5.1.1 Pathogenic bacteria (Hans Henrik Huss/Lone Gram)

Pathogenic bacteria are defined as those bacteria that that may cause illness in humans. Some pathogenic bacteria are transmitted to humans via food. Food-borne pathogenic bacteria are few among the many different types of seafood bacteria, which are causing no harm to humans. Many microorganisms are even beneficial being used in the production of food and drinks. Others are able to spoil food. Bacterial food-borne pathogens may be grouped into those that cause food intoxication and those that can result in food-borne bacterial infection.

In case of bacterial food poisoning or intoxication the causative organism multiplies in the food where it produces its toxins. A food poisoning is therefore characterized by rapid onset of the illness (typically symptoms are nausea, vomiting) as the toxins are already formed in the food before consumption. Thus ingestion of viable bacteria is not a prerequisite for the induction of the disease. Most often intoxications require that the toxin producing bacteria have grown to high numbers (10⁵ - 10⁸ cfu/g) in the food before it is eaten.

In contrast, the food merely act as a carrier for the causative organism in food-borne infections. The infectious agent may or may not have multiplied in the food, but the ingested viable bacteria continue to grow within the host's body to produce the typical symptoms (fever, diarrhoea). The number of viable bacterial cells necessary to cause disease (the Minimum Infective Dose, MID) varies considerably between bacterial species. Thus the MID is known to be high (>10⁵-10⁶ cells) for pathogenic Vibrio spp. (Twedt, 1989) and very low for some Salmonella typhi and Shigella species (Kothary and Babu, 2001).

Seafood-borne pathogenic bacteria may conveniently be divided into 3 groups according to their ecology and origin as those who are indigenous to:

• the aquatic environment (Table 5.1)
• the general environment (Table 5.2)

• the animal/human reservoir (Table 5.3).

The level of human pathogenic bacteria in fish is generally quite low as shown in Table 5.1. Highest concentrations are found in molluscs and in the intestines of molluscs' predators. The ambient temperature strongly influences the composition (quantitatively and qualitatively) of the natural micro flora present in the environment and on the fish raw material.

Table 5.1 Pathogenic bacteria indigenous to the aquatic environment and naturally present on fish (based on Huss 1997).

1. The role of Aeromonas spp. in food-borne disease is not resolved

The presence of pathogenic bacteria in the general environment is also low (Table 5.2). Furthermore it should be emphasized that all the genera of pathogenic bacteria listed in Tables 5.1 and 5.2 contain non-pathogenic environmental strains. Thus V. cholerae non-01 was detected in six samples (out of 752 samples examined) of warm-water shrimps imported to Denmark, but none of these strains contained plasmids or genes encoding cholera toxins (CT) or heat-stable enterotoxin (NAG-ST) suggesting that these organisms do not constitute a public health problem (Dalsgaard et al., 1996). However, for some organisms, such as Listeria monocytogenes, there is no known method available to distinguish between pathogenic and non-pathogenic strains.

Table 5.2 Pathogenic bacteria indigenous to the general environment and frequently present on fish (based on Huss, 1997).

The pathogenic bacteria found in the animal/human reservoir are shown in Table 5.3. They are found on outer and inner surfaces of diseased or asymptomatic carriers. Contamination of fish products is almost always due to poor hygiene (poor personal hygiene, poor processing hygiene or poor water quality).

It must be emphasized that it is nearly always possible to detect a range of human pathogenic bacteria on any fish or fish product that has not received any bactericidal treatment. Some of these pathogens may constitute part of the natural flora on the fish (pathogens from the aquatic environment) or be there as a result of unavoidable contamination (pathogens from the general environment). It is common for these pathogens that some growth in the fish products is required to produce disease in humans. This applies naturally for the intoxicating types, but as the MID for the infective environmental pathogens is high (- or higher than the natural level found in fish products), some growth is also required for these types. This means that the preventive measure for all these pathogens is prevention of growth of the organisms in the products (see Table 5.4).

Table 5.3 Pathogenic bacteria in the animal/human reservoir.

The MID for pathogens originating in the animal/human reservoir may be high or as low as (10 organisms for some Shigella and for E. coli O157 (Kothary and Babu, 2001). As these bacteria are not normally present in fish and fish products, the main preventive measure is to avoid contamination by applying good hygienic practices (GHP) and good manufacturing practices (GMP) (Table 5.4). However, for some of these bacteria, including Staphylococcus aureus, which is a toxin producing pathogen, growth in the products is required to produce disease.

Table 5.4 Seafood-borne pathogenic bacteria and disease.

1. EPEC: Enteropathogenic E. coli; ETEC: Enterotoxigenic E. coli;
EHEC: Enterohaemorragic E. coli.

The safety concerns related to pathogenic bacteria in seafood is demonstrated in Table 5.5. The mere presence (in low numbers) of pathogens from the aquatic and general environment is of no safety concern, not even in ready-to-eat (RTE) products.

In contrast, the presence of pathogens from the animal/human reservoir is a serious safety concern for products to be eaten without (further) cooking. Growth of pathogens is likewise a serious safety concern for most RTE products. For raw fish products to be eaten raw the safety concern is limited. Growth of these pathogens is only possible at elevated temperatures (>5°C) (Table 5.16), and at this condition spoilage will proceed very rapidly and the fish will probably be rejected due to off-odours and off-flavours long before being either toxic or infective organisms reach high numbers.

Table 5.5 Safety concerns related to pathogenic bacteria in seafood.

1. "+" definitely a safety concern; "(+)" limited safety concern; "-" no safety concern
2. Ready-to-eat products see Table 9.5.

Growth of pathogens in raw fish to be cooked is similarly of little safety concern. Only limited growth is possible before spoilage is causing rejection and in borderline cases, cooking will destroy the pathogen. Growth of pathogens from animal/human reservoir is of no direct safety concern in raw fish to be cooked before consumption as described above, but it may constitute a secondary hazard due to increased spread and contamination of the processing or kitchen environment with these pathogens.

5.1.1.1 Bacteria indigenous to the aquatic and general environment

Control of disease from human pathogenic bacteria occurring in the aquatic or general environment is very often ensured by preventing their growth - or destroying any organisms present. Tables 5.6 and 5.7 give overviews of growth limiting factors and heat resistance of these organisms. The D-value used to determine heat-resistance indicates the length of time (seconds, minutes) which is required at a given temperature to reduce the population to 10% of its initial count (decimal reduction).

Table 5.6 Growth limiting factors of pathogenic bacteria indigenous to the aquatic and the general environment (adapted from Huss, 1994; ICMSF, 1996).

1. Most strains of B. cereus are mesophilic with minimum temperature of approximately 8-10°C, however, psychrotrophic variants have been isolated

Table 5.7 Heat resistance of pathogenic bacteria indigenous to the aquatic and the general environment (adapted from Huss, 1994; ICMSF, 1996; Ababouch 1987).

Clostridium botulinum (Hans Henrik Huss)

Clostridium botulinum is classified into toxin types from A to G. The types pathogenic to humans (types A, B, E and F) can conveniently be divided into two groups:

• the proteolytic types A, B and F, which are also heat resistant, mesophilic, NaCl-tolerant and have the general environment as the natural habitat

• the non-proteolytic types B, E and F, which are heat sensitive, psychrotolerant, NaCl-sensitive and have the aquatic environment as the natural habitat.

a) The disease and some epidemiological aspects

Toxins produced by C. botulinum types A, B, E and F are the cause of human botulism. The disease can vary from a mild illness, which may be disregarded or misdiagnosed, to a serious disease, which may be fatal within 24 hours. In most cases, the symptoms develop within 12 to 36 hours. These are generally nausea and vomiting followed by neurological symptoms such as visual impairment (blurred or double vision), loss of normal mouth and throat function (difficulty in speaking and swallowing, dry mouth), lack of muscle coordination and respiratory impairment, which is usually the immediate cause of death.

Type E botulism tends to have most rapid onset of symptoms, while type A botulism tend to be the most severe. Early fatality rates in the first half of the 20^th century were about 50% or higher for botulism, but with the availability today of antisera and modern respiratory support systems, they have decreased to about 10% (Austin and Dodds, 2001).

The majority of botulism outbreaks in the northern and temperate regions are associated with fish, and in general type E was the responsible type. Type A and B botulism has generally been associated with meat or meat products, but fish and fish products have also been vehicle for those types. All types of fish products except raw fish to be cooked immediately before consumption have been involved in outbreaks of botulism, but the majority of outbreaks has been associated with fermented fish (Huss, 1981).

Botulinum toxin is one of the most potent of all poisons, and the amount needed to cause death in humans has been estimated to be as low as 30-100 ng (Lund and Peck, 2000). The toxin is sensitive to heat and pH above 7. For safe inactivation of any botulinum toxin at concentrations up to 10⁵ LD/g in foods, time/temperature combinations of 20 min. at 79°C or 5 min. at 85°C has been recommended (Hauschild 1989). Normal household cooking and frying of raw fish products are therefore sufficient to destroy any pre-formed toxin. This may be one of the reasons for the excellent safety record of unprocessed fish with respect to problems from C. botulinum.

While botulinum toxin is rapidly destroyed in fish products with pH>7.5, such as spoiling cod, it is extremely stable in a salty and acid environment. Thus botulinum toxin formed in the raw material will be found again or even increase in situ in the final products such as heavily salted, marinated or fermented fish (Huss and Rye Petersen, 1980). This is illustrated by the fact that many outbreaks of botulism have been traced to products, which do not support the growth of C. botulinum.

b) Prevalence in fish and fishery products

The spores of the non-proteolytic C. botulinum types, particularly type E are widely distributed in the aquatic (marine and fresh water) environment in the temperate and arctic zones. Thus, up to 100% of sediment samples from coastal areas, particularly in closed, shallow fjords and from aquaculture ponds may contain the organisms (Huss, 1980; Dodds, 1993). The distribution patterns of C. botulinum type E suggest that this is a true aquatic organism and that multiplication occurs, in situ, particularly in carrion. A much lower prevalence is found in live fish although up to 100% of fish from aquaculture and coastal waters may carry this organism. Fish caught in the high seas are generally free from C. botulinum. In warm tropical waters and in fish from these areas, other types than type E are frequently found, see Figure 5.1.

Figure 5.1 Prevalence (%-values) of Clostridium botulinum in fish. Numbers with no letter attached refer to type E; otherwise letters indicate C. botulinum types detected. ND = Not detected. For references to surveys, see Huss (1980). Cochin data from Lalitha and Surendran (2002).

The proteolytic C. botulinum are frequently found in soil and the terrestrial environment (Huss, 1980; Hauschild, 1989; Dodds, 1993). Animals, both vertebrates and invertebrates, have an important role in both the distribution and build up of botulinum spores. Spread of spores from the terrestrial environment to the aquatic environment (coastal waters and fresh waters systems) including the fish in these areas is therefore a distinct possibility as well as spread of spores to the fish processing environment. Being mesophilic, the proteolytic types do not have the same possibilities for multiplication in nature as type E.

c) Growth and survival in fish and fishery products

The main factors that control growth of C. botulinum in foods are temperature, pH, water activity (a_w), salt, redox potential and added preservatives. Maximum and minimum limits for these parameters, which would permit growth are shown in Table 5.6.

The figures quoted in Table 5.6 are used in many regulations worldwide. These figures have mostly been established at near optimal conditions in challenge studies, where C. botulinum spores have been inoculated in large numbers and as a single organism. There are at least three factors adding to the safety of fish products using the figures from Table 5.6 in the control of C. botulinum:

• the natural level of C. botulinum in fish is much lower than levels used in most challenge studies. Initiating growth and toxin production will therefore be much delayed at comparable conditions

• the associate flora in fish products may cause spoilage before the product becomes toxic. Some microorganisms may also inhibit C. botulinum

• C. botulinum is an anaerobic organism preferring a low redox potential (Eh) for growth. The Eh for fish and fish products is high (Huss and Larsen, 1979, 1980) and this may cause delay in growth and toxin production at otherwise comparable condition in bacteriological media.

The presence of an associate (spoilage) micro flora may, however, also add to the risk, as this micro flora may use oxygen and facilitate the growth and toxin production by C. botulinum type E. It is clear therefore, that using the figures in Table 5.6 in control of C. botulinum does provide considerable safety margin. It should also be emphasized, that those factors seldom function independently. Usually they act in concert often having synergetic and accumulative effects. A few examples are shown in Table 5.8.

Table 5.8 Toxin production in smoked fish inoculated with 10² C. botulinum type E spores per gram (cold smoked) or using naturally contaminated fish (hot-smoked).

1. WPS = water phase salt

The data in Table 5.8 clearly demonstrate, that a combination of salt and low temperature very effectively inhibits toxin production (and growth) of C. botulinum. A very detailed review of the effect of growth limiting factors can be found in Lund and Peck (2000) and in Eklund (1993).

Thermal inactivation of C. botulinum spores have been extensively studied. The D-value varies considerably among C. botulinum types and even among strains within the same type. The spores of the non-proteolytic types are considerably less resistant than the proteolytic types as shown in Table 5.7. The heat resistance of non-proteolytic types is particularly important for mildly heat treated, pasteurised products, where conditions for growth are excellent for surviving spores. The D-values at 82°C for these product may vary from 0.5 to 2 min as shown in Table 5.7. A minimum heat treatment of 90°C for 10 min should provide a safety factor of 10⁶ (a 6-D process or a 6-log reduction of spore count) for non-proteolytic C. botulinum as recommended by a number of advisory committees (Martens, 1999).

The spores from proteolytic C. botulinum are much more heat resistant. In general, D₁₂₁ values are in the range of 0.1-0.25 min. These spores are a particular concern in the sterilisation of low acid canned foods, and the canning industry has adopted a D-value of 0.2 min at 121°C as a standard for calculating thermal processes. For the most resistant strains, z-values (the temperature change necessary to bring about a 10-fold change in D-value) are approximately 10°C, which has also been adopted as a standard (Austin and Dodds, 2001).

d) Prevention and control

Control of C. botulinum in fishery products can be achieved by inactivation of spores or by inhibition of growth. Current guidelines regarding safety with respect to C. botulinum includes one of the following procedures (listed by Martens, 1999):

• storage at all times at <3.3°C
• storage at 5-10°C and a shelf life of <5 days
• a heat treatment of 90°C for 10 min combined with chill storage (<10°C)
• a pH (5.0 throughout the food combined with chilled storage (<10°C)

• a salt-on-water concentration (3.5% or a_w (0.97 throughout the food combined with chill storage (<10°C).

It should be noted that products where growth of non-proteolytic C. botulinum is completely inhibited (by salt or low pH) or inactivated still has a requirement for chilled storage. The reason is that the proteolytic C. botulinum may still be able to grow if temperatures are >10°C. It is a US-requirement, that vacuum packed cold smoked fish contain 3.5% NaCl (water phase salt = WPS) or 3% if combined with 100-200 ppm nitrite. For air packaged fish not less than 2.5% NaCl (WPS) in the loin muscle is required (FDA, 1998).

The canning industry has adopted a 12-D process as a minimum heat process applied to commercial canned low acid foods. The heat required to provide this "botulinum cook" or a 12-decimal reduction in proteolytic C. botulinum spores (also called F-value) is therefore equal to 12 x D₁₂₁-value or 12 x 0.2 = 2.4 min at 121°C. The highest know D₁₂₁-values is 0.25 min which gives a F-value of 12 x 0.25 = 3. Using F-values between 2.4-3 has led to safe production of canned low acid food for many decades. Often higher F values (e.g. 5) are used in commercial practice.

Refrigeration is often regarded as the primary method of preservation of fresh foods, including seafoods. At temperatures below 10°C there is no risk of toxin production by proteolytic C. botulinum types A and B. At higher storage temperature additional preservation or treatment is required to produce safe food as summarised in Table 5.9.

Table 5.9 Control of Clostridium botulinum in food.

1. WPS = Water phase salt

Vibrio species (Lone Gram)

Vibrio species belong to the Vibrionaceae family. All species are typical of marine and/or estuarine environments and most require NaCl (2-3%) to grow. Since the marine environment is their natural niche, Vibrio species are commonly isolated from fish and crustaceans. Most of the species are mesophilic and their numbers tend to increase during the warm seasons. The genus comprises 34 species of which 13 species can cause human disease, including wound infections, septicemia and gastroenteritis (Kaysner, 2000; FAO/WHO, 2001). Seafood-borne diseases are primarily caused by Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae (Oliver and Kaper, 1997). V. parahaemolyticus and V. cholerae both cause gastrointestinal disease whilst V. vulnificus causes a septicemic condition.

Vibrio species are indigenous to the aquatic environment and their presence and numbers are influenced by factors such as temperature, salinity and algal density. There is no correlation between their occurrence or numbers and faecal human pathogens or indicators of faecal human pathogens.

Vibrio parahaemolyticus (Lone Gram)

a) The disease and some epidemiological aspects

V. parahaemolyticus may cause gastroenteritis in humans and the disease has exclusively been linked to consumption of seafood, in particular raw or inadequately cooked seafoods. The incubation period ranges from 8 to 72 hours and the onset of disease is very sudden with explosive diarrhoea. Other symptoms include nausea, vomiting, headache, fever and chills (Kaysner 2000). Symptoms typically subside within 48 to 72 hours but may last up to a week and treatment of most cases primarily include rehydration. Volunteer feeding trials suggest that ingestion of 2 x 10⁵ to 3 x 10⁷ cells is required to cause disease. In these feeding trials, antacid treatment was administered to the volunteers and this probably protected the bacteria. Recent US data using epidemiological evidence indicate that doses of approximately 10 times more are required (FDA, 2000). The genus is one of the leading cause of gastroenteritis in Japan and eastern Asian countries whereas the occurrence in other countries is much lower (Table 5.10). This difference could be linked to seafood consumption patterns as the disease is mainly associated with consumption of raw seafoods.

Table 5.10 European and Japanese gastroenteritis cases caused by Vibrio parahaemolyticus (EC, 2001; CAC, 2002)

1. Associated with seafood imported from Asia
2. Associated with crayfish imported from China

The exact virulence mechanisms of V. parahaemolyticus are not known. However, at least four haemolytic components are produced. Of these, two components: a thermostable direct hemolysin (TDH) and a TDH-related hemolysin (TRH) are strongly correlated with virulence. TDH positive strains causes hemolysis of human red blood-cells and this phenomenon is known as the Kanagawa reaction. Some strains, which are TDH-negative but TRH-positive have been reported to cause gastroenteritis (EC, 2001). An elaborate serotyping system has been developed relying on O-antigens (12 types) and K-antigens (65 types) and this system is widely used in Japan. It is important to realise that the majority of environmental strains (approximately 95-99%) are not pathogenic whilst 99% of strains isolated from human cases are Kanagawa positive. Due to its mesophilic nature, incidents of V. parahaemolyticus are clearly correlated with temperature and the vast amount of cases/outbreaks occurs during the warmer months (Figure 5.2).

b) Prevalence in fish and fishery products

V. parahaemolyticus is commonly isolated from seafood products, especially in bivalve molluscs. Levels fluctuate with temperature, especially in the temperate zones, with the higher numbers being isolated in the warmer months. During colder months, the organism probably survives in sediments and is then released into the water with zooplankton when the temperature rises (EC, 2001). Also, salinity affects occurrence and the highest numbers are seen at 20-25 ppt salinity (FAO/WHO, 2001). The incidence seems to be highest in molluscan shellfish, followed by crustaceans, and lowest in finfish (Sumner et al., 2001). Numbers in oysters may range from less than one per gram to 10⁴ cfu/g but is typically less than 10 cfu/g. V. parahaemolyticus occurs less frequently in European fish and shellfish probably due to the relatively low water temperatures. During summer months, e.g. from August to October, 25 of 91 Dutch shellfish samples were positive for V. parahaemolyticus (Tilburg et al., 2000).

c) Growth and survival in fish and fishery products

Being a mesophilic, halotolerant bacteria, V. parahaemolyticus will grow well in seafoods stored at ambient temperature. The very low generation time at high temperatures (e.g. 12-18 minutes at 30°C) allows the organism to proliferate rapidly. The bacteria is also capable of proliferation in live oysters during storage. Thus numbers increased 50 fold when oysters were stored at 26°C for 10 hours and almost 800 fold after 24 hours storage. Subsequent cooling to 3°C reduced numbers by almost 10-fold during 14 days of storage (Gooch et al., 2002). In general, low temperature storage will cause numbers to decrease, however, the extent of decrease depends on food matrix, salinity and other factors.

V. parahaemolyticus is very heat sensitive and easily destroyed by cooking. D-values at 50-60°C are in the range of 0.3-0.8 min (Kaysner, 2000). Growth limits with respect to NaCl-%, temperature and pH are indicated in Table 5.6.

d) Prevention and control

Numbers of V. parahaemolyticus may be high in some live bivalves during warm months and a recent US risk assessment (FDA, 2000) demonstrated that the (initial) level in raw oysters was the most significant risk factor. However, the high infectious dose indicates that mostly growth has to take place in the product for the organism to reach hazardous levels. Thus, it is the high levels (growth) of V. parahaemolyticus and not its mere presence (in low numbers) that is the hazard. Rapid and efficient cooling (time x temperature control) is one of the most important control parameters in prevention of V. parahaemolyticus gastroenteritis. Cooling to 5°C will prevent growth. High NaCl-concentrations (>10% NaCl in water phase) or acidification as used in several semi-preserved products can prevent growth. Good Hygienic Practices (GHP) programmes should ensure that cooked products are not cross-contaminated. Depuration of molluscan shellfish has no significant effect on the level of Vibrio that may even multiply in depurating shellfish (FDA, 2000; Eyles and Davey, 1984).

Vibrio vulnificus

a) The disease and some epidemiological aspects

V. vulnificus can cause wound infections in humans and a range of fish diseases, however, it may also cause a very serious infection transmitted by seafood. As opposed to the other seafood-borne Vibrio diseases, this is a bacteremia and a septicemia not a gastrointestinal disease. Seafood-borne V. vulnificus infections are almost exclusively caused by consumption of raw bivalve molluscs such as oysters. Infections with V. vulnificus are not common in Europe but have for some years been a safety issue in the Gulf Coast area of the USA. The disease is an invasive disease causing primary septicemia, i.e. with no infectious focus. Common symptoms are fever, chills, and nausea. Symptoms occur approximately 38 hours after consumption. The disease primarily affects people in specific risk groups with underlying medical conditions such as chronic cirrhosis, hepatitis or a history of alcohol abuse (EC, 2001). Liver dysfunction is typical of several of these conditions and iron overload (typical in liver conditions) appears to facilitate infection. In particular males above 40 that have a history of alcohol consumption (and eat live oysters) are at risk (Kaysner, 2000). Mortality in risk groups may be as high as 60%.

V. vulnificus produce an extracellular cytotoxin and a battery of hydrolytic enzymes. These are probably responsible for the rapid degradation of muscle tissue seen during infection. The presence of a polysaccharide capsule is essential for infection.

Three different biotypes of V. vulnificus have been identified. Approximately 85% of strains isolated from human clinical cases are biotype 1 whereas biotype 2 mainly causes infections in eels. Biotype 3 was identified recently (Bisharat et al., 1999) and was associated with seafood mediated bacteremia.

Disease - and numbers of V. vulnificus - fluctuate with the water temperature. Most cases occur during the warm summer months. The infectious dose is not known, but shellfish with levels of 10³ V. vulnificus per gram have been implicated in disease. Using data on numbers in oysters, modelling growth between harvest and consumption, estimating number of servings based on landings and comparing this to the reported number of cases per months (Table 5.11) it becomes clear that especially high levels are likely to result in disease (FDA, 2000).

b) Prevalence in fish and fishery products

Isolation of V. vulnificus from the environment can be difficult, however, it is frequently isolated from warmer marine or estuarine waters. It appears to be associated with the Gulf Coast of the USA, although it has been isolated from other areas such as the East coast of the USA (Oliver et al., 1983) and from the Italian Adriatic coast (Barbieri et al., 1999). V. vulnificus accumulates in oysters up to 10⁴ cfu/g and can be found in levels of up to 10⁶ cfu/g in intestines of fish feeding on oysters. Just as V. parahaemolyticus, the occurrence in both water and oysters follows a seasonal pattern with high numbers (and disease) being detected in the summer-months - and 90% of the cases in the US occurring between April and October. Motes et al. (1998) found that the density in oysters was approximately 10⁴ per gram when the water was 25-30°C but dropped to below 100 per gram when the temperature decreased below 15°C. Also salinity affects its occurrence with optimal salinity at 17 ppt. V. vulnificus may multiply within the live animal and each oyster may shed up to 10⁶ bacteria per day (Tamplin and Capers, 1992).

Table 5.11 Environmental and epidemiological data for Vibrio vulnificus in the USA (modified from FDA, 2000)

1. Vv = Vibrio vulnificus

c) Growth and survival in fish and fishery products

V. vulnificus is a mesophilic bacterium and grows poorly below 15°C (minimum temperature approximately 13°C) and disease seems to be correlated to temperatures above 20°C. In seafood products stored at ambient temperature it grows rapidly and numbers can in live oysters increase with a factor 100 (2 log units) during 14 hours of storage at 24 to 33°C. Growth limiting parameters are indicated in Table 5.6.

d) Prevention and control

V. vulnificus is very sensitive to a range of food-relevant treatments. It dies rapidly during heating with D-values of approximately 78 sec at 47°C. The EU directive (EC, 1991) requires that shellfish from so-called Class C areas are heat treated at 90°C for 90 sec (or equivalent). Class C areas are areas where there is a microbiological limit on the shellfish of < 60 000 faecal coliforms/100g and the shellfish must be relayed for at least 2 months (see Chapter 11). It is more sensitive to cold-storage than V. parahaemolyticus and declines with approximately 0.04 log units per day under "normal" cold storage (FAO/WHO, 2001). The bacterium is relatively sensitive to low pH and does not grow below pH 5 (Little et al., 1997). Thus products such as pickled fish do not constitute a risk. Storage at refrigerated temperatures or below 0°C results in reduction of counts of V. vulnificus. This is either attributed to a die-off of the organism or to entrance into a so-called viable-but-non-culturable state. Frozen storage (-40°C) can result in a 4-5 log reduction over a 3 week period.

The bacterium is not removed from oysters by normal depuration and the bacterium may, as V. parahaemolyticus, actually multiply in depurating animals. In contrast, relaying in waters of high salinity does decrease numbers. Heat treatment is a very efficient way of reducing numbers. For animals with an initial low number of V. vulnificus, rapid and efficient cold-storage is crucial in preventing proliferation.

Vibrio cholerae

a) The disease and some epidemiological aspects

V. cholerae may be sub-typed into more than 130 serotypes. Of these only serotype O1 and O139 are associated with epidemic and pandemic cholera. Both produce the cholera toxin. The O1 may be further subdivided into the serogroups Ogawa or Inaba or Hikojima which is an uncommon type. O1 types may also be subdivided into two biotypes: classical and El Tor of which the latter is hemolytic. O139 strains resemble the El Tor types being also hemolytic (Kaysner, 2000).

Cholera affects only humans and the main source of the bacteria during epidemics are the faeces of acutely infected people. However, the bacteria persists in the environment and is often found attached to plankton (Chiavelli et al., 2001). V. cholerae non-O1 and non-O139 are as the other Vibrio species, ubiquitous in marine and estuarine waters. Some non-O1 and non-O139 may be pathogenic to man, causing mainly gastroenteritis, but they are not associated with the epidemic diseases. Water contaminated with sewage is the main cause of spread of cholerae but also seafood products being contaminated with cholera-containing waters have been the cause of disease. The largest recent out-break of cholera, the pandemic South American outbreak in the early 1990s was partially caused by ceviche, a raw, marinated fish product, for which contaminated water or fish was used in the preparation. This was a O1-outbreak and caused more than 400 000 cases.

Cholera is a gastrointestinal disease characterized by diarrhoea and passage of watery, voluminous (so-called rice-water) stools leaving the patient dehydrated. Treatment with salt- and sugar-water is required. One of the major virulence factors is the production of the cholerae toxin secreted by O1 and O139 serotypes. The infective dose is believed to be approximately 10⁶ cells (Kaysner, 2000) although some authors state that ingestion of as much as 10¹¹ cells are required to make up for the rapid reduction by gastric acids (Stewart-Tull, 2001).

b) Prevalence in fish and fishery products

As mentioned, an association between phyto- or zoo-plankton and numbers of V. cholerae has been observed. Water temperature and salinity also affect the occurrence and persistence of V. cholerae. Thus the highest numbers are observed at lower salinities of 2-5 ppt (Kaysner, 2000) and the natural niche of V. cholerae is estuarine waters (Oliver and Kaper, 1997). V. cholerae survives for long periods of time in river waters (FAO/WHO, 2001). Toxigenic V. cholerae have been isolated from the hindgut of crab (Huq et al. 1996) and it is believed that their chitinolytic activity, which also explains their preference for plankton aggregates, is the cause of this adherence. V. cholera is not common on fresh fish, thus none of 748 samples of warm water shrimp imported into Denmark were positive (Dalsgaard et al. 1996), nor were 131 fresh and brackish water prawn samples from Bangladesh (Balakrish Nair et al., 1991). However, in some areas of the world it may be more prevalent. V. cholerae O1 has been isolated from 3.5-18.3% of fresh fish in Mexico (Torres-Vitela et al., 1997).

c) Growth and survival in fish and fishery products

V. cholerae is very sensitive to heat, acid and cooling. Therefore, it is either eliminated by food processing treatments or its growth in foods is prevented. The majority of cases in which cholera has been linked to seafoods have involved raw products, often molluscs. Due to the involvement of ceviche in the South American epidemic, its survival in slightly acidified products has been studied and a 2-3 log reduction is seen over a 24 hour period. Limits for growth are given in Table 5.6.

d) Prevention and control

Inadequate sanitation and lack of safe water are the major causes of cholera epidemics. Therefore, cholera can only be reliably prevented by ensuring that all populations have access to adequate excreta disposal systems and safe drinking water. WHO has issued recommendations for water supply and sanitation (Table 5.12).

Table 5.12 WHO recommendations for water supply and sanitation with respect to cholera control (WHO, 1992).

Low temperature storage may reduce numbers of V. cholerae (Mitcherlich and Marth 1984, Table 5.13) but must never be relied on as a preventive measure.

Table 5.13 Survival of Vibrio cholerae (culturable cells) (Mitcherlich and Marth, 1984).

Listeria monocytogenes (Lone Gram)

Listeria monocytogenes is a Gram-positive, motile bacteria that grows well at 37°C at human body temperature but which at the same time is psychrotolerant and halotolerant (Table 5.6). Seven species of Listeria are known and of these only L. monocytogenes is pathogenic to humans (Farber and Peterkin, 2000). Listeria species are closely related to the lactic acid bacteria. L. monocytogenes is divided into 13 serovars on the basis of somatic (O) and flagellar (H) antigens, however, most isolates involved in human disease belong to three serotypes. From an epidemiological point of view, DNA-based methods such as random amplification of polymorphic DNA (RAPD), ribotyping or amplified fragment-length polymorphism (AFLP) are more discriminatory and have allowed tracing of outbreaks and contamination sources in the food industry.

a) The disease and some epidemiological aspects

Listeriosis is in its most known form an invasive disease transmitted by food products. Listeriosis is a rare disease and mostly affects people in particular risk groups where the immune defence system is reduced. This is typically elderly people, people with HIV infection, transplant patients but also pregnant women (where the immune defence is reduced to avoid rejection of the foetus). The disease infects the central nervous system and often manifests itself as meningitis. The bacterium multiplies within the macrophages and "shoots" itself from cell to cell using a tail of actin polymers. The fatality rate in the risk group is high; typically 20-40%. In infected pregnant women, listeriosis typically results in abortion. The incubation period is very variable ranging from one to 91 days and since most people do not remember their food consumption three months ago, it is often difficult to trace the food that was the source of the pathogen. If diagnosed, the disease can be treated with standard antibiotics. The incidence of listeriosis is approximately 0.5 cases per 100 000 inhabitants in the western countries. Neonates are infected since L. monocytogenes can cross the placenta and whilst the pregnant women suffer only a mild flu-like disease, the foetus is seriously affected. Recently, it has been documented that L. monocytogenes may also cause a non-invasive febrile gastroenteritis in otherwise healthy people that have eaten smoked trout (Miettinen et al., 1999). The incidence of this type of listeriosis is not known.

Listeriosis is typically caused by processed, industrialized foods that have extended shelf lives at chill temperatures and that are ready-to-eat (RTE). Thus there is no final heat treatment by the consumer. Due to its widespread occurrence, L. monocytogenes is easily isolated from several types of RTE foods. The disease was noticed initially from soft cheeses made from raw milk but has since been caused by a range of products such as paté, frankfurters, salads and RTE fish products (cold-smoked trout). Several risk assessments (Buchanan et al., 1997; FAO/WHO, 2001a; FDA, 2001) have concluded that although even low number of cells carry some risk of infection, the majority of cases (>99%) are caused by food products with high levels of the bacterium (Figure 5.3). Thus, the real risk is the growth of the organism in the product rather than its mere presence. Despite this knowledge and the understanding that low levels are unlikely to cause disease, several countries, including the United States, have regulation asking so-called zero tolerance, i.e. that the organism must not be detected in 25 grams of food.

Epidemiological evidence suggests that listeriosis has been associated with smoked mussels (Brett et al., 1998), "gravad" trout (Ericsson et al., 1997), and smoked trout (Miettinen et al., 1999). In the latter case the outbreak was not the classical invasive listeriosis, but cold-smoked trout was associated with febrile gastroenteritis in five healthy people.

b) The niche and prevalence in fish and fishery products

As indicated, L. monocytogenes is an organism indigenous to the general environment where it is typical of decaying plant material. Also, it occurs in the gastrointestinal tract and 2-6% of humans are healthy carriers. It is not typical of aquatic and marine environments. Thus the organism cannot be isolated from free open waters nor from fish caught or cultured in such waters (Table 5.14). In contrast, water close to agricultural run-off harbour the organism and in principle the bacterium must be assumed to be present, albeit in low levels on raw fish (Gram, 2001; Huss et al., 1995). In contrast to the low levels or absence on raw fish, L. monocytogenes can easily be isolated from processed fish products. Thus 3-40% of RTE seafoods are positive for L. monocytogenes (Table 5.15), but in some smoke houses as much as 80% of the samples are positive.

Table 5.14 Prevalence of Listeria spp. and Listeria monocytogenes in live or newly slaughtered fish (modified from Gram (2001)).

1. nd = not determined

Table 5.15 Prevalence of Listeria spp. and Listeria monocytogenes seafood products (modified from Farber and Peterkin (2000) and Gram (2001)).

1. nd = not determined

The bacterium is isolated at much higher frequency from RTE seafood products than from raw materials. Several studies have demonstrated that the processing environment is an important niche for L. monocytogenes (Autio et al., 1999; Fonnesbech Vogel et al., 2001). Thus using DNA-typing methods it has been shown that both slicers and salt brine harbours the types found in the product. Also, Table 5.15 shows that the bacterium is detected in heat processed products subjected to a listericidal process. Post-process contamination is the likely cause of this contamination. Cleaning and disinfection may temporarily remove the organism which is often found in more permanent niches in drains or floor mats.

c) Growth and survival in fish and fishery products

Listeria monocytogenes is halo- and psychrotolerant and can grow well in refrigerated foods. It is difficult to control it in RTE seafood products where there is no listericidal processing step and where L. monocytogenes can grow at the temperature / a_w / atmosphere conditions prevailing in the products. Several studies have demonstrated that it grows (rapidly) in brined shrimp and cold-smoked fish. Most - if not all - of these experiments were conducted with inoculated samples and growth in naturally contaminated products appear much slower. This may partly be explained by the so-called Jameson effect where the presence of a competitive associate micro flora depresses the maximum cell density of the bacterium (Figure 5.4).

The NaCl concentration is critical when evaluating growth potential, as the bacterium may grow rapidly at 3-4% NaCl but much slower with 7-8% NaCl. L. monocytogenes is of little importance in semi-preserved seafood products where 2.5% acetic acid is used. Also, use of citric acid can be used to clean floors and drains and eliminate the organism from processing environments. Nitrate, lactate, di-acetate and bacteriocins inhibit or delay growth. The limited growth illustrated in Figure 5.4 can be used deliberately as preservation by adding a bio protective competitive lactic acid bacterial flora that inhibits L. monocytogenes (Nilsson et al., 1999).

Listericidal processing consists primarily of heat treatment. The heat resistance of L. monocytogenes has been extensively studied in milk and dairy products (ICMSF, 1996). The thermal death time curve for L. monocytogenes in cod and salmon was studied by Ben Embarek and Huss (1993). The heat resistance of the bacterium is higher in salmon than in cod with D₆₀ values being 4.5 min and 1.8 min, respectively. It is assumed that the higher lipid content (approximately 13%) of salmon protected the bacterium.

d) Prevention and control

Control of listeriosis can be achieved using HACCP and GHP. A critical control point occurs when processing can include a step where L. monocytogenes is eliminated. This can only be guaranteed in products that after packaging are subjected to a listericidal process, typically a heat treatment. In many products, low levels of L. monocytogenes will occur regularly or sporadically. Control of growth in products where a listericidal process is not used can be done in several ways. Freezing of products will eliminate growth, and sufficient levels of acid and NaCl will also prevent growth. Sorbate (0.05-0.1%) or the combination of lactate (2%) and di-acetate (0.1%) has been shown to eliminate growth in frankfurters (Tompkin, 2001). As mentioned, the addition of live lactic acid bacteria may inhibit growth in some products.

Critical control points cannot be identified in the processing of a number of RTE seafood products. Therefore, control of level of contamination using GHP is of outmost importance. L. monocytogenes is sensitive to common cleaning and disinfecting agents and both chlorine, iodine, acid, anionic and quaternary ammonium-type sanitizers are effective against L. monocytogenes at concentrations of 100 ppm, 25-45 ppm, 200 ppm and 100-200 ppm, respectively. L. monocytogenes often hides in niches in the processing environment and great care must be taken to clean such niches. The processing plant must have a Listeria surveillance programme installed and procedures to be implemented when the organism is detected.

As several risk assessments have shown that low levels of L. monocytogenes are consumed daily with no adverse effect, a limit of 100 cfu/g has been suggested as a food safety objective (van Schothorst, 1998). A microbiological criteria involving 20 samples with m = 100 cfu/g and c = 0 is used has been suggested for RTE products where there is a potential for growth of the organism (van Schothorst, 1996).

Other clostridia and bacilli (Lone Gram)

Clostridium perfringens is an anaerobic, Gram-positive mesophilic spore-former widely distributed in the environment where it may be found at levels of 10³-10⁴ per gram soil. It can also be isolated from water and sediments and from faeces of healthy individuals (Adams and Moss, 2000).

If high levels of vegetative cells are eaten, a sufficient number may survive the gut passage and sporulate in the small intestine. The sporulating cells produce an enterotoxin of approximately 35 kilo Dalton (kDa). This results in nausea, abdominal pain, diarrhoea and, sometimes, vomiting 8-24 hours after ingestion. In the US, approximately 7 annual cases of C. perfringens are reported with links to seafood and it is estimated that approximately 200 seafood-caused cases occur every year (Feldhusen, 2000).

C. perfringens is typically associated with heated meat products or dishes which are temperature abused or heated slowly for long time. Due to its anaerobic nature, it prefers food with low redox potential.

C. perfringens does not grow at chill temperatures and grows only slowly below 20°C. The vegetative cells are sensitive to acid (minimum pH of 5), salt (maximum 6%) and do not grow at water activities below 0.95. Therefore controlling proliferation in seafoods is not complicated. Observing proper time-temperature conditions and avoiding cross-contamination to heated foods is essential.

Bacillus cereus strains are aerobic, Gram-positive spore-forming bacteria. As C. perfringens they are widely distributed in the environment. The spores are resistant to drying and are easily spread with dust. B. cereus can easily be isolated from many foods but typically occurs only in low numbers especially in raw foods (Granum and Baird-Parker, 2000). Heat processing will select for the spore formers.

B. cereus causes two types of disease, both caused by toxin formation. One is characterized by abdominal pain and profuse watery diarrhoea and symptoms occur 8-16 hours after ingestion. This type resembles the C. perfringens intoxication described above. The other, the so-called emetic type, has a shorter incubation period (½ to 5 hours) and nausea and vomiting are typical effects. This resembles the S. aureus gastroenteritis. The diarrhoeal type is associated with toxin formation in the gut whereas the emetic type is caused by a toxin preformed in the food. The toxin is produced in the late exponential to stationary phase and thus high numbers of B. cereus are a prerequisite for disease. The emetic type is typically related to rice, dough or other starchy products.

Most strains of B. cereus are mesophilic and do not grow below 10-15°C. However, psychrotrophic, toxin-producing strains have been isolated from foods stored at 4-6°C. Such strains must be considered for instance in the production of sous-vide products, where a mild heat-treatment is combined with subsequent cold-temperature storage. Although vacuum-packed, Bacillus species have been isolated in high numbers from sous-vide cod fillets stored at 5°C (Ben Embarek, 1994). Except for the few psychrotrophic strains, control of B. cereus is efficiently obtained by chilling.

Plesiomonas shigelloides (Lone Gram)

a) The disease and some epidemiological aspects

The genus Plesiomonas belongs to the Vibrionaceae family and consists of a single species, P. shigelloides. The species can be sub-typed and contain many serovars (Kirov, 1997). P. shigelloides can cause wound infections and septicemia but has also been suspected as cause of gastroenteritis. Thus the same serotype was found in tap water and in patients suffering diarrhoea (Tsukamoto et al., 1978) and the organism has been isolated from patients with watery (mild) diarrhoea. In the USA, P. shigelloides has mainly been linked to consumption of raw oysters. There appears to be seasonal variation, with the peak occurring during the warm summer months. The role of P. shigelloides in the described cases may be doubtful since volunteers participating in feeding studies have failed to develop diarrhoea after ingestion of 10⁹ organisms. Mild diarrhoea has, however, been induced in piglets (Kirov, 1997).

b) Prevalence in fish and fishery products

P. shigelloides is an environmental organism but is mostly associated with aquatic environments, both freshwater and marine waters (Farmer et al., 1997). The organism is mesophilic and typically growth does not occur below 8°C, however, it has been isolated from freshwater environments in cold climates (Krovacek et al., 2000). The bacterium can be isolated from different food products but is typically found in fish and seafood.

c) Growth and survival in fish and fishery products

As mentioned, P. shigelloides is a mesophilic bacteria and does not grow at chill temperatures. The organism does survive freezing. The organism is sensitive to low pH and growth is slowed at moderate salt-concentrations (> 3.5% WPS).

d) Prevention and control

The evidence of P. shigelloides as a human food-borne pathogen is not completely convincing. As an organism indigenous to the aquatic environment, it must be expected to be present on fish and shellfish. Bearing in mind the volunteer studies, it must be anticipated that some growth to high concentrations is needed for any (potential) disease to occur. Thus its control in foods is straight forward since chill storage or moderate salting/acidifying conditions will prevent growth of the organism.

Aeromonas (Lone Gram)

a) The disease and some epidemiological aspects

The genus Aeromonas is a member of the Aeromonodaceae family which was created in 1986. Formerly the genus belonged to the Vibrionaceae family. It contains species pathogenic to animals (fish) and man. In humans, Aeromonas species may cause several adverse conditions including skin or soft tissue infection through invasion via burn injury. Such infections are most commonly associated with immunosuppression (Monteil and Harf-Monteil, 1997). The taxonomy of Aeromonas is rather confusing, but the species A. hydrophila, A. sobria and A. caviae which are motile and often (but not always) mesophilic have been linked to human gastroenteritis. They are, even more commonly than P. shigelloides, isolated from patients with mild diarrhoea, often as the sole potential human pathogen. Several classical virulence factors (extracellular enzymes, exotoxins (including enterotoxins), siderophores) have been identified in these Aeromonas species. There is no evidence that toxins preformed in the food play any role (Ahmed, 1991). As for P. shigelloides, feeding high levels to human volunteers have failed to cause disease (Morgan et al., 1985) and the association between eating fish and shellfish and Aeromonas-gastroenteritis is at best circumstantial.

b) Prevalence in fish and fishery products

The Aeromonas species is a common, natural member of freshwater environments and between 33 and 100% of water samples contain the bacterium (Palumbo et al., 2000). These organisms can also be isolated from marine and estuarine environments (Knøchel, 1989). The A. hydrophila group is very commonly found in fish and fish products at levels between 10² and 10⁶ cfu/g but is also readily isolated from meat, milk, poultry and vegetable products (Palumbo et al., 2000). Several studies have implicated Aeromonas species as spoilage organisms of raw meat (Dainty et al., 1983), raw packed salmon (Gibson, 1992), fish from warm tropical waters (Gram et al., 1990) and milk (Eneroth et al., 1998). In such products the organisms may grow to 10⁷-10⁹ cfu/g.

c) Growth and survival in fish and fishery products

Although the motile aeromonads as a group are mesophilic, several studies have demonstrated that many environmental (food derived) strains grow well at chill temperatures (Knøchel, 1990; Eneroth et al., 1998). Growth is inhibited by approximately 5% NaCl (Gram, 1991) and at pH 5. The organisms are able to grow in both vacuum and modified atmosphere packed products (Palumbo et al., 2000).

d) Prevention and control

Aeromonas species are readily isolated from water, fish and shellfish and must be expected to be present. Limitation of growth requires a combination of chilling, salting and/or acidification. Growth of aeromonads will not be a problem in foods with pH below 6.5 and NaCl > 3% WPS.

5.1.1.2 Bacteria indigenous to the human/animal reservoir (Lone Gram)

Bacteria from the human/animal reservoir may, as presented in the tables in section 4.1 on statistics, cause seafood-borne diseases. Thus cases of staphylococcal enterotoxin gastroenteritis have been reported from cooked crustaceans, and oysters and other ready-to-eat products have caused salmonellosis or shigellosis. Several of these diseases are zoonotic since the major source of human illness is infected animals. The tolerance of these organisms to food-relevant preservation parameters is presented in Table 5.16.

Table 5.16 Growth limiting factors of pathogenic bacteria indigenous to the human and animal reservoir (adapted from Huss, 1994; ICMSF, 1996)

1. Some authors report growth at temperatures as low as 2°C (D'Aoust 2000)
2. Different maximum limits are reported in the literature

Salmonella species or Serovars

The genus Salmonella is a member of the Enterobacteriaceae family. Salmonellosis is a leading cause of bacterial enteric disease in both humans and animals (Brenner et al., 2000). The nomenclature is complex and causes confusion. At the Centre for Disease Control (CDC) in USA, the following scheme is used: only two species of Salmonella are recognised; the S. enterica and the S. bongori. The former occurs in 6 sub-species. Within each of these, several serotypes exists. Thus S. enterica subsp. enterica as the largest group covers approximately 1500 serotypes. Examples of such serotypes are Enteritidis, Typhimurium or Typhi (Brenner et al., 2000). The serotypes of the other sub-species are not named but identified by antigenic formula (D'Aoust, 2000).

a) The disease (adverse health effect) and some epidemiological aspects

Salmonellosis manifests itself clinically either as the enteric fever syndrome caused by typhoid or paratyphoid strains or as the nontyphoid dependent gastroenteritis. The latter may progress to a more severe systemic infection. Symptoms of the non-typhoid salmonellosis include nausea, abdominal cramps, diarrhoea with watery and possibly mucoid stools, fever and vomiting appearing 8-72 hours after exposure to the pathogen (D'Aoust, 2000). Systemic spread may occur leading to cardiac and circulatory problems. Poultry, pork and beef products are important sources of salmonellosis and eggs have, especially due to transovarian infection of the egg with S. Enteritidis, been involved in many outbreaks. Recently, also a variety of ready to eat vegetables, including bean-sprouts, have caused salmonellosis. Seafoods are relatively uncommon as causes of salmonellosis, however, the number of cases seems to be increasing. The infectious dose of salmonellae is, in general, high - typically around 10⁶ cells, however, much lower infectious doses (10-100 cells) are reported if the organism is protected against stomach acidity e.g. by fat and if the product is eaten by more susceptible groups such as children.

b) Prevalence in fish and fishery products

Salmonellae are typically mesophilic bacteria with a global distribution. However, their main reservoir is the gastrointestinal tract of man and animals, including birds. Also, environments, such as water reservoirs, contaminated with human or animal excreta may harbour Salmonella. In particular shellfish growing in contaminated waters may accumulate Salmonella and raw oysters have been the cause of salmonellosis outbreaks (Ahmed, 1991).

Open marine waters are free from Salmonella but estuaries and contaminated coastal waters may harbour the pathogen. Also, poor personal hygiene may transmit the organism. Salmonella is rarely detected in fish from temperate waters but may occur in tropical waters and on fish and shellfish from such waters. Up to 10-15% of fish samples from India and Mexico were positive of Salmonella which has also been detected in several crustacean and molluscan products from India and Malaysia (D'Aoust, 2000). There is evidence that specific serotypes of Salmonella are common in fish farms and become part of the indigenous micro flora (Feldhusen, 2000).

The integrated fish farming in some areas of South-East Asia and Asia where poultry manure and/or so-called "night soils" are sometimes used as fertilisers for the ponds may add to the Salmonella contamination. However, the use of chicken manure does not per se lead to Salmonella detection as demonstrated by Dalsgaard et al. (1995) who did not find a single positive sample out of 158 samples from shrimp production in Thailand. To further complicate the situation, Salmonella may also, in these warm climates, originate from the environment itself (Reilly et al., 1992; Bhaskar et al., 1995) and does not necessarily indicate poor hygiene. In a Japanese study, Salmonella was detected in approximately one fifth of eel culture ponds (Saheki et al., 1989).

Cooked shrimp may be post-process contaminated from the raw crustaceans or by employees and since no competing micro flora is present, it may constitute a high risk product if the bacterium is allowed to grow e.g. following temperature abuse.

c) Growth and survival in fish and fishery products

The vast majority of salmonellae are mesophilic bacteria growing from just above 5°C to approximately 45°C with an optimum at 37°C. Vegetative, unstressed cells are heat-sensitive and are easily destroyed at pasteurisation (hot-smoking) temperatures. D-values at 60°C are typically 1-3 minutes. Although Salmonella does not grow well at low water activity, it has been found to survive well in dry environments and (re)-contaminate products such as fish meal. Salmonella does not grow below pH 4.5.

Shigella

Four species of Shigella are known all of which are human pathogenic. The genus Shigella is very closely related to another Enterobacteriaceae genus, Escherichia.

a) The disease and some epidemiological aspects

Sh. dysenteriae causes the most severe condition of bacilliary dysentery whereas Sh. sonnei causes the mildest of the diseases. The infectious dose is low, approximately 10-100 cells and from 7 hours to 7 days may lapse before symptoms present themselves. These include abdominal pain, vomiting, fever and diarrhoea which may contain bloody stools. The disease is an infectious disease. Sh. dysenteriae occurs on the Indian subcontinent, in Africa and Asia whereas the mildest of the species, Sh. sonnei is the most common in the western countries (Lampel et al., 2000). In children, particularly in developing countries, the disease may be severe and Shigella diarrhoea accounts for hundreds of thousands deaths every year.

The primary route of infection is the faecal-oral route with person-to-person being the most common route of transmission. Shigellosis outbreaks follow a seasonal pattern with the largest number of outbreaks in the warm (summer) months.

b) Prevalence in fish and fishery products

Unlike Salmonella, Shigella is not associated with particular food raw materials but its presence is exclusively a question of poor hygienic handling and humans are its natural reservoir. Outbreaks have been caused by a multitude of food products, including shrimp and clams (Lampel et al., 2000). Shigella are not naturally present in water but may survive for up to 6 months in water (Wachsmuth and Morris, 1989) and may survive for long time in clams and oysters (Feldhusen, 2000). Outbreaks have typically involved contamination of raw or previously cooked foods during preparation by an infected, asymptomatic carrier with poor personal hygiene.

In the US, FDA in 1994 and 1995 reported 7 cases of shigellosis caused by seafood and estimate that the annual total number of seafood-related shigellosis cases is approximately 200 cases (Feldhusen, 2000).

c) Growth and survival in fish and fishery products

Shigella species are truly mesophilic and do not grow below 6-7°C. They are sensitive to salting and heating. As mentioned, they may survive for long periods of time in bivalves.

Escherichia coli

The genus Escherichia is a member of the Enterobacteriaceae family and E. coli is the most common aerobic organism in the intestinal tract of man and warm-blooded animals. Most of the E. coli strains are harmless commensals that colonise the intestinal tract and probably play important roles in maintaining intestinal physiology. However, some strains of E. coli are pathogenic and can cause diarrhoeal disease. E. coli strains are differentiated based on a serotyping scheme involving O (somatic), H (flagellar) and K (capsular) antigens. Pathogenic E. coli are divided into specific groups depending on virulence, clinical symptoms and distinct O:H antigens. The important groups are (Doyle et al., 1997):

• enteropathogenic E. coli (EPEC)
• enterotoxigenic E. coli (ETEC)
• enteroinvasive E. coli (EIEC)
• diffuse-adhering E. coli (DAEC)
• enteroaggregative E. coli (EAggEC)
• and enterohemorrhagic E. coli (EHEC)

• or verotoxic E. coli (VTEC).

a) The disease and some epidemiological aspects.

EPEC causes a watery type of diarrhoea accompanied by vomiting and fever. It typically occurs in infants and young children. The EIEC produced a diarrhoeal disease similar to Shigella whereas ETEC causes diarrhoea resembling V. cholerae diarrhoea. ETEC are a major cause of diarrhoea in children in developing countries and also a cause of so-called travellers' diarrhoea in adults. ETEC strains produce two types of toxin of which one resembles the cholera toxin. Also, the DAEC and EAggEC cause various variants of diarrhoea.

Due to recent outbreaks of EHEC in developed countries, much research has been directed against these organisms. By the early 1980ies it was realised that some E. coli strains caused hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Following diarrhoea and a sub-set of symptoms, EHEC may result in renal failure; the HUS condition. Whilst the disease may affect all age groups, in particular children are susceptible (Willshaw et al., 2000). E. coli O157:H7 is the most common EHEC serotype. The attachment capability appear to be important virulence factors for EHEC strains which produce two Shiga-like toxins capable of killing Vero (African Green Monkey Kidney) cells. The infectious dose is low and levels of 2 - 2 000 cells have been recorded as ingested concentrations in outbreaks.

b) Prevalence in fish and fishery products

The main source of E. coli infections have been (faecally) contaminated water and contaminated food handlers. Outbreaks by EHEC have mostly involved undercooked ground beef and raw milk. Also vegetables, such as alfalfa sprouts, washed or cultured in contaminated water have caused outbreaks. A number of famous outbreaks have been related to unpasteurised apple juice. Due to the relatively low pH, these juices were considered safe, however EHEC strains have an unusual acid tolerance and thus survived in the product.

Neither of the E. coli strains are typical of water or of aquatic products. However, poor hygiene, cross contamination by food handlers or dirty water may transfer the organism. Also, such strains may accumulate in filter feeding bivalves cultured in contaminated waters.

Whilst E. coli is not indigenous to the aquatic environment, it may survive and even multiply in warm tropical waters (Rhodes and Kator, 1988; Jiménez et al., 1989) and thus also be isolated from presumed unpolluted waters. There are no reports of isolation of O157:H7 strains from seafood products.

c) Growth and survival in fish and fishery products

All E. coli strains are mesophilic organisms with optimum growth at 37°C. They do not grow at chill temperatures and are readily destroyed by mild heating. Most isolation procedures rely on incubation at 44°C, however, EHEC strains do not grow on selective media at 44°C. In general, the organisms are sensitive to salting and acidifying. A notable exception is the acid tolerance seen in EHEC strains.

Prevention and control of mesophilic Enterobacteriaceae

Although both Salmonella and E. coli can be isolated from non-contaminated tropical waters, the main source of these organisms and Shigella are human and animal (faecal) contamination. Therefore adherence to Good Hygienic Practices with emphasis on clean water and personnel hygiene will control the organisms. As all are sensitive to heating, the GHP-programme must be particularly strict when ready-to-eat foods are processed.

Proper treatment (e.g. chlorination) of water and sanitary disposal of sewage are essential parts in a control programme.

The infectious dose of Shigella and E. coli is low and thus it is their mere presence that must be avoided. In contrast, most Salmonellae have a higher infectious dose if they are not consumed in very fatty (protective) products. Therefore their growth in the product must be avoided. Growth will be inhibited at chill temperatures and by salting.

Current levels of Salmonella in various foods and its importance in human food-borne infections underline that bacteriological testing and stringent bacteriological standards (e.g. absence) of most foods are insufficient measures in the control of salmonellosis. Even the microbiological quality of harvest water (for live bivalves) appears not to be a good predictor for Salmonella contamination, because oysters removed from closed and open beds had the same level of contamination (4%) and no correlation was observed between the presence of E. coli and Salmonella (D'Aoust et al., 1980).

Yersinia enterocolitica

As Salmonella, Shigella and E. coli, Yersinia enterocolitica is a member of the Enterobacteriaceae. It is discussed separately from the above as it is psychrotrophic and thus capable of growth at chill temperatures. The species is divided into sero- and phage types and only certain sub-types are pathogenic.

a) The disease and some epidemiological aspects

Y. enterocolitica causes a gastrointestinal disease characterized by abdominal pain, diarrhoea and mild fever. Whilst the diarrhoeal disease is self-limiting, sequela may occur resulting in arthritis and red skin lesions. These post-infection conditions may last several months. Y. enterocolitica produces an enterotoxin but its exact role in disease is not known. b) Prevalence in fish and fishery products

Y. enterocolitica is associated with pigs which are chronic carriers of the serotypes involved in human infection. Food products washed in contaminated water or contaminated milk also causes yersiniosis. The bacterium has only sporadically been detected in seafoods.

c) Growth and survival in fish and fishery products

Although seldomly occurring in seafoods, Y. enterocolitica should be considered when evaluating the risks of seafood products with long, chilled shelf lives. In particular ready-to-eat products may become hazardous if contaminated with the organisms. Jeppesen and Huss (1993) demonstrated that Y. enterocolitica serotype O3 may grow well in brined (salted) shrimp stored at 5°C.

d) Prevention and control

Proper hygienic conditions may prevent cross-contamination from agricultural sources. Due to its psychrotrophic nature, chill storage may not be sufficient to prevent growth of the bacterium in products where the competing Gram-negative spoilage flora has been eliminated. Heating (cooking) will destroy the organism as will the salting and acidifying procedures used in semi-preserved seafood products.

Campylobacter

The genus Campylobacter is the most prominent species of the family Campylobacteriaceae. The genus contains several species of which especially one, C. jejuni causes gastrointestinal disease in humans. The disease is zoonotic with several animals serving as reservoirs. Although very sensitive to a range of environmental conditions, Campylobacter can commonly be isolated from waters close to agricultural run-off and waste waters.

a) The disease and some epidemiological aspects

Campylobacter jejuni and in some cases C. coli, cause diarrhoeal disease. Symptoms develop between one and 11 days after ingestion and abdominal pain, fever and diarrhoea are the main ones. The disease is self-limiting, but in a few instances, Campylobacter has been the cause of the neurological disease, Guillain-Barrè syndrome. Although the bacteria are sensitive to acid (and thus low stomach pH), the infective dose appears to be low (< 1 000 cfu) (Nachamkin, 1997).

b) Prevalence in fish and fishery products

Although poultry appears to be the main source of Campylobacter it may be isolated from several other food products. Milk has been linked to outbreaks. Up to 14% of oyster flesh samples have been found to contain campylobacters and an outbreak in the US has been ascribed to raw clams (Adams and Moss, 2000). Campylobacters are frequently isolated from water and water supplies (Nachamkin, 1997). Whilst the bacteria die quickly in open marine waters, they may accumulate in shellfish where they appear to be protected. One study has reported that up to 42% of (Irish) shellfish were positive for mesophilic Campylobacter (cf Feldhusen, 2000).

c) Growth and survival in fish and fishery products

Campylobacter species have a narrow growth spectrum and do not grow at temperatures below 28-30°C and are sensitive to oxygen. Thus they will not grow in chill stored products but may survive under chill temperatures. The organism is sensitive to heating (D₅₅ of approximately 1 minute).

d) Prevention and control

Due to its sensitivity to food-relevant parameters, the control of Campylobacter in seafood appears simple. Avoidance of seafood from contaminated waters will control the hazard. This applies in particular to live bivalves.

Staphylococcus aureus

The Staphylococcus genus comprises several species of which especially S. aureus is associated with food-borne disease. The staphylococci are Gram-positive cocci with their primary habitat in the skin, glands and mucous membranes of warm-blooded animals including humans. Infected sores and scratches are often harbourage sites for S. aureus. The bacteria survive well in the environment and may also be isolated from a range of sources that come into contact with man and animals.

a) The disease and some epidemiological aspects

The disease caused by S. aureus is intoxication. The bacteria produce enterotoxin that upon ingestion causes nausea, vomiting, stomach cramps and, sometimes, diarrhoea. The enterotoxins are preformed in the food - thus growth of the organisms is a prerequisite for disease - and the incubation period is short, typically 2-4 hours. Seven antigenically different proteins cause the disease. All the enterotoxins have molecular weight of approximately 27 kD. The primary effect of the toxins is really a neurological (and not an enterotoxic) effect stimulating the vomiting centre in the brain. The disease is self-limiting and typically lasts only 24-48 hours, however, it may be extremely unpleasant. Due to the relatively short-lived nature of the disease, it is believed that only a small fraction (1-5%) of cases are reported. A higher frequency is seen during the warmer months and in November and December. The latter peak is probably correlated to left over holiday foods and buffets (Jablonski and Bohach, 1997).

b) Prevalence in fish and fishery products

Staphylococci may be isolated from newly caught fish, especially in warm waters (Gram and Huss, 2000). However, enterotoxigenic strains are typically transferred from food handlers with hand infections or with a cold or a sore throat. S. aureus has been isolated at levels of 2-10% in fish and bivalves but much more commonly in cooked, handled crustaceans where as much as 24-52% of samples may be positive (Jablonski and Bohach, 1997).

c) Growth and survival in fish and fishery products

Growth (to levels above 10⁶ cfu/gram) is required for toxin formation and since S. aureus is a mesophilic organism some degree of temperature abuse typically precedes intoxication. Staphylococci are poor competitors and do not grow well in the presence of other microorganisms. Although they may be detected on raw fish (and meat), they will not be able to grow to toxigenic levels. The bacterium is tolerant to high levels of salt and toxin may be produced in up to 10-15% NaCl. Growth and toxin production may occur in products such as cooked crustaceans where the heat processed meat is virtually sterile and where the hand peeling operations provides ample opportunity for contamination with staphylococci.

d) Prevention and control

Growth and toxin formation may easily be prevented by proper chilling of products. Avoidance of cross contamination of heat treated (cooked) products is also important. The toxins are compact molecules and are not degraded by gut proteases. Also, they are resistant to heat and will resist boiling for some time. Toxins have not been detected in canned foods.

EU has set a microbiological criteria for S. aureus in cooked crustaceans where none of five samples may exceed 1000 cfu/g and only two samples may exceed 100 cfu/g (EC 2001a).