Bacterial diversity and potential risk factors associated with Salmonella contamination of seafood products sold in retail markets in Bangkok, Thailand

Introduction

The global fisheries and aquaculture both inland and marine reached 171 million tonnes in 2016 (Food and Agriculture Organization of the United Nations, 2018) and consumption of fish, and fishery products per capita double from 10 kg in 1960 to greater than 20 kg in 2016 (Food and Agriculture Organization of the United Nations, 2018). In Southeast Asia, consumption of fish and fishery products varies from 6 to 64 kg per capita per year (Food and Agriculture Organization of the United Nations, 2015). In Thailand, consumption of fish and fishery products is about 31 kg per capita per year, which accounts for 12% of total protein consumption per person (Food and Agriculture Organization of the United Nations, 2015). Thailand is one of the top ten exporters of fish and fishery products, which accounted for 4% of global exports in 2016 (Food and Agriculture Organization of the United Nations, 2018).

Due to the rapid growth of global consumption of fish and fishery products, seafood safety is of high public health concern. Foodborne diseases afflict a third of the world population each year (World Health Organization, 2004), but data about the number of illnesses from seafood-borne outbreaks is limited for many parts of the world. Most examinations of seafood outbreaks have been done in the United States, where approximately 9.4 million illnesses, almost 56,000 hospitalizations, and 1,351 deaths, were associated with foodborne contamination per year (Scallan et al., 2011). Almost half (45%) of foodborne outbreaks reported in the U.S. are from bacteria, and fish are frequently implicated (Gould et al., 2013). In Europe, 5,175 foodborne outbreaks were reported in 2019. Salmonella spp. cause most of these outbreaks. There were 87,923 and 7,775 confirmed cases of salmonellosis and infections from Shiga-toxin-producing Escherichia coli, respectively (European Food Safety Authority; European Centre for Disease Prevention & Control, 2021).

Pollution, animal density, and global trading contribute to bacterial contamination of seafood products (Papadopoulou et al., 2007). The most common pathogens associated with seafood-borne diseases are Vibrio, Salmonella, Shigella, and Clostridium botulinum (Iwamoto et al., 2010). Seafood-borne outbreaks caused by V. parahaemolyticus, V. cholera serogroup O139, V. vulnificus, Salmonella serotype Weltevreden, and E. coli have been reported (Bonnin-Jusserand et al., 2019; Heinitz et al., 2000; Martinez-Urtaza et al., 2016; Raymond & Ramachandran, 2019).

In Thailand, V. parahaemolyticus and Salmonella spp. are the leading causes of foodborne diarrhea. Even though Thailand is one of the major exporters of seafood products, monitoring of bacterial pathogens of these exports is limited. Therefore, the objectives of this study were: (1) examine the prevalence of bacterial indicators, V. parahaemolyticus, V. cholerae, V. vulnificus, V. alginolyticus, and Salmonella isolated from Pacific white shrimp, oysters, blood cockle, and Asian seabass in fresh markets in Bangkok, Thailand; (2) identify serotypes of Salmonella among various seafood samples; and (3) determine risk factors for Salmonella contamination and a cutoff value for E. coli concentration that predicts the presence of Salmonella in fresh seafood.

Materials and Methods

Results

Occurrence of Vibrio and Salmonella in seafood samples

The overall prevalence for the various bacterial pathogens observed in all 335 seafood samples was 59% for V. parahaemolyticus, 18% for V. vulnificus, 19% for V. alginolyticus, 49% for V. cholerae and 36% for Salmonella. The highest prevalence of V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. cholerae, and Salmonella were observed in blood cockle (78%), Pacific white shrimp (33%), oyster (29%), Asian seabass (76%), and Pacific white shrimp (40%), respectively (Fig. 1). The lowest prevalence (<10%) of V. vulnificus was observed in blood cockle and for V. alginolyticus in Asian seabass. Moreover, shrimp were most likely to have any of the four pathogens, followed by oysters (Fig. 1). Blood cockles exhibited very high contamination of V. parahaemolyticus, while Asian seabass tended to harbor V. cholerae (Fig. 1). Based on Chi-square tests, the was a significant association between different types of samples and the occurrence of V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. cholerae, and Salmonella contamination (P < 0.0001).

Pacific white shrimp exhibited a high prevalence of V. parahaemolyticus (59%), V. cholerae (53%) and Salmonella (47%), whereas oysters were mainly contaminated with V. parahaemolyticus (45%) and Salmonella (38%). In blood cockles, they exhibited a low prevalence of V. cholerae, V. vulnificus, and V. alginolyticus. Asian seabass exhibited a high prevalence of V. cholerae (91%) and Salmonella (46%).

Matopeni (31%), Corvallis (5%), Give (5%), and Rissen (5%) were the most common serotypes of Salmonella isolated from seafood products (Table 3). Matopeni was the predominant serotype (52/56) observed from Asian seabass samples (n = 56 isolates), whereas Itami and Leith were common serovars isolated from the shrimp samples (n = 47 isolates). For oysters (n = 43 isolates) and blood cockles (n = 24 isolates), the major serotypes were Give (19%) and Rissen (33%), respectively.

Table 3:

Distribution of Salmonella serovars in seafood products sold in Bangkok, Thailand (n = 170 isolates).

Serotype	Number of positive (%)
	Shrimp	Oyster	Blood cockle	Asian seabass	Total
Agona	–	–	4 (2.35)	–	4 (2.35)
Aminatu	2 (1.18)	–	–	–	2 (1.18)
Australia	1 (0.59)	–	–	–	1 (0.59)
Bardo	–	4 (2.35)	–	–	4 (2.35)
Bonames	3 (1.76)	–	–	–	3 (1.76)
Breukelen II	–	4 (2.35)	–	–	4 (2.35)
Corvallis	–	4 (2.35)	4 (2.35)	–	8 (4.71)
Dabou	–	4 (2.35)	–	–	4 (2.35)
Dresden	1 (0.59)	–	–	–	1 (0.59)
Enteritidis	3 (1.76)	–	–	–	3 (1.76)
Farmingdale	–	4 (2.35)	–	–	4 (2.35)
Give	–	8 (4.71)	–	–	8 (4.71)
Glidji	–	2 (1.18)	–	–	2 (1.18)
Hisingen	2 (1.18)	–	–	–	2 (1.18)
Itami	5 (2.94)	–	–	–	5 (2.94)
Kentucky	3 (1.76)	–	–	–	3 (1.76)
Konongo	1 (0.59)	–	–	–	1 (0.59)
Lansing	1 (0.59)	–	–	–	1 (0.59)
Leith	4 (2.35)	–	–	–	4 (2.35)
Lexington	–	4 (2.35)	–	–	4 (2.35)
Lezennes	–	–	4 (2.35)	–	4 (2.35)
Linguere	2 (1.18)	–	–	–	2 (1.18)
Matopeni	–	–	–	52 (30.59)	52 (30.59)
Oslo	3 (1.76)	–	––	–	3 (1.76)
Paratyphi B2	–	–	–	4 (2.35)	4 (2.35)
Rissen	–	–	8 (4.71)	–	8 (4.71)
Ruiru	–	4 (2.35)	–	–	4 (2.35)
Stanley	–	–	4 (2.35)	–	4 (2.35)
Stuttgart	2 (1.18)	–	–	–	2 (1.18)
Soerenga	–	1 (0.59)	–	–	1 (0.59)
Tounouma	1 (0.59)	–	–	–	1 (0.59)
Typhimurium	1 (0.59)	–	–	–	1 (0.59)
Victoria	3 (1.76)	–	–	–	3 (1.76)
Windermere	3 (1.76)	–	–	–	3 (1.76)
Weltevreden	–	4 (2.35)	–	–	4 (2.35)
Wohlen	2 (1.18)	–	–	–	2 (1.18)
Yeerongpilly	1 (0.59)	–	–	–	1 (0.59)
II/IIIa/IV	3 (1.76)	–	–	–	3 (1.76)
Total	47 (27.65)	43 (25.29)	24 (14.12)	56 (32.94)	170 (100.00)

DOI: 10.7717/peerj.12694/table-3

ROC and area under the ROC curve

The area under the ROC curve (AUC) at 64% with standard error = 0.30 (CI [58–70%]) (Fig. 2). The ROC AUC was statistically significance (P < 0.0001) compared to the null value of AUC = 0.5. The presence of Salmonella in seafood products was predicted by a concentration of 1.3 × 10⁴ E. coli MPN/g, with a sensitivity of 84% and specificity of 61%.

Discussion

According to the Ministry of Public Health for Thailand, the concentration of E. coli should not exceed 10 MPN/g of fresh or frozen seafood and less than 3 MPN/g of seafood consumed raw; in addition, all products must not contain detectable Salmonella, V. cholerae, V. parahaemolyticus in a 25 g sample (Bureau of Quality & Safety of Food, 2020). In this study, the concentration of E. coli averaged 2 × 10⁴ MPN/g for all the seafood samples. In fact, only 18% (n = 60/335) of all seafood samples had concentrations of E. coli <10 MPN/g and only 7% (n = 6/82) of oyster samples (often eaten raw) had <3 MPN/g. Furthermore, the prevalence of Salmonella (36%), V. cholerae (49%), V. parahaemolyticus (59%) indicated widespread bacterial contamination of these seafood products, which also violates food safety standards. Therefore, implementation of basic sanitation and evaluation of microbiological contamination of seafood products sold in Bangkok are needed.

Salmonella is an important pathogen that is responsible for seafood-borne illness worldwide (Barrett et al., 2017; European Food Safety Authority, 2014). However, Salmonella is not a normal flora in finfish and shellfish products. The major sources of Salmonella contamination in seafood include aquatic and aquaculture systems, seafood processing facilities, hygiene practices during transport, storage, and handling (Amagliani, Brandi & Schiavano, 2012; Fernandes et al., 2018). In this study, the prevalence of Salmonella ranged from 14% to 47%. This prevalence was similar to the prevalence of 25% Salmonella contamination of shrimp cultured in Vietnam (Phan et al., 2005), but substantially less than the 90% to 100% prevalence of Salmonella contamination in fish (93%) and shrimp (100%) collected from a market in Indonesia (Pramono et al., 2019).

Type of seafood, sampling retail location, use of ice during retail display, regional source of seafood, and presence of E. coli and Vibrio were all significantly associated with the presence of Salmonella (Table 5). These risk factors should be interpreted with caution given the observational nature of this study and the possibility of undetected confounding in the statistical analysis. The presence of E. coli in a seafood sample was associated with a 4-fold increase in the odds of Salmonella contamination (OR = 4.0, P < 0.0001); similarly, the presence of V. alginolyticus in a seafood sample was associated with a 1.4-fold increase in the odds of Salmonella contamination (OR = 1.4, P < 0.04).

Seafood displayed on ice during retail had almost twice the odds of Salmonella contamination (OR = 1.7, P < 0.0001) than seafood not displayed on ice. This may seem counterintuitive, but ice used to chill seafood can be contaminated with pathogenic microorganisms (Falcão, Falcão & Gomes, 2004). Ice can be a vehicle for various pathogenic organisms, including diarrheagenic E. coli, Aeromonas, S. enteritidis and fecal coliforms (Falcão et al., 2002; Falcão, Falcão & Gomes, 2004; Kirov, 1993). In this study, most of the ice used to store seafood was at risk of rapidly melting due to high ambient temperatures in open air conditions. Melting ice can spread bacteria from one seafood item to nearby retail items, readily contaminating other seafood left standing in contaminated melt water.

In addition, the physical placement of seafood for display in retail markets can spread bacterial contamination between seafood items if seafood handlers do not practice proper sanitation during handling (i.e., bare hands touching multiple seafood items; not replacing latex or plastic gloves at high enough frequency during retail display placement of seafood items). Therefore, maintaining sanitary conditions during the production, storage, and use of ice to prevent microbial contamination should be closely observed. Implementation of programs for food safety and also for prevention and control of diarrheal diseases have reduced mortality and morbidity rates of diarrheal diseases and strengthened food safety in Thailand (Food Control Division, Food and Drug Administration, Thailand, 2004).

Seafood sourced from Eastern Thailand had a 3.5-higher odds of Salmonella contamination than seafood from other regions (OR = 3.5, P < 0.0001). The coastal area of Eastern Thailand has concentrated areas of industrialization, agricultural development, and tourism-related urbanization, with major concerns of increased water pollution and resource depletion (Nitivattananon & Srinonil, 2019). Wastewater quality is a major concern for this area, especially in Chonburi and Rayong Provinces due to several industrial estates. Moreover, Chonburi, Chachoengsao, and Rayong Provinces have been designated for developing the Eastern Economic Corridor (EEC), so reduction of waste and wastewater is of increasing concern.

In this study, the diversity of Salmonella serovars varied between the different seafood products. Pacific white shrimp had the greatest diversity with 21 different serovars with prevalence per serovar ranging from 1–3%. Eleven serovars from isolated from oysters, with prevalence per serovar ranging from 1–5% similar to Pacific white shrimp. In contrast, only 5 serovars were isolated from blood cockles, with a similar range of prevalence per serovar of 2–5%. Least diverse were isolates from Asian seabass. Only two serovars were recovered, with 52 of 56 of these Salmonella isolates being Matopeni and the remainder being Paratyphi B. Serotype Matopeni has been reported in aquatic pet shops (Gaulin, Vincent & Ismail, 2005) and in food supplements from Germany (European Commission, 2018). The infection of S. Matopeni has been reported in Malaysian children (Lee et al., 2003). Salmonella Paratyphi B in raw tuna sushi imported from Indonesia in 2015 caused 65 foodborne cases in the U.S. (Centers for Disease Control & Prevention, 2018). S. Typhimurium, S. Enteritidis, S. Typhi, and S. Paratyphi B were also detected in fresh fish in Iran (Rahimi, Shakerian & Falavarjani, 2013). S. Paratyphi B can be classified as d-tartrate fermenting (dT+) and d-tartrate non-fermenting (dT-) strains. The dT+ strain is less virulent and commonly reported with gastroenteritis, while the dT-strain is associated with paratyphoid fever. The dT+ strain is associated with a significant emerging disease worldwide and of public health concern (Denny et al., 2007; Hassan et al., 2018). Hence, classification of S. Paratyphi B biotype should be further investigated.

The common serovars in Pacific white shrimp were Itami (11%, n = 5/47) and Leith (9%, n = 4/47). Itami was first documented as a new serovar from a traveler to Thailand suffering from gastroenteritis (Sakazaki et al., 1981). Itami has also been reported from infected humans in Taiwan (Kuo et al., 2014). In contrast to serovars isolated during this study, serovars S. Weltevreden, S. Tennessee, and S. Dessau were isolated from shrimp from the Mekong Delta, Vietnam (Phan et al., 2005). The most common S. enterica serovar isolated from oysters was Give (19%, n = 8/43 isolates), which is different from oysters in the U.S. where Newport was the most common serotype (Brands et al., 2005). A previous study in Western Thailand found that the most common serovar in cultured oysters (C. lugubris and C. belcheri) from Phang Nga Province was Paratyphi B (Jeamsripong, Chuanchuen & Atwill, 2018). This suggests that the distribution of Salmonella serovars within Thailand depends on geographical location and type of seafood. Serovar Give is an enteric serotype usually isolated from swine and ruminants, but rarely found in humans (Higgins et al., 1997). Runoff from livestock feedlots near oyster farms could explain this contamination. S. enterica Give has been frequently reported in European national laboratories (Jansen et al., 2005). The higher virulence of the Give serovar compared to other non-typhoidal Salmonella may explain the higher hospital rate associated with human Give infections (Girardin et al., 2006).

Even though typhoid and paratyphoid salmonellosis are endemic diseases in Thailand, typhoid fever rates declined, and paratyphoid stabilized from 2003 to 2014 in this nation (Techasaensiri et al., 2018). In Thailand, S. enterica Weltevreden is commonly reported in human, frozen seafood, frozen ducks, and polluted water (Bangtrakulnonth et al., 2004). S. Weltevreden, S. Stanley, S. Anatum, and S. Rissen are frequently reported in human from northern and central Thailand (Prasertsee et al., 2019; Sirichote et al., 2010). Therefore, surveillance and monitoring of oysters due to this ~20% prevalence of Salmonella contamination, and fully cooking oysters prior to consumption are both needed to reduce the risk of food-borne Salmonella infection from Thai-cultured oysters.

In this study, the most common Salmonella serovar found in blood cockles was Rissen (33%, n = 8/24 isolates), similar to a study in India (Kumar, Surendran & Thampuran, 2009), but it should be noted that none of the five different serovars isolated from cockles had a prevalence above 5%. Seafood such as cockles can acquire Salmonella from contaminated water or other environmental matrices during aquaculture, processing, shipping, and retail display. Good hygiene and basic sanitation together with proper seafood handling and storage should be performed throughout the food chain (farm to fork) to reduce the risk of seafood-related Salmonella.

According to BQSF for Thailand seafood for human consumption should have no detectable V. parahaemolyticus and V. cholerae in 25 g of sample; however, 50–60% of samples contained these bacteria. This high prevalence is consistent with previous work demonstrating that between 2003 and 2015 the prevalence of V. parahaemolyticus was 64% in oysters, followed by clams (53%), fish (51%), and shrimp (48%) (Odeyemi, 2016). V. parahaemolyticus, V. cholerae, and V. vulnificus are important seafood-borne pathogens that cause gastroenteritis in humans. V. alginolyticus can cause ear infection and intestinal disease in humans. In this study, the main source of V. parahaemolyticus was blood cockles (OR = 9.1, P < 0.05), while V. cholerae was commonly found in Asian seabass (OR = 4.0, P > 0.05). V. parahaemolyticus and V. vulnificus have been reported in bivalves in many countries such as Thailand, China, and Korea (Changchai & Saunjit, 2014; Jiang et al., 2019; Ryu et al., 2019). In this study, shrimp and oysters were predominantly contaminated with V. vulnificus and V. alginolyticus.

Clostridium perfringens, Staphylococcus aureus, V. parahaemolyticus, and Salmonella spp. are the leading causes of foodborne illnesses in Thailand (Bureau of Epidemiology, 2019). In Thailand, human salmonellosis caused 167 illnesses per 100,000 persons in 2019. Contaminated produce and water have been indicated as important sources of Salmonella infection (Bureau of Epidemiology, 2019). The trend of V. cholerae infection decreased from 2.51 to 0.02 cases per 100,000 persons during 2010–2019, and contaminated water and seafood, poor sanitation, and dense housing have been blamed as sources of contamination (Bureau of Epidemiology, 2019).

In this study, the determination of bacterial prevalence and abundance was made using culture-dependent methods. For Salmonella spp. and Vibrio spp. detection this approach has high accuracy and sensitivity compared with certain molecular techniques (Almeida et al., 2013; Eriksson & Aspan, 2007; Hara-Kudo et al., 2001; Mainar-Jaime et al., 2013; Yeung & Thorsen, 2016). However, these methods can fail to detect viable but nonculturable state (VBNC) strains. VBNC bacteria can preserve metabolic activity and generate virulent proteins (Alleron et al., 2013; Morishige, Fujimori & Amano, 2015). Hence, molecular techniques are recommended to determine bacterial contamination, but unfortunately such equipment, needed supplies and training required to implement these molecular techniques are not readily available to many food safety officers, even in the developed world. In the absence of molecular equipment, the use of an appropriate MPN assay is justified and provides an estimate of bacterial load, along with ease of interpretation of the results. In this study, the determination of bacterial species was made using bacterial phenotyping methods. This culture-dependent method was justified given our goal of developing a highly sensitive assay using readily available reagents.

E. coli concentrations appeared predicted Salmonella contamination of seafood. Based on the Youden index (Ruopp et al., 2008), a cutoff value for E. coli was 1.3 × 10⁴ MPN/g can be implemented for both monitoring seafood for Salmonella contamination and to establish threshold control measures at processing or during retail storage. This cutoff is much higher than the microbiological criteria set forth in the Commission Regulation (EC) No 2073/2005 (European Commission, 2005), and the BQSF, Thailand (Bureau of Quality & Safety of Food, 2020). This reflects high concentrations of E. coli in this sample collection. Given that the detection of Salmonella and Vibrio spp. is of similar expense and technical difficulty as quantifying E. coli concentrations in seafood matrices, it may be more expeditious and more accurate to focus seafood safety monitoring protocols on Salmonella and Vibrio spp. detection rather than rely on indicator bacteria like E. coli that invariably suffer from false-positive and false-negative signals.

Conclusions

Finfish and shellfish products sold in Bangkok are contaminated with diverse Salmonella serovars and species of Vibrio. Although the concentration of E. coli predicted Salmonella contamination for these seafood samples, the high cutoff value (1.3 × 10⁴ MPN/g) for maximal test accuracy will likely prevent this method from being adopted as a food hygiene surveillance tool. We recommend measuring Salmonella and Vibrio directly to assess the risk of food poisoning. Bacterial contamination varied by seafood commodity, with substantial differences between Asian seabass, oysters, blood cockle, and Pacific white shrimp. This may reflect different aquaculturing, harvesting, processing, and retail display practices.

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