Detection of Vibrio spp.

in Raw Maguro (Bluefin Tuna) Sashimi From Citra Mina Seafood Market
Anna Margarita P. Tongco
Department of Food Science and Nutrition, College of Home Economics, University of the Philippines, Diliman, Quezon City 1101, Philippines Received 08 March 2011

Abstract

This study involves the analysis for the presence of Vibrio spp. in raw maguro (bluefin tuna) sashimi, as well as its overall microbial quality. The method of detection utilized was analysis of growth on Thiosulfate Citrate Bile Salts Sucrose (TCBSS) agar, and consequent determination of total plate count (TPC) from said growth. The final TPC value obtained was reported as 633.48 CFU/g, which falls below the literature value of 103 CFU/g from previous studies for microbial quality of fresh and frozen fish products. These results indicate that the raw bluefin tuna sashimi analyzed is of good microbial quality and is fit for human consumption.

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1. Introduction 1.1. Maguro sashimi The market for fish products and other types of seafood is very large and diverse. Globally, over 63.5 million tons of seafood are caught and consumed each year. In particular, fish is a popular food product due its high protein content, as well as its high level of omega-3 fatty acids, which are known to reduce cholesterol levels (Feliciano et al, 2010). In order to preserve much of the nutritional value of the fish, many have taken to finding ways to consuming fish that is as minimally processed as possible. The least processed way to eat fish is when it is raw and freshly caught. One food that utilizes raw fish is sushi. It has long been known as a traditional Japanese food, but the practice of eating it has spread around the globe. Sushi is made with vinegared rice, vegetables, seaweed, and raw fish. One particular type of sushi, known as sashimi, is defined as raw fish served alone, without any other ingredients. (Wallner et al, 2006). The fish product of choice often used for sashimi is maguro, or bluefin tuna. It is, quite possibly, the best-known and most commonly eaten fish in all of sushi dining. Approximately 80% of the worlds bluefin tuna catch is used for sashimi and other sushi styles. The part of the tuna that is most prized for sashimi is the belly, because it is the fattiest portion of the tuna and provides the silkiest mouthfeel when eaten raw (The Sushi FAQ, 2000).

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1.2. Microbial contamination of raw fish Raw foods can contain bacteria and, if handled incorrectly, the population of bacteria present in the food product can increase. It is also susceptible to cross-contamination due to poor handling and storage. As such, special procedures and principles must be observed to ensure that the food product is safe for human consumption (NSW Food Authority, 2009). Fish carry normal microflora in their skin, scales, and digestive tracts (Ray et al, 2008). However, it is the overall sanitary quality of the water from which the fish are taken that has the largest effect on the microbial quality of the finished product (Jay et al, 2005). Different steps in processing scaling, skinning, cutting can also add to the microbial load of the product. Raw fish is of particular concern, because its high water and protein content favors the growth of pathogens, and because there is a lot of hand contact involved in its preparation (FEHD, 2001). It also undergoes very few processing steps and therefore, any means of sanitation before it is consumed. Due to this lack of antimicrobial processing, raw fish is classified as a potentially hazardous food (NSW Food Authority, 2007), and as such must be subjected to more stringent types of storage to prevent contamination and ensure food safety (Wallner et al, 2006). Low temperature storage is the most commonly used method of storing raw fish and preventing additional microbial contamination to the product. The low temperature is the key factor in maintaining the quality of fresh fish, since this would slow down and therefore limit the microbial and enzymatic activity which causes deterioration of the fish (Trazo, 2001). In particular, chilling with the use of ice the reduction of temperature to some point below (-2 to -4 C) or above (0 5 C) the freezing point of water is effective because, as ice
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melts, it absorbs large quantities of heat before the temperature of the ice changes (Trazo, 2001). Studies have also shown that the use of sanitized ice in the storage of fish products can reduce bacterial load on the fish as the ice melts, therefore reducing the potential for crosscontamination (Feliciano et al, 2010). To avoid microbial contamination, fish and other marine products should be harvested from unpolluted water sources, and proper sanitation and hygiene practices must be observed throughout the entire handling process. All products should also be stored properly to prevent any further microbial growth and contamination (Ray et al, 2008). Despite these sanitary measures, there are still many recorded outbreaks of foodborne illnesses from marine products (Botana, 2008). Many microorganisms are identified with the contamination of raw fish; Pseudomonas, Salmonella, Escherichia coli, Staphylococcus aureus, and Vibrio are important examples (Gabriel, 2010).

1.3. Vibrio Vibrio is a genus of Gram-negative bacteria, identified by their characteristic curved rod shape with polar flagella. During cultivation on agar, however, vibrios develop lateral flagella not as long or as thick as the polar flagellum which are associated with increased motility (Cruickshank et al, 1973). Most members of the family Vibrionaceae are facultative anaerobes, halophilic, oxidasepositive, and all use D-glucose as their primary energy source (Willey et al, 2008). They are most commonly found in marine and estuarine environments, but some species of Vibrio have been found in freshwater environments as well (Jay et al, 2005).
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Several vibrios are noted as important pathogens. Vibrio cholerae causes cholera, a dangerous disease which has caused seven pandemics throughout recorded history (Willey et al, 2008). Vibrio vulnificus is a highly lethal pathogen which can invade the bloodstream, causing septicemia through food poisoning and wound infection. The disease it causes has a mortality rate of over 50%, and is responsible for 95% of all seafood-related deaths (Todar, 2008). Vibrio parahaemolyticus is an invasive microorganism which affects the digestive tract, and causes gastroenteritis due almost entirely to the consumption of contaminated seafood. Vibrio parahaemolyticus is the prime focus of this study, due to its association with seafood.

1.4.Vibrio parahaemolyticus Vibrio parahaemolyticus is one of the best-described pathogenic vibrios, first reported in Japan in 1951 by Dr. Fujino (Jay et al, 2005; Ray et al, 2008). It is the causative organism of vibriosis, the gastroenteritis associated with the consumption of raw or undercooked seafood (Sakazaku, 2003; Jay et al, 2005). Studies have shown that 60-100% of seafood samples in the US are contaminated with the organism (Curtis, 2006), and accounts for 40-70% of the total bacterial foodborne diseases in Japan (Sakazu, 2003; Ray et al, 2008). V. parahaemolyticus is a Gram-negative, motile, non-sporulating, curved rod. They are generally oxidase- and catalase-positive. All strains can grow in media containing glucose without gas production, but are unable to ferment sucrose and lactose (Curtis, 2006; Ray et al, 2008). V. parahaemolyticus is commonly found in marine environments, but can only be detected at certain water temperatures. They, however, can be killed off in distilled water (Jay et al, 2005). They can grow over a temperature range of 5-42 C, with an optimum temperature for

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growth at 30-37 C. They have a pH range of 4.8 to 11.0, with 7.6 to 8.6 being the optimum pH for growth (Jay et al, 2005; Curtis, 2006; Ray et al, 2008). Studies have shown that some strains are capable of resuscitating and surviving even after initial methods of antimicrobial treatment (Chiang et al, 2009). Not all strains of V. parahaemolyticus are clearly enteropathogenic for man. The foodborne pathogenic strains can cause hemolysis because of the presence of a heat-stable hemolysin, and thus are designated as Kanagawa-positive from the Kanagawa phenomenon in which isolated samples of V. parahaemolyticus display hemolysis on high-salt Wagatsuma blood agar (Cruickshank et al, 1973; Ray et al, 2008). Most strains isolated from natural sources are not hemolytic on this medium, and are therefore Kanagawa-negative. Vibriosis, the gastroenteritis caused by V. parahaemolyticus, can occur after ingesting raw or contaminated seafood. An individual must consume 105 107 cells of a Kanagawapositive strain for symptoms to develop. Incubation time is 4 96 hours, and symptoms appear 10 24 hours after ingestion of the cells and can last for 1 7 days. Symptoms include vomiting, abdominal cramps, nausea, diarrhea, fever, headache, and chills (Ray et al, 2008). Though it can cause significant discomfort, vibriosis is rarely fatal. However, it can spread easily through the fecal-oral route, or through wound infection (Curtis, 2006). Due to its virulent nature, the detection of V. parahaemolyticus, and its differentiation from other related microorganisms, is of high priority in seafood safety (Su et al, 2007). There are several ways to distinguish the microorganism.

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V. parahaemolyticus performs enzyme activities on certain amino acids. This, along with other biochemical reactions, can be used to distinguish it from Aeromonas and Plesiomonas.

Test Oxidase Catalase Lysine decarboxylase Glucose (acid) Mannose (acid) Indole Growth in: 0% NaCl 6% NaCl Motility Gelatinase

Vibrio + + + + + + V V + +

Aeromonas + + + + + + + +

Plesiomonas + + + + + + + -

Campylobacter + V + + -

Aerobic growth + + + Table 1. Representative biochemical reactions of Vibrio, Aeromonas, Plesiomonas, and Campylobacter. (Cruickshank et al, 1973)

V. parahaemolyticus can also be differentiated from other vibrios through the use of certain biochemical tests.

Test Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Acid from: Lactose Sucrose Arabinose Voges-Proskauer Growth in: 0% NaCl 3% NaCl 6% NaCl 10% NaCl

V. cholerae + + (+) + V + + V -

V. parahaemolyticus + + V + + -

V. alginolyticus + + + + + + +

V. anguillarium + + V + + V -

V. metschnikovi + V V + + + + -

Group F Vibrio + + + V + + -

V. vulnificus + V + + -

Table 2. Representative biochemical reactions of Vibrio species (Cruickshank et al, 1973)

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Once V. parahaemolyticus has been detected and identified, action must be taken immediately. Several factors need to be considered in the control of gastroenteritis from V. parahaemolyticus. Seafood harvested should be assumed to contain the microorganism, and should be treated with the proper processes accordingly. In improperly stored or inadequately heated seafood, the cells can rapidly multiply and produce toxins. Even heat treatment cannot eliminate the toxins once the pathogenic strains grow to infective numbers. Thus, the best way to prevent the proliferation of V. parahaemolyticus in raw seafood is to avoid temperature abuse (Ray et al, 2008).

1.5 Objectives of the study This study was conducted to analyze for the presence of Vibrio spp. in raw bluefin tuna slices intended for use in sashimi. The results of this analysis may give additional information about the microbial aspect of specific minimally processed foods, and how these products should be handled properly for consumption.

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2. Materials and Methods 2.1. Procurement of sample Pre-cut slices of raw bluefin tuna belly were procured from the Citra Mina Fresh Seafood Market, found at #8 Presidents Avenue, Tahanan Village, Sucat, Paraaque City. The bluefin tuna belly was already pre-packaged in sealed plastic packaging, and stored in a freezer. It could be seen through the glass doors that the shelves holding the fish products are neat and clean.

Figure 1. Exterior of Citra Mina Seafood Market.

Figure 2. Freezers housing the fish products.

The raw tuna belly slices were purchased the evening before the analysis was performed. A small sample of the tuna belly slices was transferred to a Ziploc bag and stored in a refrigerator before analysis was done 10 hours later.

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2.2. Microbial analyses The sample was aseptically prepared by manual crushing in a Ziploc bag. 25 grams of the crushed sample was measured onto a sterile Petri plate (Pyrex) using a triple beam balance. This sample was then transferred to an Erlenmeyer flask (Pyrex) containing 225.00 mL of 0.1% Peptone water (High Media, India). Afterwards, the flask was placed in the incubator, set at 37 C, and left for 4 and a half hours. After the incubation period, 1.00 mL aliquots were obtained from the diluent in the Erlenmeyer flask. This was transferred onto a sterile test tube containing 9.00 mL peptone water. Sequential 1.00 mL aliquots were obtained from the tubes until a dilution of 10-4 was attained. 0.100 mL aliquots were obtained from the diluted samples and aseptically inoculated onto duplicate plates (Pyrex) containing pre-prepared and solidified Thiosulfate Citrate Bile Salt Sucrose (TCBSS) agar (High Media, India), using spread plate method. Due to the inadequate amount of media available for laboratory use, only dilutions 10-1 10-3 were given duplicate plates. The 10-4 dilution was given only a single plate. The plates were incubated at 37 C for 24 hours to test for the presence of Vibrio spp. in the sample.

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3. Results and Discussion

3.1. Microbial quality of collected samples of raw maguro sashimi

Day 1 5

Date 4-Feb 8-Feb

10-1 A 0 2

10-1 B 1 3

10-2 A 1 3

10-2 B 4 5

10-3 A 0 1

10-3 B 0 0

10-4 A 0 0

Table 3. Recorded CFU of Vibrio counted.

The results of microbial analyses for the raw maguro sashimi samples are summarized in Table 3. Serial dilutions of the sample were spread-plated onto Thiosulfate Citrate Bile Salt Sucrose (TCBSS) agar, and the resulting growths were analyzed after 1 day and subsequent. Of the 7 plates prepared, only 4 displayed the growth of colonies after 1 day, and that growth per plate was minimal enough (<25 CFU) to be considered negligible. Even after 5 days, only 5 of the 7 plates displayed growth, and the bacterial colony count per plate was still below 25 CFU. Altogether, the final CFU/g was recorded as 633.48 CFU/g, or 6.33 x 102 CFU/g. The literature value given by the Food and Drug Administration in the Philippines for the maximum CFU/g of Vibrio parahaemolyticus in fresh and frozen fish is 103 CFU/g (Gutierrez, 2004), and the recorded value from this study falls below that. Thus, it can be concluded that the experiment yielded negative results. However, it must be noted that there were other microorganisms observed in the plates. They were not the distinctive moist, opaque yellow, nucleated colonies that identify Vibrio spp (Sakazaku, 2003). Some of the observed colonies in the plates were pale yellow string-like growths, in a swarming pattern. These colonies are possibly of the microorganism Proteus, of the Enterobactericeae family (Jay et al, 2005). As Vibrionaceae the family under which Vibrio
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spp. is listed is closely related to Enterobactericeae (Willey et al, 2008), it is also possible for members of this family to grow in TCBSS agar.

3.2. Vibrio spp. growth in collected samples of raw maguro sashimi It is expected that there would be practically no bacterial growth from the raw maguro sashimi sample. As the raw fish product was obtained from a very clean source, and was stored in a freezer prior to sampling and analysis, it is safe to assume that several means have already been taken to ensure the decrease of the microbial load in the sample even before microbial analysis was performed. Vibrio parahaemolyticus, in particular, cannot grow at temperatures below 5 C (Ray et al, 2008), and their numbers can be greatly reduced at temperatures of -20 C (Saxena et al, 1985). It is also possible that the conditions of the experiment were not ideal for the growth of Vibrio spp. Vibrio is known to be a Viable but Non-culturable (VBNC) microorganism, making analysis of Vibrio spp. through direct plate count more difficult (Wong et al, 2004). Several adjustments must be made in order to grow Vibrio spp. at room temperatures, especially if the sample source came from low temperature storage (Mizunoe et al, 2000).

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4. Summary and Recommendations This study analyzed for the presence of Vibrio spp in raw maguro (bluefin tuna) sashimi slices, purchased from Citra Mina Seafood Market in #8 Presidents Avenue, Tahanan Village, Sucat, Paraaque City. The basis of microbial quality is the CFU/g sample level obtained from the inoculation of serially diluted samples into Thiosulfate Citrate Bile Salt Sucrose (TCBSS) agar plates. Only 5 out of the 7 plates yielded positive results. The final CFU/g value recorded was 6.33 x 102 CFU/g. Based on the literature value from previous studies, wherein a CFU/g sample value of 103 indicates relatively high and possibly unsafe levels of microbial contamination in the food, it can be concluded that the raw bluefin tuna belly samples analyzed are safe for human consumption. This can be attributed to the minimal processing and sanitizing undergone by the fish product prior to purchase and analysis. It is recommended that additional biochemical tests be performed to identify the species of Vibrio present in the sample, minimal as it may be. The hygiene and food handling practices of the establishment from which the fish was bought should also be noted, so other establishments may follow in their lead.

5. Acknowledgements The author would like to thank Sir Al Gabriel for his assistance, guidance, patience, and good humor throughout this study, Maam Kristia Marte for watching over me in Sir Als absence, Mang Jimmy and Kuya Mike for assisting in the use of the laboratory, the graduate students who graciously shared the use of their laboratory in every step of the experiment, my parents, and God Almighty, without whom none of this would be possible.
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6. References

Print References Botana, L.M. (2008). Seafood and Freshwater Toxins, 2nd ed. Florida: CRC Press Taylor & Francis Group LLC. Chiang, M.; Chou, C. (2009). Survival of Vibrio parahaemolyticus under environmental stresses as influenced by growth phase and pre-adaptation treatment. Food Microbiology 26(4): 391-395. Cruickshank, R.; Daguid, J.P.; Marmion, B.P.; Swain, R.H.A. (1973). Medical Microbiology, 7th ed. New York: Longman Group Ltd. Feliciano, L.; Lee, J.; Lopes, J.A.; Pascall, M.A. (2010). Efficacy of sanitized ice in reducing bacterial load on fish fillet and in the water collected from the melted ice. Journal of Food Science 75(4): M231-237. Gabriel, A.A. (2010). Intrinsic and extrinsic characteristics of foods affecting microbial growth. Unpublished Presentation, Department of Food Science and Nutrition, College of Home Economics, University of the Philippines, Diliman, Quezon City. Gutierrez, L.B.B. (2004). Guidelines for the assessment of microbiological quality of processed foods. Bureau Circular No. 01-A s. 2004. Republic of the Philippines Department of Health: Bureau of Food and Drugs. Jay, J.M.; Loessner, M.J.; Golden, D.A. (2005). Modern Food Microbiology, 7th ed. New York: Springer Science + Business Media, Inc.
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Mizunoe, Y.; Wai, S.N.; Ishikawa, T.; Takade, A.; Yoshida, S. (2000). Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation. FEMS Microbiology Letters 186(1): 115-120. Ray, B.; Bhunia, A. (2008). Fundamental Food Microbiology, 4th ed. Florida: CRC Press Taylor & Francis Group LLC. Saxena, M.P.; Kulshrestha, S.B. (1985). Effect of physicochemical factors on the survival of Vibrio parahaemoloyticus on fish. Aquaculture 47(4): 369-372. Sakazaki, R. (2003). Vibrio parahaemolyticus. Tokyo: Japan Institute of Biological Sciences. Su, Y.; Liu, C. (2007). Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiology 24(6): 549-558. Trazo, E.S. (2001). Tuna belly quality changes during shipment. Unpublished Undergraduate BS Food Technology Thesis, College of Home Economics, University of the Philippines, Diliman, Quezon City. Willey, J.M.; Sherwood, L.M.; Woolverton, C.J. (2008). Prescott, Harley, and Kleins Microbiology, 7th ed. New York: McGraw-Hill Higher Education. Wong, H.; Wang, P.; Chen, S.; Chiu, S. (2004). Resuscitation of viable but non-culturable Vibrio parahaemolyticus in a minimum salt medium. FEMS Microbiology Letters 233(2): 269275.

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Web References Curtis, L. (2006). Vibrio parahaemolyticus. Food Safety Watch. Available from

http://www.foodsafetywatch.com/public/984.cfm. Accessed 06 March 2011. Food and Environmental Hygiene Department. (2001). Food Safety Plan Sashimi/Sushi Training Course. Public Health Laboratory, Kowloon. Available from

http://docs.google.com/viewer?a=v&q=cache:KDNThfYWLfQJ:www.cfs.gov.hk/english/progra mme/programme_haccp/files/sushi.ppt+sushi+vibrio&hl=en&gl=ph&pid=bl&srcid=ADGEESi6 Owyb1F3s1pvuxuR90uZaeslk_RyWLCOJbb8xOdRKZuqvlAI0Dvdu6CuzYsSlswB3Bc2rMYYyEhh4FOQbdQp_BKg7n26rwddYahp9oXqMs4CBxqS9SK9h8QIepcoMtI GuNfn&sig=AHIEtbRIHGvbMqjnttPzt9iiAwUssEACUg. Accessed 06 March 2011. NSW Food Authority. (2007). Food safety guidelines for the preparation and display of sushi. Available from http://www.foodauthority.nsw.gov.au/_Documents/industry_pdf/Sushi-

Guidelines-Eng.pdf. Accessed 05 March 2011. NSW Food Authority. (2009). Microbial quality of sushi 2009. Available from http://www.foodauthority.nsw.gov.au/_Documents/science/sushi_survey_2009_report.pdf. Accessed 05 March 2011. The Sushi FAQ. (2000). Sushi Items Maguro (Tuna). Available from

http://www.sushifaq.com/sushi-items/sushi-items-tuna-maguro.htm. Accessed 06 March 2011. Todar, K. (2008). Todars Online Textbook of Bacteriology. Available from

http://www.textbookofbacteriology.net/V.vulnificus.html. Accessed 06 March 2011.

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Wallner, S.; Schroeder, M.; Kendall, P. (2006). Sushi: minimizing the food safety risk. Colorado State University: SafeFood News 10(3). Available from

http://www.ext.colostate.edu/safefood/newsltr/v10n3s01.html. Accessed 04 March 2011.

7. Appendices 7.1. Tables

Test

Vibrio

Aeromonas

Plesiomonas

Campylobacter

Oxidase + + + + Catalase + + + V Lysine decarboxylase + + Glucose (acid) + + + Mannose (acid) + + Indole + + + Growth in: 0% NaCl V + + + 6% NaCl V Motility + + + + Gelatinase + + Aerobic growth + + + Table 1. Representative biochemical reactions of Vibrio, Aeromonas, Plesiomonas, and Campylobacter. (Cruickshank et al, 1973)

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Test Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Acid from: Lactose Sucrose Arabinose Voges-Proskauer Growth in: 0% NaCl 3% NaCl 6% NaCl 10% NaCl

V. cholerae + + (+) + V + + V -

V. parahaemolyticus + + V + + -

V. alginolyticus + + + + + + +

V. anguillarium + + V + + V -

V. metschnikovi + V V + + + + -

Group F Vibrio + + + V + + -

V. vulnificus + V + + -

Table 2. Representative biochemical reactions of Vibrio species (Cruickshank et al, 1973)

Day 1 5

Date 4-Feb 8-Feb

10-1 A 0 2

10-1 B 1 3

10-2 A 1 3

10-2 B 4 5

10-3 A 0 1

10-3 B 0 0

10-4 A 0 0

Table 3. Recorded CFU of Vibrio counted.

7.2. Figures

Figure 1. Exterior of Citra Mina Seafood Market.

Figure 2. Freezers housing the fish products.

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