological role of foods. In addition, most countries does not have the regulation to enforce the reporting of foodbornediseases to the public health authorities. According to Bryan (1980), "fish" has been most frequently involved, followed bybivalve mollusks and crustacea. Bacterial pathogens in the aquatic environment could be categorized into three general groups:the normal components of the aquatic environment (Vibrio spp., Listeria monocytogenes, Aeromonas hydrophila, etc ..), theenteric bacteria which are present due to faecal contamination, and bacterial contamination during processing. Variousresearchers have reported on that these pathogens have been been isolated from fish and shellfish, and the danger of infectionsis higher because these pathogens are resistant to various antibiotics. This study reports on the isolation of Aeromonas speciesfrom raw fish, Listeria monocytogenes from fermented fish, Salmonella Enteritidis from raw fish, and Vibrio vulnificus fromcockles, shrimp and seawater.Materials and MethodsRaw fish, cockles, shrimp and fermented fish samples were purchased from the wet market in Selangor, whereas the seawatersamples were collected from the coastal area around Pulau Kapas, Terengganu. The general procedure used in isolating thesepathogens from fish, cockles and shrimp samples were as follows: Twenty-five grams of the samples were homogenized with225 ml of the appropriate enrichment buffer (alkaline peptone water for Aeromonas spp., Salmonella Enteritidis and Vibriovulnificus; Listeria enrichment broth for Listeria monocytogenes). After incubation at 37°C for 6-18 hours, the samples wereserially diluted in peptone water and 0.1 ml of the appropriate dilutions were spread plated onto glutamate starch agar(Aeromonas species), thiosulfate citrate bile salts agar (Vibrio vulnificus), Palcam agar (Listeria monocytogenes) andSalmonella-Shigella agar (Salmonella Enteritidis). For the seawater samples, direct dilution were made and spread plated inthe same manner. After an overnight incubation at 37°C, suspected colonies for the various pathogens from the selected plateswere picked and subjected to standard biochemical tests for identification to species level.Results and DiscussionA total of 87 market fish samples representing five types of fish were evaluated for the presence of Aeromonas spp. Of thesamples examined, 69%, 55%, 11.5% and 2.3% harbored Aeromonas species, A. veronii biovar sobria, A. hydrophila and A.caviae, respectively. More specifically, they were isolated from 72%, 60%, 67%, 50% and 50% of 32 ikan tilapia, 20 ikan keli,15 ikan terubuk, 10 ikan merah and 20 ikan puyu samples, respectively. Though the occurrence of foodborne infections due toAeromonas has not been recognized in Malaysia, it has been suggested in many countries in association with consumption ofvarious foods. In Malaysia, fish is usually eaten after being cooked, and therefore, fish may be a low risk food, despite thepresence of Aeromonas species.Forty of the 148 cockle samples, 30 of the 433 shrimp samples and all the 57 samples of seawater were positive for Vibriovulnificus. V. vulnificus biotype I and 2 were isolated from all three types of samples. Biotype I has been associated withhuman infection while biotype 2 was identified as a pathogen for eel or fish. The high percentage of cockles positive for V.vulnificus is not surprising as filter feeding mollusks such as oysters, clams, mussels and scallop have high concentration of thebacteria. In Malaysia, the consumption of half-cooked cockles is very popular, and this will increase the risk of infection. Iaddition, the isolation of V. vulnificus from seawater from a recreational resort area can pose a health hazard to visitors whoused the beach for recreational purposes.In general, Listeria species were detected in 14 (56%) of the 25 samples of fermented fish from the wet market. However,Listeria monocytogenes were isolated from 3 (12%) of the 25 samples examined. The major outbreaks of listeriosis has beenassociated with consumption of foods of animals and fish origin. with case-fatality rates of about 20-30%. Thus the detectionof all the Listeria species and especially the Listeria monocytogenes in the fish samples examined is of great concern since fishhave been reported as possible vector of Listeria monocytogenes.Salmonella Enteritidis is one of the most common causes of nontyphoidal salmonellosis after S. Typhimurium, thus it is ofimportance to determine the prevalence of this pathogen in popular foods, such as fish. In this study, S. Enteritidis has beenisolated from lO of 23 samples of tilapia (Tilapia mossamblcai fish obtained from the wet market. Since tilapia is a popularfish served in restaurants as well as at food stalls in Malaysia, there is a need for consumer protection against infection by323UPM Research Report 1997-2000, Vol II,Section 2-Extended Abstractspotential pathogenic strains of the bacteria from fish. Though foods are normally prepared thoroughly by cooking, there isalways the possibility of cross-contamination at the processing, preparation and service steps. In addition, the consumption ofready-to-eat raw fish is getting popular in Malaysia.ConclusionsThis study confirms the present of the various species of human bacterial pathogens from the different types of samplesexamined, and reinforce the notion that foodbome pathogen can be a potential health hazard. There is no proper surveillancesystem for these pathogenic bacteria in Malaysia. The ingrained tradition of not taking infections seriously such as having about of stomach-ache after consumption of foods could contribute to the under reporting of foodbome diseases in Malaysia.Benefits from the studyThis study provide an insight into the prevalence of these human bacterial pathogens in the foods and environmental samplesexamined. In addition, several postgraduate students have been trained in the isolation, identification and molecularcharacterization of these pathogens.Patent(s}, if applicable:NilStage of Commercialization, if applicable:NilProject Publications in Refereed JournalsI. Son Radu, Noorlis A., Foo HL and Reezal A. 2003. Prevalence and characterization of Aeromonas species from retail fishin Malaysia. International Journal of Food Microbiology 81: 261-2662. Lesley MB, Son Radu, Laurence J, Vickneswaran Y, Cheah YK, Wong CMYL and Nishibuchi M. 2002. Vibrioparahaemolyticus from local cockles (Anadara granosa). The Medical Journal of Malaysia 57 (Suppl. D): 137.3. Son Radu, Tg Ahbrizal TA and Sahilah AM. 2002.Characterization of Vibrio vulnificus isolated from retail cockles and shrimps by plasmid profiling and antibiotic susceptibility.Journal of Tropical Agriculture and Food Science 30: 31-37.4. Zaiton H, Endang P, Son Radu, Raha AR and Rusul G. 2001. Prevalence of Listeria spp. and Listeria monocytogenes inmeat and fermented fish in Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 32: 402-4075. Endang P, Son Radu, Zaiton H, Ooi WL and Raha AR. 2000. Plasmid-mediated streptomycin resistance of Listeriamonocytogenes. Malaysian Journal of Medical Sciences 7: 48-516. Tg, Ahbrizal, Son Radu, Abdul Reezal AL, Rahim AM, Rusul G, Nasreldin E and Yuherman. 2000.Random amplified polymorphic DNA analysis of Vibrio vulnificus strains isolated from cockles and shrimps. MalaysianJournal of Medical Sciences 7: 41-467. Son Radu, Yuherman, Rusul, G, Lum KY and Nishibuchi M. 2000. Detection and molecular characterization of Vibriovulnificus from coastal waters in Malaysia. Southeast Asian Journal of Tropical Medicine and Public Health 31: 668-6738. Son Radu, Sahilah AM, Rusul G, Samuel L, Zuraini MI and Nasreldin E. 2000. Comparison of arbitrarily primed PCR,antibiotic resistance and plasmid profiling for differentiating Salmonella enteritidis isolated from fish. Asian Fisheries Science13: 13-20Project Publications In Conference ProceedingsI. Son Radu, Cheah YK, Lesley M, Yickneswaran Y, Gwendolyne BT, Nasreldin EH, Yuherman, Chien HC and NishibuchiM. 2002. Detection of Vibrio cholerae and other Vibrio spp. from environmental sources in Malaysia. In: Proceedings of theUS-JAPAN Cooperation Medical Science Program Conference. 17-19 December. Laguna Garden Hotel, Naha, Okinawa,Japan2. Son Radu and Nishibuchi M. 2002. Detection of toxigenic Vibrio cholerae 0139 and 01 in the seafood marketed inMalaysia. In: Proceedings of the US-JAPAN Cooperation Medical Science Program Asian Region Collaboration ResearchProject Meeting. 21-23 January 2002. International MedicalCenter, Tokyo, Japan.3. Lesley MB, Son Radu, John L, Cheah YK, Belinda E and Nishibuchi M. 2003. Improved method and rapid identification ofVibrio parahaemolyticus strains from local cockles (Anadara granosa) using CHROMAgar™ Vibrio and PCR targeted thetoxR gene. In: Proceedings of the 13th Scientific Meeting of the Malaysian Society for Molecular Biology and Biotechnology.19-21 May 2003. PutraJaya, Malaysia. p44.4. Lesley MB, Son Radu, Raha AR, John L, Cheah YK, Belinda E and Nishibuchi M. 2003.Antibiotic susceptibility of Vibrio parahaemolyticus isolated from cockles (Anadara granosa). In: Proceedings of the 7thSymposium of Applied Biology. 3-4 June 2003. Selangor, Malaysia. p64.5. Gwendolyne BT, Son Radu and Nishibuchi M. 2002. Identification of Vibrio parahaemolyticus strains by CHROMagarTMVibrio and detection of thermostable direct hemolysin gene (tdh), thermostable direct hemolysin-relata! itrh) and toxk geneusing PCR based method. In: Proceedings of the Malaysia Science and Technology Congress 2002. 12-i4 December 2002.Kuching, Sarawak, Malaysia.324Agricultural Sciences6. Nasrcldin H, Son Radu, Rusul G, Zaiton H, Yousr A, Rcczal A, Lum K, Nishibuchi M. 200 I. Prevalence of pathogenicVibrio species in seafood. In: Proceedings of the 24th Symposium of the Malaysian Society for Microbiology. September 9.12,2001. Cherating, Pahang. p23.277. Noorlis A, Son Radu, Foo HL, Reczal A, Yuherman and Sahilah AM. 2001. Characterisation of strains of Aeromonas spp,by whole cell protein fingerprints. In: Proceedings of the 24th Symposium of the Malaysian Society for Microbiology.September 9·12, 200 I. Cherating, Pahang. p43-448. Yousr A, Zaiton H, Son Radu, Rusul G, Nasreldin EH, Samuel Land Apinya V. 2001. Incidence of Listeria spp. in shrimpavailable in Selangor wet markets. In: Proceedings of the 24th Symposium of the Malaysian Society for Microbiology.September 9·12, 200 I. Cherating, Pahang. p 164·1679. Nasreldin, E. H., Son Radu, Rusul, G., Hassan, Z., Reezal, A. and Lurn, K. Y. 2000. Application of polymerase chainreaction (PCR) and DNA colony hybridization in detection of Vibrio cholerae genes isolated from seafood marketed inMalaysia. In: Proceedings of the 23rd Symposium of the Malaysian Society for Microbiology. November 19·21, 2000.Langkawi, Malaysia. p29·3210. Hassan, Z., Purwati, E., Son Radu, Raha, A. R. and Rusul, G. 2000. Incidence of Listeria species from food sources inMalaysia. In: Proceedings of the 23rd Symposium of the Malaysian Society for Microbiology. November 19·21, 2000.Langkawi, Malaysia. p33·36II. Tg Ahbrizal, Son Radu, Reezal A, Mutalib AR, Nasreldin E and Yuherman. 2000. SDS·PAGE analysis of whole cellproteins for analyzing the clonal lineage among Vibrio vulnificus strains isolated from cockles iAnadara granosay and shrimps(Paneus indicus). In: Proceedings of the 23rd Symposium of the Malaysian Society for Microbiology. November 19·21,2000.Langkawi, Malaysia. p 160·16212. Tg Ahbrizal TA, Son Radu, Reezal A, Mutalib AR, Rusul G, Nasreldin E and Yuherman. 2000. Strain typing of Vibriovulniflcus biotype I in cockles and shrimps by random amplified polymorphic DNA assay. In: Proceedings of the 4th UNESCONational Workshop on Promotion of Microbiology in Malaysia, 26 August, 2000, University Malaya. p32.13. Noorlis A, Son Radu, Ling FH, Latif ARA, Haryanti T and Ooi WL. 2000. Random amplified polymorphic DNA (RAPD)analysis of Aeromonas spp. from fish. In: Proceedings of the 6th National Conference on Medical Science, 10·11 May 2000,Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia. p II14. Nasreldin EH, Son Radu, Rusul G, Khair J, Hassan Z, Latif ARA, Nimita F and Sahilah AM. 2000. Prevalence of Vibriospp. in shrimp (Penaeus indicus) marketed in Malaysia. In: Proceedings og the 3rd National Symposium on Health Sciences,29·30 Apri12000. Putra World Trade Center, Kuala Lumpur. pl44·14815. Endang P, Hassan Z, Son Radu, Raha AR, Rusul G, Salasiah K, Sahilah AM, Samuel Land Nasreldin E. 2000. Studies onthe multiple antibiotic resistance of Listeria monocytogenes. 6th National Conference on Medical Science, to·I I May 2000,Universiti Sains Malaysia, Kota Bharu, Kelantan, Malaysia. p88Graduate ResearchNameGraduateof Research Topic Field of Expertise Degree Awarded Graduation YearYuherman Molecularcharacterization ofV. chalerae, V.parahaemolyticusand V. vulniflcusisolated frommarine sourcesMolecular Biology Ph.D. 2001Nasreldin Elhadi Molecular Molecular Biology Ph.D. 2001Hussein characterization ofVibrio cholerae andother Vibrio spp.from seafoods andclinical sources.Endang Purwati Prevalencemolecularcharacterization ofListeria spp.isolated from foodand Molecular Biology Ph.D. 2003sources.325UPM Research Report 1997-2000. Vol II. Section 2-Extended AbstractsTg Ahbrizal Isolation and Molecular Biology MSFarizal Tg molecularAhmad characterization ofVibrio vulnificusfrom shrimps.Noorlis Ahmad Isolation and Molecular Biology MSmolecularcharacterization ofAeromonas spp,from seafoods.Lesley Maurice Isolation, Molecular Biology MSidentification andmolecularcharacterization ofListeria species.200120012002IRPA Project number09-02-04-OI72UPM Research Cluster:AFF326

Recovery of pathogenic bacteria from aquatic environment, fish and cockles

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Recovery of pathogenic bacteria from aquatic environment, fish and cockles

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