II Initial microflora

The population of microorganisms associated with living fish reflects the microflora of the environment at the time of capture or harvest, but is modified by the ability of different microorganisms (mainly bacteria) to multiply in the sub-environments provided by the skin/shell surfaces, gill areas, and the alimentary canal. Shellfish taken from waters near human habitations will tend to have higher bacterial loads and a more diverse microflora compared with those taken from isolated areas (Faghri et al., 1984). The muscle tissue and internal organs of freshly caught, healthy finfish and molluscan shellfish are normally sterile, but bacteria may be found on the skin, chitinous shell, gills of fish, as well as in their intestinal tract (Baross and Liston, 1970; Shewan, 1977). The circulatory system of some crustaceans is not “closed” and the hemolymph of crabs can harbor substantial levels of bacteria, particularly members of the genus Vibrio. Examples of the relative populations of various aerobic bacteria on finfish and crustaceans from different sources are shown in Table 3.3.

Microbial levels vary depending on water conditions and temperature. Finﬁsh and crustaceans from colder (<10–15 ◦

C) waters generally yield counts of 10 2 –10 4 cfu/cm 2 of skin and gill surface, whereas animals from warm waters have levels of 10 3 –10 6 cfu/cm 2 . Tropical shrimps carry higher numbers of bacteria, 10 5 –10 6 cfu/g, than cold-water species, 10 2 –10 4 cfu/g. Counts for intestinal contents vary widely from as low as 10 2 cfu/g in non-feeding ﬁsh to 10 8 cfu/g in actively feeding species. Counts in molluscs show marked variation with water temperature from ≤10 3 cfu/g in cold unpolluted water to ≥ 10 6 cfu/g in warm waters or when bacterial pollution levels are high.

A Saprophytic microorganisms After capture or slaughter and death, ﬁnﬁsh are normally stored in crushed ice or chilled brines, giving

rise to changes in the microflora. The most important environmental factor influencing composition of fish microflora is temperature. Typically, bacterial populations on finfish and shellfish from temperate waters are predominantly psychrotrophic, reflecting water temperatures of ≤10 ◦

C in the main water mass. However, surface water temperatures may rise during prolonged hot weather and under those conditions pelagic species of finfish (e.g. mackerel and herring) may show increased skin counts and elevated levels of mesophilic bacteria. Pelagic finfish and other surface dwelling creatures in the tropical oceans typically have higher levels of mesophilic bacteria. Both psychrotrophs and mesophiles grow well at ambient temperature (20–35 ◦ C), and spoilage occurs in 1–2 days at temperatures above 15 ◦ C. Although psychrotrophs are present in significant proportions on fish from warm tropical waters (Gram et al ., 1989), iced storage of tropical fish typically leads to a long shelf life (Lima dos Santos, 1981; Deveraju and Setty, 1985; Gram et al., 1989).

The microflora of marine finfish and shellfish is often incorrectly referred to as being predominantly halophilic. In most cases, the microflora are not true obligate halophiles. Instead, the microorganisms are predominantly halotolerant; able to grow over a wide range of salt concentrations, but displaying optimal growth at sodium chloride concentrations of 1–3%. This is enhanced by the common use of ice

Table 3.3 Bacterial genera associated with various raw finfish and crustaceans, and their percentage of the total microflora

or not Fish type

Acinetobacter– Flavobacterium–

Gram-

positive

Bacillus identiﬁed Reference Marine ﬁsh (temperate)

Pseudomonas Vibrionaceae

Moraxella

cytophaga

negative Coryneforms

cocci

North Sea Fish 1932

Shewan (1971) North Sea Fish 1960

Shewan (1971) North Sea Fish 1970

Shewan (1971) Haddock (North Atlantic)

Laycock and Regier (1970) Flatﬁsh (Japan)

Simidu et al. (1969) “Pescada” (Brazil)

3 Watanabe (1965) Shrimp (North Paciﬁc)

3 7 4 8 Harrison and Lee (1969) Scampi (UK)

3 Walker et al. (1970) Marine ﬁsh, (tropical)

Mullet (Australia)

Gillespie and Macrae (1975) Prawn (India)

Surendran et al. (1985) Sardine (India)

11 Surendran et al. (1989) Shrimp (Texas Gulf )

2 Vanderzant et al. (1970) Shrimp (Texas, pond)

15 25 12 43 3 0.5 1 Vanderzant et al. (1971) Freshwater ﬁsh, (temperate)

Pike (Spain) 10 15 55 10 5 5 Gonz´alez et al. (1999) Brown trout (Spain)

Trout (Spain, reared) 11 7 26 5 45 6 Gonz´alez et al. (1999) Trout (DK, reared)

Spanggaard et al. (2001) Freshwater ﬁsh, (tropical)

Nile perch (Kenya)

Gram et al. (1990) Catﬁsh (India)

Venkataranan and Sreenivasan (1953) Carp (India)

Venkataranan and Sreenivasan (1953)

FISH AND FISH PRODUCTS

Table 3.4 Culturable microorganisms on gills, skin, and in the intestinal tract sampled from a total of 49 rainbow trout

Number of isolates 26 Skin

Total number Group/genera

38 Gill

33 Gut

of isolates Pseudomonas spp.

27 96 23 146 Acinetobacter/Moraxella

163 Other Gram-negative

1 018 Modiﬁed from Spanggaard et al. (2001).

to chill fish and shellfish, which exposes the bacterial population to decreasing salinity during storage, favoring the survival and growth of halotolerant species. One example of the effect of salinity is the bacteria found in the intestines of finfish, halotolerant Vibrio spp. often being reported as dominant in marine species, with Aeromonas spp. dominant in fresh water fish. These genera alternate in anadromous fish, which spawn in fresh water, but spend their adult life in the ocean.

The number and variety of microorganisms in the intestine are determined by the quantity and origin of food consumed by fish. Non-feeding fish have very low levels of bacteria in the intestine. During feeding, fermentative Gram-negative bacteria often become dominant. Bacteria living on the surfaces of marine animals are phenotypically capable of living on carbon sources such as amino acids, peptides, and other non-carbohydrate sources (Table 3.4). Utilization of these substrates typically leads to the production of slightly alkaline conditions in stored fish products. Members of the Enterobacteriaceae commonly found in the intestine of warm-blooded and reptilian species are not normally isolated from finfish captured away from coastlines.

Typically, bacteria from skin and gills are predominantly aerobic, although facultative bacteria, particularly Vibrio spp., may occur in high numbers on pelagic fish (Simidu et al., 1969). Obligately anaerobic bacteria are uncommon on the surface of fish but can occur in significant numbers in the intestine (Matches and Liston, 1973; Matches et al., 1974; Huber et al., 2004). Lactic acid bacteria, in particular carnobacteria, are also commonly isolated from fish gut (Ringø and Gatesoupe, 1998).

The bacteria on finfish and shellfish are predominantly Gram-negative for fish from temperate waters. A higher proportion of Gram-positive cocci and Bacillus spp. can be found on some fish from warm, tropical, waters and some studies report as much as 50–60% of the microflora being of these types (Shewan, 1977). However, the microflora of fish from warm, tropical, waters may also be dom- inated by Gram-negative bacteria (Table 3.3, Gram et al., 1990). The microflora of living fish from temperate waters is remarkably consistent, and commonly includes members of the genera Psychrobac- ter, Moraxella , Pseudomonas, Acinetobacter, Shewanella (previously Alteromonas), Flavobacterium, Cytophaga , and Vibrio/Aeromonas. Corynebacterium and Micrococcus. The Gram-negative bacteria on warm water finfish are similar to those on cold-water fish. Fresh-water fish show similar patterns except that Aeromonas replaces Vibrio. Psychrobacter–Acinetobacter–Corynebacterium, and Micro- coccus dominate on crustaceans with lesser proportions of Pseudomonas. The microflora of molluscs is similar to that of fish, but Vibrio spp. are more prominent and often dominate the microflora of oysters. As molluscs are commonly associated with inshore environments, their microflora may reflect terrestrial influences, and Enterobacteriaceae and Streptococcacae occur. Run-off from land can have consequences for the microflora of fish cultured close to land. Thus, whilst Listeria monocytogenes cannot be detected on newly caught fish from open waters, the incidence of the bacterium may be high

MICROORGANISMS IN FOODS 6

on ﬁsh caught in waters with agricultural run-off (Ben Embarek, 1994a; Jemmi and Keusch, 1994; Huss et al ., 1995).

In addition to bacteria, yeasts, such as Rhodotorula, Torulopsis, Candida spp., and occasionally fungi, are reported from finfish and shellfish (Table 3.4). However, literature on their occurrence on living fish is scant (Morris, 1975; Sikorski et al., 1990). Yeasts are fairly widespread in fresh and salt waters but are present at much lower levels than bacteria. Fungi, except for some specialized planktonic forms, appear restricted mainly to estuarine and fresh waters. Their occurrence on fish is probably adventitious except in a few instances where they are parasitic, e.g. for salmonid fish (usually when debilitated by high water temperatures or spawning) and crustaceans by chitinolytic fungi, especially Fusarium solani , have been reported.

B Pathogens and toxicants Fish and ﬁsh products may cause a variety of food-borne diseases in humans and accounted for 19% of

967 food-borne outbreaks (with known cause) in the United States from 1993 to 1997 (Table 3.5; Olsen et al ., 2000). No food commodity was identified in almost two-third (1 784 of 2 751) of the outbreaks. Shellfish (molluscs and crustaceans) caused as many cases (∼1 900) as did poultry, which is consumed in much larger quantities. The major etiological agents are bacteria, virus, parasites, aquatic toxins, and biogenic amines (Table 3.6). Viral diseases and shellfish toxins are typically carried by shellfish, whereas intoxication by the marine toxin, ciguatera, and biogenic amines are the major causes of disease from fin fish.

In Japan, seafoods were involved in several reported outbreaks (Table 3.7). In particular, molluscan shellfish were causing disease being responsible for two-third of the diseases caused by seafood. Also, several cases of pufferfish toxin poisoning caused deaths (Japanese Ministry of Health, Labour and Welfare, 2002). These cases were typically associated with preparation of pufferfish in the home and not in restaurants. The etiological agents identified included especially Norwalk virus (and other small round structured viruses) and Vibrio parahaemolyticus. Also, in 1998, soy sauce marinated salmon roe caused a major outbreak of Escherichia coli O157:H7 (IARS, 1998).

Table 3.5 Food implicated in food-borne disease in the United States 1993–1997

Deaths Food

Outbreaks

Cases

Number % Meat

2.2 0 0.0 Other meat

0.7 2 6.9 Shellﬁsh

0.4 3 10.3 Dairy products

0.4 1 3.4 Ice cream

1.4 0 0.0 Bakery goods

1.0 0 0.0 Fruits and vegetables

2.8 0 0.0 Several foods

29.8 1 3.4 Total (known foods)

68.5 16 55.2 Known food

68.5 16 55.2 Unknown food

31.5 13 44.8 TOTAL

181 Table 3.6 Etiological agents implicated in disease from ﬁsh and

FISH AND FISH PRODUCTS

shellﬁsh (crustaceans and molluscs) from 1993 to 1997 Number of outbreaks (% of total) caused by Agent

Fin ﬁsh Bacteria

Shellﬁsh

0 (0%) Aquatic biotoxins

140 (100%) Modiﬁed from Olsen et al. (2000).

Table 3.7 Food implicated in reported food-borne diseases in Japan 1999–2001. Number of reported outbreaks, cases and deaths modiﬁed from Japanese Ministry of Health, Labour and Welfare (2002)

Cases a Deaths a Food

Outbreaks

Outbreaks a Cases

% NO % Meat, poultry, pork

2.7 2 826 3.0 0 0.0 Fish, total

5.5 5 749 6.1 1 6.7 Pufferﬁsh

0.1 126 0.1 5 33.3 Fish products

2.4 2 479 2.6 0 0.0 Dairy products

13.6 4 246 4.5 0 0.0 Cereal, grain

1.7 1 792 1.9 0 0.0 Several foods

38.1 39 720 42.1 2 13.3 Total known

94 383 100.0 15 100.0 a Excluding a major outbreak (10 000 cases) from year 2000 of staphylococcal enterotoxin caused by milk powder.

Bacteria. Pathogenic microorganisms reported to be associated with seafood are listed in Table 3.8. These microorganisms are categorized according to whether they originate in the aquatic environment (i.e. potentially on the raw material), the general environment, or whether they are the result of contam- ination/pollution from the human/animal resevoir.

Some bacteria are both frank pathogens for humans and indigenous members of the normal microﬂora of the marine environment or marine animals. These include psychrotrophic types of Clostridium botulinum and various Vibrio species (Hackney and Dicharry, 1988). Plesiomonas shigelloides and Aeromonas hydrophila are both aquatic bacteria that have been associated with gastroenteritis in humans. Although they can be isolated in high numbers from some cases of diarrhea, feeding studies with volunteers have failed to reproduce symptoms and their precise role in human illness remains unclear.

Cl. botulinum is derived most commonly from sediments, and can be assumed to be present on whole fish (Dodds, 1993; Dodds and Austin, 1997; Pullela et al., 1998). Disease is caused by a neurotoxin and the different types of toxin are used to distinguish Cl. botulinum serovars; from type A to type G. Serotype E and non-proteolytic strains of types B and F can be isolated from the intestine and occasionally from the skin of marine fish (Hobbs, 1976). Psychrotrophic Cl. botulinum are typically present only in low numbers on marine fish, but has been reported to occur at relatively high levels in pond-raised

MICROORGANISMS IN FOODS 6

Table 3.8 Pathogenic agents potentially transmitted to man from ﬁsh and ﬁsh products

Agents of disease from ﬁsh and ﬁsh products

Natural habitat Bacteria

Aquatic toxins Biogenic amines Aquatic sources

Virus

Parasites

Cl. botulinum

Ciguatera, Enterobacteriaceae, V. parahaemolyticus,

E (B and F),

Nematodes, (Anisakis,

Tetrodotoxin, Photobacterium V. cholerae, V. vulniﬁcus,

Pseudoterranova),

PSP, ASP, Aeromonas spp.,

Cestodes, Trematodes

DSP Plesiomonas General

L. monocytogenes, environment

Cl. botulinum

A and B

Animal–man- Staph. aureus, Salmonella,

Enterobacteriaceae resevoir

Noro, Hep

Shigella, E. coli

A, B, SRV, Rotavirus

Modified from Huss et al. (2001). themselves die of botulism as a result of consuming dead finfish (Eklund et al., 1982b). In animals raised

by aquaculture, poor management practices aggravate the occurrence of pathogenic microorganisms,

e.g. increased incidence of Cl. botulinum in trout raised in ponds with earth bottoms (Cann et al., 1975), and botulism epidemics in young salmon populations associated with cannibalism (Eklund et al., 1982b). Increased incidence of botulism in ﬁnﬁsh often appears to be related to excessive feed levels.

Mesophilic Vibrio species have been isolated from both pelagic and bottom dwelling fish (Simidu et al ., 1969; Baross and Liston, 1970; Sera and Ishida, 1972; Joseph et al., 1982). Among the potentially pathogenic Vibrio occurring naturally on finfish and shellfish, Vibrio parahaemolyticus is most widespread. During recent years, serotypes causing seafood-borne disease have changed and for in- stance serotype O3:K6 is now commonly associated with disease (CAOF, 2000; Chowdhury et al., 2000). During cholera outbreaks, Vibrio cholerae can be isolated from fish, but it is also an indogenous marine species and can be isolated from waters with no outbreaks (Rogers et al., 1980; Feachem, 1981, 1982; Feachem et al., 1981). The mesophilic Vibrio species are most commonly found in in-shore waters with reduced salinity. For example, V. vulnificus is commonly found in estuarine fish, particularly bottom feeders, but is less common in off-shore fish (DePaola et al., 1994). Vibrio is the genus most often implicated in diseases of bacterial origin resulting from eating contaminated shellfish (Janda et al., 1988; Levine et al., 1993).

The incidence and levels of mesophilic vibrios present on marine animals is greatly affected by water temperature (Kaneko and Colwell, 1973; Kelly, 1982; Williams and LaRock, 1985; West, 1989; O’Neil et al ., 1990). Typically, they multiply rapidly at temperatures between 20 ◦

C. This is reﬂected in the large numbers of the organisms isolated from molluscan shellﬁsh when water temperatures rise to

C and 40 ◦

C, and their virtual absence from molluscs taken from cold waters (IOM, 1991), although they have also been isolated from crabs taken from cold waters (Faghri et al., 1984). Vibrio parahaemolyticus is widespread when water temperatures exceed 15 ◦

C (Kaneko and Colwell, 1973; Liston and Baross, 1973) and incidence of disease is highly seasonal (Figure 3.2). Vibrio levels in the estuarine environment are dependent on the time of day, depth, and tidal levels (Koh et al., 1994). Vibrio parahaemolyticus,

V. cholerae , and V. vulnificus are routinely part of the microflora of crustaceans captured from estuarine waters (Davis and Sizemore, 1982; Faghri et al., 1984; Molitoris et al., 1985; Varma et al., 1989). In Western countries, seafood-related illness caused by pathogenic Vibrio species is commonly associated with crustaceans or molluscan shellfish, whereas finfish are a common vehicle for outbreaks in Japan and other Asian countries. Disease caused by V. vulnificus is particularly associated with the consumption of live oysters but a Japanese outbreak caused by squilla (a crustacean) has also been reported (Ono et al ., 2001). The major form of the disease is primary septicaemia, i.e. septicaemia with no apparent infectious focus (Levine and Griffin, 1993; Oliver and Kaper, 1997). Other presentations are wound in- fections or gastrointestinal infection. Of these disease forms gastrointestinal infection is very rare,

FISH AND FISH PRODUCTS

Number of incidents

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

II Initial microflora