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AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: 2151-7517, ISSN Online: 2151-7525, doi:10.5251/abjna.2012.3.9.360.364 © 2012, ScienceHuβ, http://www.scihub.org/ABJNA Micriobiological quality of smoke-dried mangrove oysters (Crassostrea gasar) sold in Port Harcourt, Nigeria Odu1, N.N, Njoku1, H.O. and Mepba2 H.D 1 Department of Microbiology, University of Port Harcourt, Nigeria Department of Food Science and Technology, Rivers State University of Science & Technology, Port Harcourt, Nigeria. [email protected] 2 ABSTRACT The microbiological quality of smoke-dried oysters (Crassostrea gasar) sold in Port Harcourt, Nigeria was investigated. The microbial load and pathogens in smoke-dried oysters from four major fishing ports: Kalabari (Ka), Bille (Bi), Okrika (Ok) and Nembe (NB) that supply the Port Harcourt market were evaluated and studied. Highest total viable counts were recorded in oysters from Bille and Nembe fishing ports and values ranged from 7.18-7.2log10 cfu/g and 6.7-6.9log10 cfu/g, respectively, while lowest counts were obtained in oysters from Okrika fishing port (5.75-5.9log10 cfu/g. The bacteria pathogens isolated were in the order of Staphylococcus aureus (45%), Bacillus cereus (25%), Micrococcus virans (15%), Klebsiella spp.(10%) and E.coli (5%). Among the predominant fungal isolates were Aspergillus niger, Aspergillus flavus, Penicillium spp, Neurospora spp. and Saccharomyces spp. Thus bacteria of aerial origin and storage fungi were isolated from smoke-dried oysters from the four major fishing ports that supply the fish market in Port Harcourt, Nigeria. The microbial load and pathogens in the smoke-dried oysters could be attributed to poor handling practices and faulty storage temperature by the fish vendors. Key words: Microbiological quality, oysters, fishing ports, bacterial isolates, pathogens. INTRODUCTION Molluscan bivalves such as oysters are seafoods that are widely consumed. They are excellent sources of protein but are highly perishable as fresh seafoods. Their short shelf life thus pose serious practical problems of their storage and distribution (Frazier and Westhoff, 2000). Oysters are consumed raw (ingested whole) in some parts of the world thereby leading to the transmission of pathogenic organisms (CDC, 2006). Thus various bacteria that are aetiologic agents of shellfish-related food infection such as salmonellosis, shigellosis, Vibrio and Hepatitis A virus had been isolated from oysters (CDC, 2006). Additionally, septicaemia had been reported among consumers of raw oysters from the Gulf of Mexico (FAO/WHO, 2005). Smoke-dried oysters account for over 75% of the animal proteins in the diets of people in the Niger Delta region of Nigeria. Traditional curing methods such as sun-drying and smoking are used in preserving seafoods that cannot be sold by fishermen (Eboh et al., 2006). Smoking was originally used for meat preservation whereby surface bacteria were killed by the heat of the smoke and regrowth prevented by dehydration and inhibitory chemicals such as formal dehyde in wood smoke (Potter and Hotchkiss, 2000). Smoked-dried oysters have unique flavour and are considered as delicacies in the Niger Delta region. They are eaten as snacks or with fermented tubers without subjecting them to further cooking. Although this post-harvest technology had remarkably reduced wastage and improved the keeping and eating qualities of seafoods (FAO, 1999; Adebayo-Tayo and Ogunjobi, 2008) the public health implication is underscored. The present study evaluates the microbial load and pathogens in smoke-dried oysters from local fishing communities in the Niger Delta region of Nigeria. MATERIALS AND METHODS: Samples source and preparation: Smoke-dried mangrove oysters (Crassostrea gasar) were purchased from Creek Road Market, a popular seafood market in Port Harcourt, during the months of April-June, 2010. The oysters had been harvested and traditionally smoke-dried at four fishing ports in Rivers State; (3 locations) and Bayelsa State (1 location). Among the fishing ports in Rivers State were: Kalabari (Ke boko Borikiri), Bille (Tuma Borikiri) and Okrika (Anchichukama Borikiri). In Bayelsa State was Nembe (Toniboko Borikiri). All samples were transported to the laboratory in sterile polythene bags and analysed within 2 hours of purchase. Microbiological Analysis: Triplicate samples (25g each) were asceptically transferred to a sterile stomacher (Model BA 6021, Seward Medical, UK) and homogenised with 225 ml sterile 0.1% peptone water (Oxoid CM 509) for 2 min. Serial ten fold dilutions of homogenates were made by transferring 1ml to fresh sterile diluent. Aliquots of appropriate dilutions were transferred separately to plates of surface-dried Nutrient Agar (Oxoid CM 3), for total bacteria count, Mannitol salt Agar (Oxoid) for staphylococcal count, Salmonella-Shigella agar (Oxoid) for salmonellae count. A pre-enrichment step described by APHA (2001) was followed for isolation of salmonellae. All Agric. Biol. J. N. Am., 2012, 3(9): 360-364 o (15%), Klebsiella spp. (10%) and E.coli (5%). Among the predominant fungal isolates were Aspergillus niger, Aspergillus flavus, Penicillium spp, Neurospora spp, and Saccharomyces spp (Table 3). plates were incubated 37 C for 48 hours. The 5-tube MPN method (Feng et al., 2002) was adopted for the enumeration of total coliforms, faecal coliform and E.coli. o The tubes for total coliforms were incubated at 37 C for 24 o hrs while those for faecal coliform were incubated at 45.5 C for 24-48hrs. Positive tubes showing acid and gas production were streaked on Eosin Methylene Blue Agar o and incubated at 37 C for 24 hrs. Estimation of bacterial numbers was by the plate count and the Most Probable Numbers (MPN) technique describe by Frazier et al., 1999. -1 For isolation of bacterial types, 0.1 ml of the 10 dilution of the sample was spread on appropriate media and incubated. Typical colonies on the media were picked, streaked out to obtain pure cultures and finally maintained on Nutrient Agar slopes in the refrigerator. Pure isolates were characterized and identified following morphological and biochemical tests such as motility, catalase, oxidase, gram stain, spore stain, indole production, citrate utilization, Methyle Red (MR), Voges Proskauer (VP), urease, coagulase and carbohydrate fermentation test. The isolates were identified following the method of Cowan and Steel (2003). Yeast and moulds were characterised and identified using the schemes of Frazier and Westhoff (2000). RESULTS Kalabari Bille Okrika Nembe 8 7 Log Cfu/g of Meat 6 5 4 3 2 1 The microbial load of smoke-dried oysters from the various fishing ports are expressed graphically in Fig 1. Highest total viable counts were recorded in samples from Bille and Nembe fishing ports and ranged from 7.18-7.2log10 cfu/g and 6.7-6.9log10 cfu/g, respectively while lowest total bacterial counts were obtained in samples from Okrika fishing port (5.75-5.9log10 cfu/g. Smoke-dried oysters from Kalabari and Bille fishing ports had highest staphylococcal counts (3.49-3.59log10 cfu/g and 3.49log10 – 3.6log10 cfu/g, respectively. The staphylococcal counts in samples from Okrika and Nembe ranged from 3.3log10 cfu/g – 3.4log10 cfu/g and 3.3log10 cfu/g-3.5log10 cfu/g, respectively. Smokedried oysters from Kalabari fishing ports had significantly (P≤ 0.05) higher fungal counts than samples from Bille, Okrika and Nembe fishing ports. Mean total fungal counts in smoke-dried oysters from Bille, Okrika and Nembe ranged from 3.33-3.31 log10 cfu/g. Data in Table 1 give the mean total/faecal coliform counts per gram of samples from the four fishing ports. Total coliform counts for smoke-dried oysters from Kalabari, Bille, Okrika and Nembe fishing ports ranged from 14.2 ± 1.0 to 24.6 ± 1.6 coliforms/g while < 3 faecal coliforms/gram were isolated on samples from each of the fishing ports. No significant difference (P>0.05) was observed in the total coliform counts (MPN/g) of samples from Kalabari and Nembe fishing ports. Smoke-dried oysters from Bille fishing ports had a significantly (P≤ 0.05) higher total coliform count than samples from other fishing ports. Table 2 presents data on the morphological and biochemical characteristics of bacterial isolates in smokedried oysters from the four fishing ports. The bacterial pathogens isolated were in the order of Staphylococus aureus (45%), Bacillus cereus (25%), Micrococcus virans 0 NA MSA SDA Fig. 1 Histogram distribution showing Microbial Load of Smoke dried Oysters from fishing ports in the Niger Delta, Niger DISCUSSION: Highest total viable counts were obtained in smoke-dried oysters from Bille, Nembe and Kalabari fishing ports (Fig 1.) The high bacterial counts on smoke-dried oysters from these fishing ports could be attributed to the frequent contamination of water with sewage in addition to the endogenous microflora of the oysters. Molluscs are transported live to the point of sale or processing where the flesh can often be contacted by hand. Although contamination may occur at this stage, the significant public health problems associated with shellfish arise from their ability to concentrate viruses and bacteria from the surrounding waters (Adams and Moss, 2005). The initial microbial load on ready-to-eat foods is important, however, factors such as, processing, storage and display may influence the microbiological load of ready-to-eat foods at the point of sale (Angelidis et al, 2006; Beuchat and Ryu, 2004). Although smoke-drying reduces water activity and destroys bacteria through the agency of heat, postprocessing contamination can and do occur especially during handling and transportation of processed foods to the point of sale (Mepba et al., 2002). Besides, the high relative humidity of the riverine communities, the exposure of the smoke-dried oysters and the unacceptable direct contact with hands of purchasers and hawkers alike during haggling for price (Mepba et al., 2008) could significantly contribute to the microbial load of the oysters. 361 Agric. Biol. J. N. Am., 2012, 3(9): 360-364 Table 1. Total/faecal coliform counts (MPN/g) LOCATIONS Kalabari TOTAL COLIFORMS (MPN/g) 24.6b ±1.6 FAECAL COLIFORMS (MPN/g) 2.0a ± 0.01 Bille 24.0a ± 7.0 2.0a ± 0.01 Okrika 14.2bc ±1.0 2.0a ± 0.01 b Nembe 2.0a ± 0.01 41.0 ± 2.0 Values are means ± S.D. of triplicate determinations. Means in the same columns not followed, by the same superscripts differ significantly (P≤ 0.05) using Duncan’s New Multiple Range Tests (DNMRT). Oxidase Citrate Urase Voges proskauser Indole Glucose Lactose Maltose - - + - + - - - + - A A - - Staphylococcus aureus + + + + - - - - - + - - A - - Bacillus spp. Bi, Ka, NB, Ok. Cocci + - - + - - - + - + - - A/G - - Micrococcus spp Bi, NB, Ka, Ok. Rods - + - + - - - - - + - - A/G A/G + Enterobacter spp Bi, NB, Ka, Ok. Rods - - - - - - + - - + - - A/G A/G + Klebsiela spp Bi, NB, Ka, Ok. Rods - + - + - - - - + - + - A/G A/G + E.coli H2s Coagulase + Rods Methyl red Catalase Cocci Bi, NB, Ka, Ok. Motility Ka, Bi, Ok, NB. LOCATION Gram stain Spores Cell morphology Table 2. Morphology and biochemical characteristics of bacterial isolates from smoke-dried oysters IDENTITY OF GENUS Ka = Kalabari, Bi = Bille, Ok = Okrika, NB = Nembe, A = Acid, A/G = Acid and Gas. Table 3. Morphological characteristics of predominant fungal isolates from smoke-dried oysters from fishing ports in the niger delta. LOCATION COLOUR MICROSCOPIC DESCRIPTION IDENTITY OF ORGANISM. Bi, Ka, NB, Ok Yellowish-green mycelium Brush-like condidia, septate branching conidiphore was smooth/rough walled. Penicillium spp. Ka, Bi, NB, Ok. Effuse black colony Simple septate and branched conidia in chains. Aspergillus niger NB, Bi, Ka, Ok Greenish mycelium Conidiophores with vesicles, unbranched, conidiohores in chains Aspergillus flavus NB, Ok, Ka. Pink Septate hyphae with conidiophores arranged at the tips Neurosphora spp. Ok, NB. Dirty, cream Spheroid with budding cells and one ascus Saccharomyces spp Bi = Bille, Ka = Kalabari, NB = Nembe, Ok = Okrika 362 Agric. Biol. J. N. Am., 2012, 3(9): 360-364 Data in Fig 1. also show that the staphylococcal counts in oyster samples from Okrika and Nembe fishing ports ranged from 3.29-3.36log10 cfu/g and 3.18-3.5log10 cfu/g, respectively. Staphylococcus aureus has been isolated in fish (Sokari and Anozie 2005), and in street foods (Mepba et al., 2008). Other ready-to-eat foods such as salads and sandwiches have also been implicated in foodborne illness outbreaks. These foods are often prepared by hand and this direct contact may lead to an increased incidence of contamination with potential foodborne pathogens, such as Staphylococcus (Christiansen and King, 2007; Colombari et al., 2007; Nguz, 2007). The presence of small numbers of Staphylococcus aureus in food is not uncommon but not a health threat because they are eliminated by cooking or pasteurization (Jay et al., 2005). Additionally, large numbers, typically > 106g-1 are required for production of enough toxin to cause illness. Thus contamination is necessary but is not alone sufficient for an outbreak to occur (Adegoke, 2010). (ICMSF, 1986). Christianson et al., (2008) reported about 5-6log cfu/g of coliforms and E.coli on meat- and cheese-based filled baguettes and salads containing meat in Johannesburg, South Africa. It has been suggested that several factors may contribute to the presence of E.coli in ready-to-eat foods like smoke-dried oysters, including poor handling practices by food handlers, or cross contamination from food contact surfaces or high storage temperatures as in the tropics (Fang et al., 2003). The predominant bacteria pathogens isolated from the smoke-dried oysters were of aerial origin (Table 2). The incidence of potential foodborne pathogens including S. aureus (ca. 3log cfu/g); B. cereus (ca. 2log cfu/g), E.coli (< 3.cfu/g) on the ready-to-eat smoke-dried oysters was relatively low. Studies have suggested that the presence of S. aureus on ready-to-eat food may be as a result of improper handling, cross contamination and poor temperature control (Synder, 2009; Christison et al., 2008). The predominance of Bacillus spp. could be attributed to the display of the dried oyster meat without any form of packaging. Being frequently displayed uncovered the meat became liable to contamination to bacteria of aerial origin. Some strains of Bacillus (e.g. B. cereus and Staphylococcus aureus are known enterotoxin producers (Bryant, 2007). The inherent danger in the association of B. cereus and S. aureus with or without their metabolic products in various foods, without further heat treatment is the possible outbreak of serious foodborne illness. Smoke-dried oysters from Kalabari fishing ports had significantly (P ≤ 0.05) higher fungal counts than the rest of the tested oysters (Fig. 1). Post-processing fungal contamination of oysters from the fishing ports was expected given the poor handling practices often exhibited by the fishermen and the high relative humidity of the storage environment. Besides, the flesh of molluscs such as cockles, mussels, oysters and clams differs from that of free swimming fish by containing appreciable amounts (about 3.0%) of carbohydrate in the form of glycogen (Adams and Moss, 2005). Though many of the same organisms are involved, spoilage is therefore glycolytic rather than proteolytic. Coliforms are still considered indicators for assessing general hygienic status of food contact surfaces, the contamination of oysters in the present study could be attributed to poor handling practices and improper storage temperatures (Jackson et al., 2005). The high total coliform counts (Table 1) of smoke-dried oysters from the fishing ports could be attributed to poor handling practices by the fishermen. Besides, the seas around coasts are influenced by inputs of terrestrial and fresh water microorganisms and by human activities. For instance, the seas in the Niger Delta has become convenient dump for sewage and other waste products (Eboh et al., 2006). Many shellfish used for food grow in polluted waters and majority feed by filtering out particles from large volumes of sea waters. These waters contaminated with sewage pose the risk that enteric organisms from infected individuals may be present and concentrated by the filter feeding activities of shellfish. However, a large infective dose of enteropathogenic E.coli (EEC) is required for either the enterotoxigenic or invasive illness to occur. Thus foods must be highly contaminated or inadequately preserved to allow for prolific growth (Adams and Moss, 2005). Among the predominant fungi isolated from the smokedried oysters were Penicillium spp., Aspergillus niger, Aspergillus flavus and Neurospora spp. (Table 3). The high humidity and warm environmental temperatures in the coastal areas of the Niger Delta predispose to growth of storage fungi such as penicillia and aspergilli that could result in mycotoxin production in the smokedried oysters. It has been suggested (Jay et al., 2005) that conditions of high humidity and warm temperature predispose to high levels of a flatoxin in foods, often exceeding the upper limit initially established by the Food and Agriculture Organization (FAO)and World Health Organisation (WHO) of 30 μgkg-1. Although aflatoxin may be considered amongst the most caranogenic of natural products for some animals, it is still not clear whether it is a carcinogen for man (Adams and Moss, 2005). Results from the present study show that E.coli counts did not differ significantly (P≤ 0.05) and were > 3.0 cfu/g of meat which did not exceed the maximum recommended E.coli count for good quality product 363 Agric. Biol. J. N. Am., 2012, 3(9): 360-364 common commercially available fish species in Oron Local Government, Nigeria.Food Chem. 97:490-497. CONCLUSION: Bacteria of aerial origin and storage fungi were isolated from smoke-dried oysters from the four major fishing ports that supply the fish market in Port Harcourt, Nigeria. Good hygienic practise aimed at minimizing the microbial load of these ready-to-eat foods must be considered. Proper packaging to prevent direct handcontact and contamination by bacteria of aerial origin, control of relative humidity and warm temperature that favour fungal growth is advocated. Fang T.J., Wie, Q., Liao, C., Hung, M. and Wang, T. 2003. Microbiological quality of 180C ready-to-eat food product sold in Taiwan.Int. J. Food Microbiol. 80:241-250 FAO (1999). Food and Agriculture Organisation. Studies on traditional and improved method of smoking fish. FAO Experts panel. Consultants on fish technology in Africa. Rome, Italy. FAO/WHO. 2005. Risk assessment of Vibrio vulnificus in raw oysters. Interpretative summary and technical report . Food and Agriculture Organization of the United Nations/World Health Organization, Geneva, Switzerland. REFERENCE: Adams, M.R and Moss, M.O. 1999. Microbiology of primary food commodities: In, Food Microbiology, 3rd Edn. The Royal Society of Chemistry, Cambridge, U.K. Pp 122. Feng, P., Weagant, S.D. and Grant, M.A. 2002. Enumeration of Escherichia coli and coliform bacteria. In: Bacteriological Analytical Method. 8th edn. Adebayo-Tayo, B.C and Ogunjobi, A.A 2008. Comparative effect of oven drying and sun drying on the microbiological, proximate and mineral composition of Tympanotonus spp. (Periwinkle) and Crassostrea spp. Elect. J. Agric and Food Chem, 7 (4): 2856-2862. Frazier, W.C and Westhoff, D.C. 2000.Classification and identification of molds/yeasts and yeast-like fungi. In Food Microbiology, 4th Edn.McGraw-Hill Book Company, Singapore. pp. 17-39. Adegoke, G.O. 2010. Bacteria in foods; In, Understanding Food Microbiology, 2nd Edn. Alleluia Publishers, Ibadan, Nigeria. P.45. Frazier, W.C. and Westhoff. D.C. 2000.Contamination, preservation and spoilage of fish and other seafood: In, Food Microbiology, 4th Edn. McGraw-Hill Book Company, Singapore. Pp. 243-253. Angelidis, A.S. Chronis, E.N, Papageorgiou D.K., azakis,I .I., Arsenoglou, K.C and Stathopoulos, G.A. 2006. Nonlactic acid containing flora in ready-to-eat foods: A potential quality index. Food Microbiol, 23:95-100. Frazier, W.C., Marth, E.H., Deibel, R.H. 1999. Principles of quantitation. In, Laboratory Manual for Food Microbiology. Burgess Publishing Company, Minneapolis, Minn. American Public Health Association (APHA) 2001.Compendium of methods for the Microbiological Examination of Foods. Vol.4, Washington DC. Pp.1219. Beuchat, L,R. and Ryu, J.H.2004. Produce handling and processing practices, Emerging Infectious Diseases, 3:459-465. International Commission on Microbiological Specifications for food (ICMSF) 1986, Recommended microbiological limits for seafoods. In, Microorganisms in Foods. 2. Sampling for Microbiological Analysis: Principles and Specific Applications, 2nd Edn. University of Toronto Press, Buffalo, N Y. Bryant, R.G. 2007. Selective enterotoxin production by a Staphylococcus aureus strain implicated in foodborne outbreak.J. Food Protect, 151:130-131. Jackson, J.M., Loessner, M.J. and Golden, A.A. 2005. Modern Food Microbiology, 7th edn. Aspen publishers Inc, Gaithersburg, Maryland U.S.A. CDC, 2006. Surveillance for foodborne disease outbreaksUnited States 1998-2002. Morb-Mortal Wkly Report.Surveill Summ, 55/SS-10.Pp. 1-42. Jay, J.M., Loessner, M.J. and Golden, A.A. 2005. Modern Food Microbiology (7th edn.). Aspen publishers Inc, Gaithersburg, Maryland U.S.A. Christiansen, L.N and King, N.S. 1991.The microbial content of some salads and sandwich at retail outlets.J. of Milk and Food Technol. 34;289-293. Mepba, H.D. 2002. Effect of processing methods and storage on the Qualities of coconut milk. Discov. Innov, 14 (3/4): 179-185. Christanson, C.A., Lindsay, D. and von Holy, A. 2008. Microbiological survey of ready-to-eat foods and associated preparation surfaces in retail delicatessens, Johannesburg, South Africa.Food Control, 19:727-733. Mepba, H.D, Achinewhu, S. C., Aso, S.N. and Wachukwu, C.K. 2008. Microbiological quality of selected street foods in Port Harcourt, Nigeria. J. Food Safety, 27:208-218. Nguz, K. 2007. Assessing food safety system in sub-Saharan countries: An overview of key issues.Food Control, 18:131 -134. Conlombari, V, Mayer, M.D, Laicini, Z.M., Mamizuka, E, France, B.D, Destro, M.I. 2007. Foodborne outbreak caused by Staphylococcus aureus: Phenotypic and genotypic characterization of strains of food and human sources. J. Food Protect. 70: 489 – 493. Potter, N.N. and Hotchkiss, J.H. 2000,Food Science, 5 th edition, CBS Publishers, New Delhi. Sokari, T.G and Anozie, S.O 2005. Occurrence of enterotoxin Producing strains of S. aureus from fish and related sources.Discov. Innov. 3: 69-71. Cowan, M and Steel, M. 2003. Manual for the identification of medical bacteria. 6th Edn. Cambridge University Press, London Pp.331. Synder, O.P. 2009. Hand washing for retail food operations-A review. Dairy, Food and Environmental Sanitation, 18:149-162. Eboh, L., Mepba, H.D. and Ekpo, M.B. 2006. Heavy metal contaminants and processing effects on the composition, storage stability and fatty acid profiles of five 364