FOODBORNE PATHOGENS AND DISEASE Volume 9, Number 2, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2011.0989
Prevalence of Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella spp. in Seafood Products Using Multiplex Polymerase Chain Reaction Mehdi Zarei,1 Siavash Maktabi,1 and Masoud Ghorbanpour 2
Although several etiological agents can be transmitted through seafood consumption, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella spp. are considered among the most important pathogens in terms of public health and disease. In this study, multiplex polymerase chain reaction (PCR), as a rapid and cost-effective method, was used to determine the prevalence of these pathogens in 245 samples of raw/fresh, frozen, and ready-to-eat (RTE) seafood products marketed in Iran. The prevalence of L. monocytogenes in raw/fresh fish and shrimp samples was 1.4%, whereas 2.9% of the raw/fresh fish and 7.1% of the shrimp samples were contaminated with V. parahaemolyticus. No contamination with L. monocytogenes and V. parahaemolyticus was found in frozen and RTE seafood products. The prevalence of S. aureus was found to be higher than other investigated pathogens. S. aureus was detected in 5% of the raw/fresh samples of fish and shrimp, 17.5% of the frozen, and 12.3% of the RTE samples. Further, our findings indicate that 2.9% of the fish samples, 4.3% of the shrimp samples, and 1.5% of the RTE samples were contaminated with Salmonella spp. Owing to the potential hazard of these pathogenic bacteria, multiplex PCR can provide a rapid and cost-effective method for the surveillance of these pathogens in seafood products.
Introduction
T
he microbiological safety of seafood is a significant concern of consumers, industries, and regulatory agencies all over the world. The rapid and accurate identification of bacterial pathogens is important, both for quality assurance and to trace bacterial pathogens within the food supply (Bhagwat, 2003; Germini et al., 2009). During the last few years, international standards have agreed on the use of polymerase chain reaction (PCR) for the detection of foodborne pathogens and legislations are implementing new types of analyses as the accepted official methods. For example, European regulation EC 2073/2005 allows for the use of alternative detection methods if based on certified analyses according to international standards (EU, 2005). Multiplex PCR is a relatively new method that can simultaneously amplify template mixture and decrease the detection cost, conquering the weakness of single PCR detecting only one target (Kim et al., 2007; Germini et al., 2009). In studies of seafoodborne pathogens, four major pathogens have emerged as being of significant importance in terms of human health and disease. These include Listeria mono-
cytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella spp. (Feldhusen, 2000). L. monocytogenes has been isolated from fish and seafood products all over the world. The contamination rate of seafood products with L. monocytogenes can vary from 0% to more than 50% (Ben Embarek, 1994; Jinneman et al., 1999). Contamination of seafood products with L. monocytogenes depends on many factors such as cleaning and processing procedures, working habits, and the existence of surfacepersistent L. monocytogenes types in processing facilities. Moreover, raw materials contaminated with Listeria may also be a reason for the contamination of the final product (Rørvik et al., 1997; Miettinen and Wirtanen, 2005). V. parahaemolyticus is a human pathogen that occurs naturally in the marine environments and is frequently isolated from a variety of seafood including fish, shrimp, crab, lobster, scallop, and oyster (Austin, 2010). This pathogen is a common cause of foodborne illnesses in many Asian countries, including Taiwan, China, and Japan, and is recognized as the leading cause of human gastroenteritis associated with seafood consumption in the United States ( Jaksic et al., 2002; Su and Liu, 2007).
Departments of 1Food Hygiene and 2Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
108
FOODBORNE PATHOGENS IN SEAFOOD PRODUCTS S. aureus is one of the most prevalent causes of gastroenteritis worldwide, which is caused by the ingestion of food that contains preformed toxins. Humans are common carriers of S. aureus in the nose, throat, and skin infections. Therefore, the organism can be easily transferred to seafood during handling ( Jablonski and Bohach, 2001). The contamination of seafood could be the result of a combination of improper and unsanitary handling, improper storage, and cross contamination (Huang et al., 2001; Jablonski and Bohach, 2001). Salmonella can occur both in water, especially of contaminated coastal regions or ponds, and in the fresh fish from these areas. The incidence of salmonellosis caused by the consumption of contaminated seafood, such as raw shellfish, is a primary concern of public health agencies in many countries (Feldhusen, 2000). The present study was, therefore, carried out to determine the prevalence of L. monocytogenes, V. parahaemolyticus, S. aureus, and Salmonella spp. in raw/fresh, frozen, and ready-toeat (RTE) seafood products marketed in Iran using multiplex PCR technique. Materials and Methods
109
FIG. 1. Multiplex polymerase chain reaction applied to single and multiple pathogen detection. M: 50 bp DNA ladder; 1: multiple pathogen; 2: Listeria monocytogenes ATCC 7644 (454 bp); 3: Vibrio parahaemolyticus ATCC 35118 (375 bp); 4: Salmonella Typhimurium ATCC 35987 (678 bp); 5: Staphylococcus aureus ATCC 12599 (264 bp). visualized following gel electrophoresis on 2% agarose gels using ethidium bromide staining.
Sampling, preenrichment, and enrichment procedure
Limits of detection of multiplex PCR reactions
During a 10-month period, a total of 245 samples were taken from various seafood products such as raw/fresh fish and shrimp, frozen fish fillets, frozen peeled and undeveined (PUD) shrimp, fish nuggets, breaded shrimp, shrimp burgers, and Kentucky shrimp. All samples were placed in cold portable insulated boxes, transported to the laboratory in ice, and processed within 1 h of collection. Twenty-five grams each of the seafood samples were aseptically taken and blended for 60 sec in a stomacher (Bagmixer 400W; Interscience) with 225 mL of Universal preenrichment broth (Atlas, 2006) and incubated at 35C for 6 h. For enrichment of each pathogen, 10 mL of preenrichment broth was added to an equal volume of double-strength enrichment broths; Listeria enrichment broth, Staphylococcal enrichment broth, alkaline peptone water, and Rappaport-Vassiliadis enrichment broth for L. monocytogenes, S. aureus, V. parahaemolyticus, and Salmonella, respectively (Atlas, 2006). Enrichment broths were incubated for 18–20 h at 35C, except for Salmonella, which incubated at 42C.
The sensitivity of multiplex PCRs was assessed on artificially contaminated seafood samples. From the three main categories of seafood products used in this study; raw/fresh, frozen PUD, and breaded shrimp were selected. Twenty-five grams of samples was homogenized in 225 mL of UPB. Subsequently, the homogenates were spiked simultaneously with 1 mL of serial decimal dilutions of the four pathogens. The bacterial concentration of the contaminating cultures was determined by plating on tryptic soy broth (TSA). For each bacterium and product used, three levels of contamination were tested (Table 1). A nonspiked seafood sample was used as negative control. Seafood homogenates were incubated at 35C for 6 h and then enriched as described earlier. DNA extraction and multiplex PCR were performed in the same way as the seafood samples.
DNA extraction and multiplex PCR DNA extraction was performed through boiling. One milliliter of sample of the enrichment broths was centrifuged at 14,000 rpm for 3 min. Bacterial pellets were resuspended in 1 mL of sterile saline solution (0.85% NaCl). Following centrifugation in the same way, the supernatants were replaced with 50 lL of sterile distilled water and incubated at 100C for 10 min to release the bacterial DNA. After boiling, the clear supernatants obtained after 5 min centrifugation at 14,000 rpm were collected and stored at - 20C until use. DNA extracted from each specific enrichment broth, assuming equal amounts of DNA, was mixed together and used in multiplex PCRs. The oligonucleotide primers used in this study and multiplex PCR were carried out according to Kim et al. (2007) (Fig. 1). PCRs were performed in a thermal cycler (Eppendorf, Mastercycler Gradient) and PCR products were
Results and Discussion In this study, multiplex PCR-based method was used for detection of organisms after enrichment of bacterial cultures. The sensitivity and specificity of this multiplex PCR assay were explained by Kim et al. (2007). They also explained the validity of the multiplex PCR assay for samples of ham, milk, and water. In the present study, sensitivity of multiplex PCR was assessed on artificially contaminated seafood samples (Table 1). As shown, after enrichment, for L. monocytogenes, V. parahaemolyticus, and Salmonella spp., there were no differences in the detection limit between raw/fresh, frozen PUD, and breaded shrimp samples, whereas for S. aureus in raw/ fresh shrimp it was higher than in frozen PUD and breaded samples. It is possible that DNA from normal microflora of raw/fresh samples may decrease the sensitivity of multiplex PCR assay for detecting S. aureus and cause underestimation of prevalence. Overall, the sensitivity of this method together with its ability to give rapid and cost-effective results make it a suitable method to be implemented in control laboratories for the detection of these pathogens in seafood samples.
110
ZAREI ET AL. Table 1. Limits of Detection of Multiplex Polymerase Chain Reaction After Enrichment Fishery product
Level of contamination (CFU/25 g)
PCR resultsa
Raw/fresh shrimp Frozen PUD shrimp Breaded shrimp Raw/fresh shrimp Frozen PUD shrimp Breaded shrimp Raw/fresh shrimp Frozen PUD shrimp Breaded shrimp Raw/fresh shrimp Frozen PUD shrimp Breaded shrimp
0/33/79/112 0/33/79/112 0/33/79/112 0/21/56/97 0/21/56/97 0/21/56/97 0/42/91/172 0/42/91/172 0/42/91/172 0/18/73/88 0/18/73/88 0/18/73/88
-/+/+/+ -/+/+/+ -/+/+/+ -/+/+/+ -/+/+/+ -/+/+/+ -/-/-/+ - /( + )/ + / + -/+/+/+ - /( + )/ + / + -/+/+/+ -/+/+/+
Pathogen used Listeria monocytogenes Vibrio parahaemolyticus Staphylococcus aureus Salmonella spp.
a
Triplicate experiments. + : Presence of PCR product; - : Absence of PCR product; ( + ): weak PCR product. PUD, peeled and undeveined; PCR, polymerase chain reaction.
In the present study, L. monocytogenes was detected in 1.4% of the raw/fresh fish and shrimp samples. However, contamination with this pathogen was not observed in frozen and RTE seafood samples at the retail level (Table 2). Only a few reports on the prevalence of L. monocytogenes in seafood products in Iran have been previously published. According to Basti et al. (2006), the prevalence of L. monocytogenes in fresh fish samples was 2.6%. However, L. monocytogenes was not found in 85 samples of fresh and frozen fish and shrimp analyzed by Jalali and Abedi (2008). L. monocytogenes contamination of seafood varies with product category. Jorgensen and Huss (1998) demonstrated that the highest prevalence of L. monocytogenes is in cold-smoked fish (34%–60%), whereas the lowest is in heat-treated and cured seafood (4%–12%). In general, L. monocytogenes is not usually found on fish captured from open waters. However, contamination may take place long before the fish raw material reaches retail trade or processing factories. Potential sources of L. monocytogenes on fishing vessels include contamination from water and ice, soiled surfaces, and boxes as well as from human and avian sources. As L. monocytogenes is commonly found in coastal waters and in surface waters of lakes, fish captured or cultivated in these waters may possibly carry this microorganism (FAO, 1999). Our findings indicate that 2.9% of the raw/fresh fish and 7.1% of the shrimp samples were contaminated with V.
parahaemolyticus. However, no contamination was found in 105 samples of frozen and RTE seafood products (Table 2). This pathogen has been isolated and detected from seafood, particularly shellfish or bivalve mollusks all over the world. In Iran, V. parahaemolyticus was detected in 9.3% of the shrimp samples analyzed by Rahimi et al. (2010). In Croatia, during the summer of year 2000, 117 samples of sea fish, shrimp, and bivalve mollusks were sampled and V. parahaemolyticus was found in 9.4% of the total samples ( Jaksic et al., 2002). A very high level of contamination with V. parahaemolyticus has been reported in cockle (Anadara granosa) in Malaysia. According to Bilung et al. (2005), of the 100 cockle samples tested, 62 were positive for the presence of V. parahaemolyticus. Considering the widespread distribution in marine environments, short generation times, and low infectious doses of pathogenic strains (Kaysner and De Paola, 2000), intensive and continuous monitoring of potentially pathogenic V. parahaemolyticus is strongly recommended to evaluate human health risk arising from seafood consumption. The prevalence of S. aureus was found to be higher than other investigated pathogens. S. aureus was detected in 5% of the raw/fresh samples of fish and shrimp, whereas the prevalence of this bacterium was 15% in frozen and RTE samples (Table 2). Some reports have presented the incidence of S. aureus in seafood: 7% in shrimp, 4% in frozen cuttlefish, 4% in fish (Rodma et al., 1991), 68% in frozen fishery products
Table 2. The Prevalence of Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella spp. in Seafood Samples Tested Samples with (%) Product category Raw/fresh Frozen Ready-to-eat
Fishery products
Samples tested
L. monocytogenes
V. parahaemolyticus
Salt water fish Shrimp Fish fillet PUD shrimp Fish nugget Shrimp burger Breaded shrimp Kentucky shrimp
70 70 20 20 10 10 30 15
1 (1.4) 1 (1.4) 0 0 0 0 0 0
2 (2.9) 5 (7.1) 0 0 0 0 0 0
S. aureus 3 4 3 4 1 0 5 2
(4.3) (5.7) (15) (20) (10) (16.7) (13.3)
Salmonella 2 (2.9) 3 (4.3) 0 0 0 1 (10) 0 0
FOODBORNE PATHOGENS IN SEAFOOD PRODUCTS (Sanjeev et al., 1986), and 8% in fish and shellfish (Ayulo et al., 1994). It is well documented that freshly caught seafood are free from S. aureus, and contamination takes place upon handling (Shewan, 1962; Bryan, 1980). Several conditions, such as delay in processing, inadequate refrigeration, poor personal hygiene, and postprocess contamination, are associated with staphylococcal contamination, especially in frozen and RTE samples. Therefore, it is recommended to use sanitary gloves for handling RTE seafood to reduce the problem of S. aureus contamination. As shown in Table 2, of the 245 samples investigated in this study, Salmonella was found in 6 samples. About 2.9% of the fish samples, 4.3% of the shrimp samples, and 0.95% of the frozen and RTE samples were contaminated with Salmonella spp. The overall prevalence of Salmonella spp. in seafood samples tested was lower than that reported elsewhere. The occurrence of Salmonella in brackish water ponds was monitored over a 2-year period in one of the major shrimp-exporting countries in Southeast Asia. Salmonella were present in 16% of shrimp and 22.1% of mud/water samples (Reilly and Twiddy, 1992). In addition, Salmonella species were present in 21% of eel culture ponds in Japan (Saheki et al., 1989). The presence of Salmonella in water depends on some factors, for example, the pollution from a nearby poultry farm or animal manures. Considering the disease surveillance reports from public health authorities in some countries, human salmonellosis associated with the consumption of seafood occurs very rarely compared with those associated with other foods, for example, poultry products (Reilly, 1998). In conclusion, although classical microbiological methods offer reliable and standardized procedures for the detection of foodborne pathogens (e.g., ISO standards), they often result in time-consuming analyses not always compatible with the need for rapid results. This study shows that multiplex PCR is a very useful tool for the detection of seafoodborne pathogens, namely, L. monocytogenes, V. parahaemolyticus, S. aureus, and Salmonella spp. The method represents a rapid and cost-effective method for the detection of these pathogens in seafood products. Moreover, the results of the present study can provide a significant contribution to both regulatory agencies and seafood industries. The surveillance of these pathogenic bacteria in harvested seafood and seafood products marketed is crucial to safeguarding public health. Acknowledgment This study was supported by a research grant provided by the Shahid Chamran University of Ahvaz. Disclosure Statement No competing financial interests exist. References Atlas RM. Handbook of Microbiological Media for the Examination of Food, 2nd edition. Boca Raton, FL: CRC Press, 2006. Austin B. Vibrios as causal agents of zoonoses. Vet Microbiol 2010;140:310–317. Ayulo AMR, Machado RA, and Scussel VM. Enterotoxigenic Escherichia coli and Staphylococcus aureus in fish and seafood from the Southern region of Brazil. Int J Food Microbiol 1994;24:171–178.
111 Basti AA, Misaghi A, Salehi TZ, and Kamkar A. Bacterial pathogens in fresh, smoked and salted Iranian fish. Food Cont 2006;17:183–188. Ben Embarek PK. Presence, detection and growth of Listeria nomocytogenes in seafoods: a review. Int J Food Microbiol 1994;23:17–34. Bhagwat AA. Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. Int J Food Microbiol 2003;84:217–224. Bilung LM, Radu S, Bahaman AR, Abdul Rahim R, Napis S, Ling MW, Tani GB, and Nishibuchi M. Detection of Vibrio parahaemolyticus in cockle (Anadara granosa) by PCR. FEMS Microbiol Lett 2005;252:85–88. Bryan FL. Foodborne diseases in the United States associated with meat and poultry. J Food Prot 1980;43:140–150. [EU] European Union. Commission Regulation (EC) No. 2073/ 2005 of 15 November 2005, Microbiological Criteria for Foodstuffs. European Union, 2005. [FAO] Food and Agriculture Organization. Fisheries Report No. 604. Expert Consultation on the Trade Impact of Listeria in Fish Products. Amherst, MA: FAO, 1999. Feldhusen F. The role of seafood in bacterial food-borne diseases. Microbes Infect 2000;2:1651–1660. Germini A, Masola A, Carnevali P, and Marchelli R. Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR. Food Cont 2009;20:733–738. Huang SL Weng YN, and Chiou RY. Survival of Staphylococcus aureus and Escherichia coli as affected by ethanol and NaCl. J Food Prot 2001;64:546–550. Jablonski LM and Bohach GA. Staphylococcus aureus. In: Food Microbiology: Fundamentals & Frontiers. Doyle MP, Beuchat L, and Montville T (eds.). Washington, DC: ASM Press, 2001, pp. 411–433. Jaksic S, Uhitil S, Petrak T, Bazulic D, and Karolyi LG. Occurrence of Vibrio spp. in sea fish, shrimp and bivalve mollusks harvested from Adriatic Sea. Food Cont 2002;13:491–493. Jalali M and Abedi D. Prevalence of Listeria species in food products in Isfahan, Iran. Int J Food Microbiol 2008;122:336–340. Jinneman KC, Wekell MM, and Eklund MW. Incidence and behavior of Listeria monocytogenes in fish and seafood products. In: Listeria, Listeriosis, and Food Safety. Ryser ET and Marth EH (eds.). New York: Marcel Dekker, 1999, pp. 601–630. Jorgensen LV and Huss HH. Prevalence and growth of Listeria monocytogenes in naturally contaminated seafood. Int J Food Microbiol 1998;42:127–131. Kaysner CA and De Paola A. Outbreaks of Vibrio parahaemolyticus gastroenteritis from raw oyster consumption: assessing the risk of consumption and genetic methods for detection of pathogenic strains. J Shellfish Res 2000;19: 657–660. Kim JS, Lee GG, Park JS, Jung YH, Kwak HS, Kim SB, Nam YS, and Kwon ST. A novel multiplex PCR assay for rapid and simultaneous detection of five pathogenic bacteria: Escherichia coli O157: H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus. J Food Prot 2007;70: 1656–1662. Miettinen H and Wirtanen G. Prevalence and location of Listeria monocytogenes in farmed rainbow trout. Int J Food Microbiol 2005;104:135–143. Rahimi E, Ameri M, Doosti A, and Gholampour AR. Occurrence of toxigenic Vibrio parahaemolyticus strains in shrimp in Iran. Foodborne Path Dis 2010;7:1107–1111.
112 Reilly PJA. Emerging food safety issues and the seafood sector, 26th Session of the Asia Fisheries Commission, September 24– 30, China, 1998. Reilly PJA and Twiddy DR. Salmonella and Vibrio cholerae in brackish water tropical prawns. Int J Food Microbiol 1992;16:293–301. Rodma P, Satjapala T, and Suwanvitaya P. Staphylococcus aureus occurrence in frozen foods. Food 1991;21:197–204. Rørvik LM, Skjerve E, Knudsen BR, and Yndestad M. Risk factors for contamination of smoked salmon with Listeria monocytogenes. Int J Food Microbiol 1997;37:215–219. Saheki K, Kobayashi S, and Kawanishi T. Salmonella contamination of eel culture ponds, Nippon Suisan Gakk 1989;55:675–679. Sanjeev S, Iyer KM, Rao CCP, and James AM. Occurrence of enterotoxigenic Staphylococci in frozen fishery products. Fishery Technol 1986;23:164–166.
ZAREI ET AL. Shewan JM. Food poisoning caused by fish and fishery products. In: Fish as Food. Borgstrom G (ed.). New York: Academic Press, 1962, pp. 443. Su YC and Liu C. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol 2007;24:549–558.
Address correspondence to: Mehdi Zarei, Ph.D. Department of Food Hygiene Faculty of Veterinary Medicine Shahid Chamran University of Ahvaz Ahvaz 61355–145 Iran E-mail:
[email protected]
