Risk Assessment Studies
Report No. 20

VIBRIO SPECIES IN SEAFOOD

August 2005
Food and Environmental Hygiene Department
The Government of the Hong Kong Special Administrative Region

¡@¡@Vibrio Species

¡@¡@¡@¡@V. cholerae
¡@¡@¡@¡@V. parahaemolyticus
¡@¡@¡@¡@V. vulnificus

¡@¡@Vibrio spp. and Seafood

¡@¡@¡@¡@Factors affecting growth and survival of vibrios in seafood

¡@¡@V. parahaemolyticus Food Poisoning
¡@¡@Cholera Cases

Discussion

¡@¡@Advice to Trade
¡@¡@Advice to Public

References

Table 1: V. parahaemolyticus Food Poisoning by Food Group (1999 to 2003)

The aims of this paper are to evaluate the local situation of Vibrio species (spp.) in seafood products and to make recommendations to reduce risk associated with the consumption of seafood.

2. Seafood is a nutritious food that constitutes one of the desirable components of a healthy diet. Nevertheless, there are health risks associated with the consumption of seafood. One of the major risks involves the consumption of raw or undercooked seafood that may be naturally contaminated by foodborne pathogens present in the marine environment. Such risk is further increased if the food is mishandled during processing where pathogens could multiply exponentially under favourable conditions.

3. In contrast to most other foodborne pathogens, Vibrio spp. have the aquatic habitat as their natural niche. As a result, vibrios are most commonly associated with seafood as natural contaminants. Foodborne infections with Vibrio spp. are common in Asia [1], including Hong Kong.

4. Vibrio spp. are Gram-negative, facultatively anaerobic motile curved rods with a single polar flagellum. Among the members of the genus, 12 species have so far been reported to be pathogenic to humans, where eight of these may be associated with foodborne infections of the gastrointestinal tract.[2] Most of these foodborne infections are caused by V. parahaemolyticus and V. cholerae, and to a lesser extent by V. vulnificus.[3] The following paragraphs highlighted the characteristics of these three vibrios.

V. cholerae

5. Among the vibrios, V. cholerae is of most concern because of its ability to cause cholera. V. cholerae can be divided into serogroups on the basis of the O antigen. Of the more than 200 V. cholerae serogroups that exist, only O1 and O139 are associated with the epidemiological features and clinical syndrome of cholera. However, organisms of V. cholerae serogroups other than O1 and O139 (non-O1 non-O139 serogroups) have been associated with sporadic cases of foodborne outbreaks of gastroenteritis, but have not spread in epidemic form.[4] The most important virulence factor associated with V. cholerae O1 and O139 serogroups is the cholera toxin. Non-O1 non-O139 serogroups are generally nontoxigenic.

6. Cholera remains a public health threat globally causing hundreds of thousand cases every year. However, this threat is much reduced in places with safe water supply and good standards of hygiene and sanitation. Cholera is an acute intestinal infection. Its incubation period ranges from a few hours to five days, usually two to three days. Although asymptomatic infection is more common, clinical illness may be exhibited. Symptoms include a sudden onset of profuse painless watery diarrhoea that can quickly lead to rapid dehydration, acidosis, circulatory collapse, hypoglycaemia in children, renal failure and death if treatment is not promptly given. Nausea and vomiting also occurs early in the course of illness. Cholera is transmitted through ingestion of food or water contaminated with the bacterium, especially via faeces or vomitus of infected persons, directly or indirectly. Human volunteer feeding studies utilising healthy individuals have demonstrated that the infective dose is approximately one million organisms. However, conditions which decrease acidity in the stomach such as antacid consumption markedly lowers the infective dose.[5]

7. Gastroenteritis caused by V. cholerae non-O1 non-O139 serogroups is milder than cholera. The incubation period ranges from 12 to 24 hours in outbreaks[4] and is characterised by diarrhoea and abdominal cramps. About 70% of infected individuals will have fever. Nausea and vomiting are reported in 21% and bloody diarrhoea in 25% of cases.[6] Cases of non-O1 non-O139 gastroenteritis are usually linked to consumption of raw or undercooked seafood, particularly shellfish. It is suspected that large numbers (more than one million) of the organism must be ingested to cause illness.[7]

8. V. cholerae is a mesophilic organism that grows in the temperature range of 10 to 43^oC, with optimum growth at 37^oC. The pH optimum for growth is 7.6 although it can grow in the pH range of 5.0 to 9.6. V. cholerae can grow in the salt range of 0.1 to 4.0% NaCl, while optimum is 0.5% NaCl.6

V. parahaemolyticus

9. V. parahaemolyticus was first identified as a foodborne pathogen in Japan in the 1950s. By the late 1960s and early 1970s, V. parahaemolyticus was recognised as a cause of diarrhoeal disease worldwide, although most common in Asia and the United States.[3] In Hong Kong, V. parahaemolyticus continued to be the top causative agent among all the reported food poisoning outbreaks in recent years.[8],[9],[10],[11],[12]

10. The illness caused by V. parahaemolyticus food poisoning is a gastroenteritis characterised by watery diarrhoea and abdominal cramps in most cases, with nausea, vomiting, fever and headache. The incubation period is usually between 12 and 24 hours[4] and the disease usually resolves in three days.[13] The infection is typically acquired through consumption of contaminated seafood. These could be raw or inadequately cooked, or that have been cross-contaminated by improper handling. Poor temperature control of storage favours bacterial proliferation. The total dose of greater than one million may cause disease. This dose may be markedly lowered by coincident consumption of antacids or presumably by food with buffering capacity.[14] One volunteer feeding study done in Japan estimated that 2 x 10⁵ to 3 x 10⁷ cells have to be ingested for disease.[15]

11. V. parahaemolyticus is a slightly halophilic bacterium. The optimum growth NaCl concentrations range from 2 to 4% and poor growth is exhibited in media below 0.5% NaCl. The bacterium is inactivated rapidly in distilled water and growth at levels of 10% NaCl is inhibited.[16] The organism grows at a temperature range between 5 and 43^oC, with optimum growth at 37^oC. The optimum pH range for growth is 7.8 to 8.6, although it can grow in the pH range of 4.8 to 11.[13]

V. vulnificus

12. V. vulnificus is an opportunistic pathogen that can cause wound infections and primary septicaemia. This bacterium has less often been described as a cause of gastroenteritis, and its role as a primary cause of gastrointestinal disease remains to be determined.[17]

13. Wound infections occur in connection with puncture wounds after handling of raw seafood or trauma and exposure to saline environments that harbour the organism.[18]

14. The primary septicaemic form is the major form of infection with V. vulnificus which involves a rapidly progressing septicemia with few gastrointestinal signs. The incubation period is from seven hours to several days. The most frequent symptoms are fever, chills, nausea and cardiovascular hypotension. This form of disease is predominantly associated with the consumption of raw bivalve shellfish containing the organism by individuals with underlying chronic disease, particularly liver disease.[18] In these individuals, the microorganism enters the blood stream, resulting in septic shock, rapidly followed by death in many cases (about 50%). Over 70% of infected individuals have distinctive bulbous skin lesions. For predisposed persons, septicemia can presumably occur with doses of less than 100 total organisms.[19]

15. V. vulnificus is very similar to V. parahaemolyticus in cultural characteristics and sensitivity to processing procedures. It differs principally in salt requirement and tolerance, growing in media containing between 0.1 and 5% NaCl. Same as V. parahaemolyticus, the organism grows optimally at 37^oC although it can grow at a temperature range between 8 and 43^oC. The pH range for growth of V. vulnificus is 5 to 10, with an optimum at 7.8.[20]

16. Vibrios are abundant in the aquatic environment. Most of them require 2 to 3% NaCl or a seawater base for optimal growth.[15] Vibrios are associated with live seafood as they form part of the indigenous microflora of the environment at the time of seafood capture or harvest. Healthy live fish is protected by its immune system and therefore bacteria cannot grow in its flesh. When the fish dies, the immune system no longer functions and the bacteria present are able to proliferate freely.[21] In addition, bacteria may be found on the skin, chitinous shell, gills as well as the intestinal tracts of fish or shellfish.[22] If subsequent handling is improper and that there is no or inadequate pathogen reduction step (e.g. cooking) afterwards, the level of bacteria in the final product may increase to such an extent that may present a health risk to consumers.

17. Molluscan bivalves are filter feeders and they tend to accumulate microorganisms in the surrounding waters which may also contain vibrios. They are usually grown and harvested in shallow, near-shore estuarine waters and are therefore likely to harbour high concentrations of pathogenic organisms including pathogenic vibrios. As they often are eaten raw or after a very mild heat treatment, they constitute a significant health risk to the consumers.[21]

18. Among the potentially pathogenic vibrios occurring naturally on fish and shellfish, V. parahaemolyticus is the most widespread. Endogenous marine species of V. cholerae can also be isolated from fish during cholera outbreaks. It has been suggested that vibrios are the most common bacterial causative agents in food poisoning resulting from the consumption of shellfish.[22]

19. Water temperature can greatly affect the vibrio levels in seafood. Vibrios can multiply rapidly between 20 and 40^oC. Growth at the optimum temperature (37^oC) can be very rapid and generation times of 9 to 10 minutes have been reported.[6] V. parahaemolyticus is primarily associated with coastal inshore waters rather than the open sea. It is rarely isolated from water with temperatures below 15^oC.[23],[13]

Factors affecting growth and survival of vibrios in seafood

i. Effect of temperature

20. Growth of pathogenic vibrios occurs optimally at around 37^oC although the maximum and minimum growth temperatures are 43^oC and 5^oC respectively.[23] All vibrios are sensitive to heat. In shellfish, heating to produce an internal temperature of at least 60^oC for several minutes appears sufficient to eliminate the pathogenic vibrios.[17] Chilling and refrigeration are critical control measures to prevent growth of these microorganisms.

ii. Effect of pH and other factors

21. Vibrios are acid sensitive and grow best at pH values slightly above neutrality, i.e. 7.5 to 8.5.[23] They are also sensitive to drying. While V. parahaemolyticus has an absolute Na⁺ ion requirement and shows optimal growth at about 2 to 4% NaCl, freshwater inactivates this organism.

22. Food poisoning and cholera are notifiable infectious diseases under the Quarantine and Prevention of Diseases Ordinance, Cap.141, Laws of Hong Kong. The following paragraphs summarised the figures concerning local food poisoning outbreaks due to V. parahaemolyticus and local cholera cases.

V. parahaemolyticus food poisoning

23. V. parahaemolyticus ranked first as the most common causative agent of food poisoning outbreaks in Hong Kong in recent years. According to the figures provided by the Department of Health (DH), 552 confirmed V. parahaemolyticus food poisoning outbreaks affecting 2725 persons were reported during 1999 to 2003. Table 1 showed the breakdown of these cases by food group.

Table 1: V. parahaemolyticus Food Poisoning by Food Group (1999 to 2003)

* Percentages may not add up to 100% due to rounding of figures.

24. Among all the food groups, seafood is the most frequently incriminated food which caused V. parahaemolyticus food poisoning outbreak. It accounted for 56.7% of the total number of confirmed cases. Table 2 summarised the various seafood items which caused V. parahaemolyticus food poisoning.

Table 2: V. parahaemolyticus Food Poisoning Outbreaks Due to Consumption of Seafood (1999 to 2003)

* Percentages may not add up to 100% due to rounding of figures

25. Of the 313 confirmed V. parahaemolyticus food poisoning outbreaks due to consumption of seafood during 1999 to 2003 (Table 1), inadequate cooking was the main contribution factor which accounts for 59.7% of the cases, and this was followed by contamination by raw food which accounts for 23.6% of the cases. These two factors contributed to more than 80% of all the cases. Other factors included other reasons for cross contamination, improper storage, inadequate reheating and food prepared too far in advance. (Table 3)

Table 3: Primary Contributing Factors* of V. parahaemolyticus Food Poisoning Outbreaks Due to Consumption of Seafood (1999 to 2003)

* More than one factor may be involved in an outbreak.
† Percentages may not add up to 100% due to rounding of figures.

Cholera cases

26. Information from the DH showed that there were totally 49 local cholera cases reported during 1999 to 2003. The suspected food item was identified in about half of the cases, of which, seafood accounted for about 80% (Table 4)

Table 4: Local Cholera Cases by Food Group (1999 to 2003)

27. Following the isolation of V. cholerae in fish tank water of two retail outlets selling live seafood in 2003, there were calls to further strengthen the control the microbiological quality of fish tank water and abstraction of seawater for keeping live seafood. The limited number of local study on ecology of V. cholerae suggested that the non-O1 non-O139 serogroup were isolated in both local seawater and seafood samples.[24],[25] However, these studies were limited by small sample numbers and limited coverage of sampling locations.

28. In light of the above, a one-year study was conducted in 2004 by an interdepartmental task force (TF) which comprised representatives from the Food and Environmental Hygiene Department (FEHD), Agriculture, Fisheries and Conservation Department (AFCD), Environmental Protection Department (EPD) and Department of Health (DH) in 2004 to fill in the existing data gaps. The objectives of this study are to determine: (i) the occurrence and distribution of V. cholerae in local waters and its association with environmental factors including the E. coli count; (ii) the occurrence of V. cholerae in live fish kept in fish culture zones and its association with other parameters.

29. A total of 90 water samples were taken from 15 selected sampling sites of four categories (Annex I). The V. cholerae count, E. coli count, salinity and water temperature were determined. Twenty-four live fish samples that were mature live fish ready for market were collected at the four selected fish culture zones.

30. Results showed that no V. cholerae O1 or O139 serogroups were detected in any of the samples. During the 12-month study period, 14 (16%) out of all the 90 water samples were tested positive for V. cholerae non-O1 non-O139 serogroups. Among these 14 samples, eight (57%) were from typhoon shelters, five (36%) were from shorelines close to the wholesale fish markets and one (7%) was from a fish culture zone. There was significant difference among the four sampling site categories in terms of isolation of V. cholerae in water samples (c² test, p < 0.001). The isolation rate of V. cholreae in water samples from typhoon shelters and shoreline (13 out of 42 water samples, or 31%) is significantly higher than that from fish culture zone (1 out of 24, or 4%) (c² test, p = 0.01) and that in open waters (0 out of 24, or 0%) (c² test, p = 0.002).

31. Water samples which were tested positive for V. cholerae were found to contain significantly higher E. coli count (Mann-Whitney U Test, p < 0.001). When analyses were conducted within the samples collected at typhoon shelters and shoreline waters, those water samples which were tested positive for V. cholerae were found to contain significantly higher E. coli (Mann-Whitney U Test, p = 0.049).

32. No correlation between V. cholerae counts and water temperature (p = 0.96) and salinity of seawater (p = 0.89) was found in the study.

33. Twenty-four water samples were taken from the sampling sites within the open waters category and none of them were found positive with V. cholerae.

34. As for the 24 live fish samples taken from fish culture zones, none of the fish samples were tested positive for V. cholerae even though one water sample taken from a fish culture zone was found positive with the non-O1 non-O139 V. cholerae.

35. While vibrios are natural habitants of seawater, it is not surprising that the majority (56.7%) of V. parahaemolyticus food poisoning outbreaks are caused by the consumption of seafood (Table 1). Among all the seafood items, crustaceans, including crab, shrimp/prawns and lobster, represented the highest percentage (28.7%) of incriminated seafood involved in V. parahaemolyticus outbreaks (Table 2). Nearly 60% of all the V. parahaemolyticus food poisoning cases by seafood occurred in 1999 to 2003 were due to the consumption of inadequately cooked food (Table 3). The second and third major contributing factors for V. parahaemolyticus food poisoning was due to the consumption of food contaminated by raw food and the consumption of contaminated raw food respectively. Thorough cooking and avoidance of cross contamination hold the key to successful prevention of V. parahaemolyticus food poisoning.

36. Because of the availability of safe water supply to the population and a reasonably high standard of sanitation locally, cholera no longer appears in an epidemic form in Hong Kong. However, since V. cholerae are widely distributed in temperate and tropical aquatic environment, in particular estuarine waters and that cholera is endemic in this part of the world, cholera still appears in Hong Kong as sporadic diseases. For the local cholera cases in which a suspected food item was identified, most of them were related to the consumption of seafood (Table 4).

37. In the Study on the Ecology of V. cholerae in Marine Water and Live Seafood, water samples taken from typhoon shelters and shoreline waters were found to have higher chance of detecting V. cholerae than that from open waters and fish culture zones. Typhoon shelters and shoreline waters (especially the shorelines in urban areas) are sites which are considered more polluted. Water samples which were tested positive for V. cholerae also have higher E. coli counts. The results implied that if V. cholerae is the concern when considering abstracting seawater for keeping live seafood, both the site where the water is abstracted and the E. coli count are important parameters to be considered. Abstraction of seawater from typhoon shelters and shoreline in urbanised areas for this purpose is therefore not recommended.

38. To minimise the potential risk of cholera and vibrio food poisoning due to the consumption of seafood products, good manufacturing and handling practices should always be observed. Attention should also be paid to the hygienic quality of fish tank water for keeping live seafood. Source of the seawater is of particular importance. The followings are some recommendations:

Advice to Trade

(A) Handling of raw materials

1. Purchase raw materials from reputable and reliable suppliers.
2. Buy only those shellfish which are fresh, with intact shell and free from abnormal odour.
3. Do not abstract seawater from shoreline in urbanised areas and typhoon shelters for keeping live seafood. Use of synthetic seawater is more desirable than the use of natural seawater.
4. Keep live fish and shellfish separately in different fish tanks equipped with proper filtration and disinfection systems. These systems should be regularly maintained. References can be made to FEHD¡¦s Guidelines on the Filtration and Disinfection Facilities for Fish Tank Water.[26]
5. Scrub and rinse shellfish in clean water. Remove internal organs of shellfish.

(B) Manufacturing and storage

1. Adopt a first-in-first-out principle to store raw materials and keep them at appropriate temperatures.
2. Avoid holding chilled ingredients and finished products at above 4^o C for more than 2 hours.
3. Reserve a specific portion of a refrigerator or a designated refrigerator for storage of seafood to be eaten raw, such as sashimi and oysters. Seafood shall be properly wrapped or covered before storage.
4. Remove the shells of shellfish, as far as possible, before cooking as it impedes heat penetration.
5. Cook food thoroughly before consumption. High-risk food such as oysters shall be cooked in boiling water for not less than five minutes.
6. Avoid preparing ready-to-eat dishes and raw foods at the same time.
7. Avoid preparing dishes in large quantities at one time and too far in advance.
8. Display seafood at 4^o C or below or 60^o C or above in buffet settings.

(C) Equipment, utensil and personal hygiene

1. Establish a clean-up and disinfection programme to clean and sterilise equipment and utensils including fish tanks, refrigerators, chopping boards, choppers, containers and mixers.
2. Observe good personal hygiene. Food handlers should wash their hands with soap and potable water thoroughly before preparing food and after every interruption in food preparation, particularly after having used the toilet.
3. Prevent cross-contamination between fish tank water, raw seafood and other food.

Advice to Public

(A) Purchase

1. Buy food from reputable and reliable suppliers.
2. Do not patronise illegal hawkers as their source of supply may not be safe.
3. Buy only those shellfish which are fresh, with intact shell and free from abnormal odour.

(B) Preparation

1. Wash seafood thoroughly before cooking.
2. Scrub and rinse shellfish in clean water. Remove internal organs of shellfish.
3. Remove the shells of shellfish, as far as possible, before cooking as it impedes heat penetration.
4. Cook food thoroughly before consumption. High-risk food such as oysters shall be cooked in boiling water for not less than five minutes.
5. Discard leftovers or otherwise store them properly in the refrigerator at 4^o C or below. Reheat leftovers thoroughly before consumption.
6. Observe good personal hygiene and prevent cross-contamination.

(C) Consumption

1. Consume seafood as soon as possible.
2. If seafood is not consumed immediately, they should be:
   • packed and stored at 4^o C or below.
   • separated from raw food.
   • consumed within 1 to 2 days.
3. Avoid consuming the internal organs of shellfish.
4. The elderly, children, pregnant women and persons with lowered immunity should be careful when choosing food especially high risk food, such as sashimi and oysters to be eaten raw.

* Designated fish culture zones governed by AFCD. Sampled by AFCD during even months.
† Designated water quality monitoring stations under the Marine Water Quality Monitoring Programme of EPD. Sampled by EPD during odd months (Typhoon Shelters) and even months (Open Waters).
‡ Designated wholesale fish markets governed by the Fish Marketing Organization, a statutory body established under the Marine Fish (Marketing) Ordinance, Cap 291 which was administered by AFCD. Sampled by AFCD during odd months.
¡± A popular seawater abstraction point where seawater was less likely to be polluted as suggested by EPD. Sampled by FEHD during odd months.