Submission Date	: June 2,2015
Acceptance Date	: June 19,2015
Publishing Date	: June 30,2015
Type of Paper	: Research Article
Mode of Access	: Open Access
Author	:S.Velmurugan1*, Shani T.John1, Sheta Alemu2 and D. Senthil Nagaraj2
Affiliation	1 Department of Biology, Madawalabu University, Post Box No: 247, Bale Zone, Robe, Ethiopia.2Department of Chemistry, Madawalabu University, Post Box No: 247, Bale Zone, Robe, Ethiopia.
Country	:India
Contact	:  velkas.cas@gmail.com

Abstract

The sea food is considered highly nutritious and less harmful when compared to other red meats. In the present study, the total heterotrophic bacterial population density was recorded in four different site fish samples. The microbial load of freshly caught fish was low, ranged from 1.21×10³ to 1.42×10³, followed by fish after two hours 7.62×10³ to 8.83×10³, fish at landing center were 2.0×10⁴ to 3.22×10⁴ and fish at market ranged from 4.91×10⁴ to 6.07×10⁴. The total microbial load was increased along with pathogenic microbes. In this study totally 25 strains were isolated from fish samples, of these isolates 9 genera were identified based on the biochemical characters. The names of the bacterial genera were Vibrio (17.5%), Pseudomonas (22.5%), Shigella (5.75%), Bacillus (15.25%), and Escherichia coli (15.5%), Klebsiella (8.5%), Staphylococcus(7%), Corynebacterium (5%) and Salmonella (3%).

Key words

Contamination, Pathogens, Total Heterotrophic Bacteria, Sea food.

Introduction

Fish and seafood constitute an important food component for a large section of world population (Bark et al., 2011; Sakthivel and Fernando, 2012; Trivedi et al., 2012; Ozcan et al., 2013; Varadharajan et al., 2013). Fishery products can also be a source of various food borne diseases, (Darlington and Stone, 2001). It acts as a vehicle for all important species of food borne pathogens. Environmental conditions play an important role on the pathogens count in fresh fish (Bark et al., 2011). Shellfish may be contaminated with food borne pathogens which are naturally present in aquatic environments, such as Vibrio spp. or derived from sewage contaminate water such as Salmonella (Ali and Hamza, 2004). In  the  developing  world,  food borne  infection  leads  to the  death  of  many  children,  as  well  as  resulting  in diarrheal  disease  which  can  have  long-term  effects  on children’s growth as well as on their physical development  and  it  also  heavily  affects  the  healthcare systems (Adak et al., 2005).

According  to  Clarence  et al.,  (2009),  food  borne diseases  are  diseases  resulting  from  ingestion  of bacteria,  toxins  and  cells  produced  by  microorganisms present in food. The intensity of the signs and symptoms may vary with the amount of contaminated food ingested and susceptibility of the individuals to the toxin. The quality of fresh fish and seafood products is rapidly reduced as a consequence of various microbial, biochemical and chemical breakdown processes. The initial quality loss is mainly due to the post mortem autolytic activity and chemical degradation processes, such as lipid oxidation. The rate of quality loss depends directly on the nature of the fish species in question, as well as handling and storage conditions. Analyses of the microbial load and diversity are used to determine the amount of specific spoilage bacteria in a sample as well as the total bacterial count. For marine fish stored aerobically on ice, the bacterial flora is well studied, and dominated by Pseudomonas spp. (Gram and Huss, 1996). Knowledge of spoilage organisms and their specific activity in various fish species at different storage conditions has led to more precise shelf-life predictions and facilitated modeling of spoilage (Gram and Dalgaard, 2002). ¬ The main purpose of this study was to determine and evaluate the microbial contamination of fish Mugil cephalus collected from catching point to fish vendors.

Materials and Methods

Sampling Procedure

A total number of 90 samples of fish Mugil cephalus were collected from Parangipettai coast, South East coast of India.

Fish from different sites, such as freshly caught fish, after two hour fish, fish at landing centre and fish at local market were collected. After collection, the samples were individually transferred to clean and unused polythene bags and transported to the laboratory in a portable ice chest box. Aseptic procedures were adopted to minimize secondary contamination. Seafood with visible signs of deterioration, injury and disease was discarded.

Bacteriological Analysis

Enumeration of Total Heterotrophic Bacteria (THB)

Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar was used for the selective isolation and culture of Vibrios. Eosin Methylene Blue (EMB) Agar and Salmonella-Shigella Agar (SSA) were used for E.coli and Salmonella, Shigella. MacConkey agar with 0.15 % bile salt, crystal violet and NaCl has been used in accordance with USP/nfxi (1) for the detection, isolation and enumeration of coli forms. Cetrimide agar was used as a selective medium for the isolation of Pseudomonas aeruginosa. The plates were used for different samples and the plates were incubated at 37°C for 24-48 hr for evidence of growth. Pure isolates of resulting growth were identified using biochemical methods as described by Jolt et al. (1994).

Results

Total heterotrophic bacterial population

Total heterotrophic bacterial population of the fish from four different sites were analyzed and recorded. The microbial load of freshly caught fish was low, ranged from 1.21×10³ to 1.42×10³, followed by fish after two hours 7.62×10³ to 8.83×10³, fish at landing center were 2.0×10⁴ to 3.22×10⁴and fish at market ranged from 4.91×10⁴ to 6.07×10⁴. The total microbial load was increased along with pathogenic microbes (Table.1).

Generic composition of total heterotrophic bacteria

The total number of 25 strains were isolated from four different site fish samples and randomly selected, sub-cultured and identified up to generic level. The total of 9 genera were recorded from the samples such as Vibrio (17.5%), Pseudomonas (22.5%), Shigella (5.75%), Bacillus (15.25%),Escherichia coli (15.5%), Klebsiella (8.5%), Staphylococcus (7%), Corynebacterium (5%) and Salmonella (3%) (Fig.1). The result showed the presence of more number of gram negative genera than the gram positive genera. Totally 6 genera recorded namely Vibrio, Pseudomonas, shigella, E. coli, Klebsiella and Salmonella belong to the gram negative group.

Table 1. THB population in four groups of fish samples

Fig.1. Percentage composition of microorganisms

Discussion

Microbes play an important role in determining the quality of any aquatic environment. In the marine environment, they balance characteristically in different ecosystem in which they live. In this study total bacterial population was recorded from in freshly caught fish was low, ranged from 1.21×10³ to 1.42×10³, followed by fish after two hours 7.62×10³ to 8.83×10³, fish at landing center were 2.0×10⁴ to 3.22×10⁴ and fish at market ranged from 4.91×10⁴ to 6.07×10⁴. Prabakaran et al., (2011) reported the total plate count showed maximum in the processing area samples in fish samples (65×10⁴ CFU.g-1). The Higher microbial counts in some samples may be attributable to handling during harvest or processing.

The total bacteria count on fish rarely indicate the quality of the fish but it gives an indication of the risk of spoilage induced since each of these organisms had different ways of effecting health conditions of consumers of such contamination fish (WHO, 2007). Montville et al. (2002) have similarly concluded that, during handling and preparation, bacteria may be transferred from contaminated hands of food workers to food and subsequently to other surfaces (including food contact surfaces). In this study total of 9 genera were recorded from the samples such as Vibrio, Pseudomanas, Shigella, Bacillus, Escherichia coli, Klebsiella, Staphylococcus, Corynebacterium and Salmonella. Venugopal (2002) studied the contamination of fish particularly by pathogens such as Salmonella sp., Staphylococcus aureus, Campylobacter jejuni, Escherichia coli, Vibrio parahaemolyticus, Yersinia enterocolitica, and Listeria monocytogenes, may occur prior to harvest, during capture, processing, distribution and storage. The contamination of fish from enteric bacteria of human and animal origin may also be responsible for various food spoilages (Empikpe, 2011). During handling and storage, or while transporting quality deterioration of fresh fish rapidly occurs and limits the shelf life of the product, Adebayo-Tayo et al. (2012b). The quality of fish degrades; due to a complex process in which physical, chemical and microbiological form of deterioration is implicated (Adedji, 2012). Huss et al. (2000) have pointed out that some pathogenic bacteria are naturally present in the aquatic (Clostridium botulinum type E, pathogenic Vibrio sp., Aeromonas) and the general environment (C. botulinum, type A and B, Listeria monocytogenes) and may therefore be found on live or raw fish. More fascinatingly, fish at market and fish at landing center has recorded more number of pathogenic bacterial populations and comparatively very low population density of E. coli and total absence of the other bacteria have been noticed freshly caught fish. The study done by Reij et al. (2004) attributed poor hygiene, particularly deficient or absence of hand washing as the causative mode of transmission and contaminated surfaces has been observed in many cases and unclean, insufficiently or inadequately cleaned equipment have been identified as a source of bacterial contamination in seafood.

Conclusion

The present study suggested that the contamination level was found increased from the catch point to the consumer end because of unhygienic handling and time taken for selling in the market resulted in deterioration. The present investigation concluded that microbial load increased based on the time duration and improper handling of the vendors. To avoid these problems fish should be washed properly with the natural antibacterial compounds like salt, turmeric and tamarind, after washing the fish should be boiled at a maximum temperature. This process of washing and boiling would be helpful in eradicating the pathogens from the sea food before consumption by the human beings.

Acknowledgement

The authors are thankful to the authorities of Annamalai University for the facilities provided and CAS in Marine Biology for the encouragement and support. The authors are sincere thanks to Madawalabu University, Bale Robe, Ethiopia.

References

1. Adak G K, Meakins S M, Yip H, Lopman B A, O’Brien S J (2005). Disease risks from  foods,  England  and  Wales,  1996–2000.  Emerging Infectious Diseases.(Retrieved on 2013, 04 20).
2. Adebayo-Tayo B C, Ody N N, Anyamele L M, et al. 2012. Microbiological and physiochemical changes and its correlation with indices of tilapia fish (Oreochromis niloticus) sold in Itu and Uyo markets in Akwa Ibom State,Nigeria. Nature and Science, 5(4): 38-45
3. Adedji O B, Okerentugba P O, Innocent-Adiele H C, and Okonko I O. 2012. Benefits, Public health hazards and risks associated with fish consumption. New York Science Journal, 5(9): 33-61
4. Ali M A, and Hamza M I E. 2004. Prevalence of seafood borne pathogens in shellfish at retail level.2004. First Annual Conference Faculty Veterinary Medicine, Moshtohor, Egypt
5. Bakr W M K, Hazzah W A, and Abaza A F.2011. Detection of Salmonella and Vibrio species in some seafood in Alexandria. Journal of American Science, 7(9): 663-668
6. Clarence  Y,  Obinna  C N and  Shalom  NC  (2009).  Assessment  of bacteriological  quality  of  ready  to  eat  food  (Meat  pie)  in  Benin  City metropolis, Nigeria. Afr. J. Microb. Res.3 (6): 390-395.
7. Darlington L G, and Stone T W.2001. Antioxidants and fatty acids in the amelioration of rheumatoid arthritis and related disorders. British Journal of Nutrition, 85(3): 251-269
8. Emikpe B O, Adebisi T, and Adedji O B.2011. Bacteria load on the skin stomach of Clarias gariepinusand Oreochromis niloticusfrom Ibadan South west Nigeria: Public health implications. Journal of Microbiology and Biotechnology Research, 1(1): 52-59
9. FDA, 1998. Bacteriological Analytical manual, 8th ed. Chapter.3
10. Gram, L. and Dalgaard, P. 2002. Fish spoilage bacteria-problems and solutions. Current Opinion in Biotechnology. 13: 262-266.
11. Gram, L. and Huss, HH. 1996. Microbiological spoilage of fish and fish products. International Journal of Food Microbiology. 33: 121-137.
12. Huss, H H, Reilly, A. and Embarek, P.K.B., Food Control, 2000, 11: 149-156.
13. Jolt, JG, Krieg, N R, Sneath, P H A, Stanley, J T, and Williams, S.T. 1994. Bergey’s manual of systematic bacteriology, 9th ed. Williams & Wilkins Co. Baltimore, Maryland, p786.
14. Montville, R., Chen, Y., and Schaffner, D.W. 2002. Risk assessment of hand washing efficacy using literature and experimental data. International Journal of Microbiology. 73: 305-313.
15. Ozcan T, Erdogan H, and Ozcan G. 2013. A contribution to a knowledge ofthe freshwater decapods of Hatay region, Turkey. Arthropods, 2(1): 42-44
16. Prabakaran P, Sendeesh Kannan K., Anand, M.and Pradeepa V .2011. Microbiological quality assessment in a fish processing plant at Mandapam, Ramanathapuram District. Archives of Applied Science Research, 3 (2):135-138
17. Reij, M W, Den Aantrekker, E D, and ILSI Europe Risk Analysis in Microbiology Task Force 2004. Recontamination as a source of pathogens in processed foods. International Journal of Food Microbiology. 91:1-11.
18. Sakthivel K, and Fernando A. 2012. Brachyuran crabs diversity in Mudasal Odai and Nagapattinam coast of south east India. Arthropods, 1(4): 136-143
19. Trivedi J N, Gadhavi M K and Vachhrajani K D. 2012. Diversity and habitat preference of brachyuran crabs in Gulf of Kutch, Gujarat, India. Arthropods, 1(1): 13-23
20. Varadharajan D, Soundarapandian P and Pushparajan N. 2013. The global science of crab biodiversity from Puducherry coast, south east coast of India. Arthropods, 2(1): 26-35
21. Venugopal, V. 2002. Biosensors in fish production and quality control. Biosensors and Bioelectronics. 17: 147-157.
22. WHO (World Health Organization). 2007. The world health report, a safer future. Global public health security in the 21 st century. Geneva, Switzerland