Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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or persons who are immunocompromised in some

way. The route of entry of the organism in these

cases is not well defined, although foods containing

the organism would be a likely source.

Treatment/Prevention

0026 As with epidemic V. cholerae, treatment of diarrhea

is dependent on adequate rehydration. Septicemia

requires aggressive antibiotic therapy and supportive

care. Prevention in the developed world is based on

the avoidance of raw or undercooked shellfish. In the

developing world prevention is similar to that for

other enteric pathogens, including avoidance of raw

or undercooked foods of all types.

See also: Contamination of Food; Diarrheal

(Diarrhoeal) Diseases; Shellfish: Contamination and

Spoilage of Molluscs and Crustaceans; Vibrios: Vibrio

parahaemolyticus; Vibrio vulnificus

Further Reading

Albert MJ, Neira M and Motarjemi Y (1997) The role

of food in the epidemiology of cholera. World Health

Statistics Quarterly 50: 111–118.

Blake PA, Allegra DT, Snyder JD et al. (1980) Cholera – a

possible endemic focus in the United States. New

England Journal Medicine 302: 305–309.

Franco AA, Fix AD, Prada A et al. (1997) Cholera in Lima,

Peru, correlates with prior isolation of Vibrio cholerae

from the environment. American Journal of Epidemi-

ology 146: 1067–1075.

Karaolis DKR, Somara S, Maneval DR Jr, Johnson JA and

Kaper JB (1999) A bacteriophage encoding a patho-

genicity island, a type-IV pilus and a phage receptor

in cholera bacteria. Nature 27: 375–379.

Morris JG, Jr (1990) Non-O group 1 Vibrio cholerae:a

look at the epidemiology of an occasional pathogen.

Epidemiology Review 12: 179–191.

Morris JG Jr, Takeda T, Tall BD, et al. (1990) Experimental

non-O group 1 Vibrio cholerae gastroenteritis in

humans. Journal of Clinical Investigation 85: 697–705.

Mujica OJ, Quick RE, Palacios AM et al. (1994) Epidemic

cholera in the Amazon: the role of produce in disease

risk and prevention. Journal of Infectious Diseases 169:

1381–1384.

Nair GB, Ramamurthy T, Bhattacharya SK et al. (1994)

Spread of Vibrio cholerae O139 Bengal in India. Journal

of Infectious Diseases 169: 1029–1034.

Pollitzer R (1959) Cholera. Geneva: World Health Organ-

ization.

St Louis ME, Porter JD, Helal A et al. (1990) Epidemic

cholera in West Africa: the role of food handling and

high-risk foods. American Journal of Epidemiology 131:

719–728.

Twedt RM, Madden JM, Hunt JM et al. (1981) Character-

ization of Vibrio cholerae isolated from oysters. Applied

Environmental Microbiology 41: 1475–1478.

Wachsmuth IK, Blake PA and Olsvik O (eds) (1994)

Vibrio cholerae and Cholera. Washington, DC: ASM

Press.

Waldor MK and Mekalanos JJ (1996) Lysogenic conversion

by a filamentous phage encoding cholera toxin. Science

272: 1910–1914.

Vibrio parahaemolyticus

R Sakazaki, Japan Institute of Biological Sciences,

Chiyodaku, Tokyo, Japan

Introduction

0001Vibrio parahaemolyticus, the causative organism of

gastroenteritis associated with the consumption of

raw or undercooked seafish, shellfish, and other con-

taminated foods, is one of the best described of patho-

genic vibrios. Although several synonyms have been

suggested for it, the name V. parahaemolyticus has

been formally accepted.

Characteristics

0002V. parahaemolyticus is a short, slightly curved or

straight, Gram-negative, facultatively anaerobic,

motile rod. Sometimes it reveals polymorphism with

coccoid cells, especially in older strains. In broth cul-

tures, cells each exhibit a single polar, sheathed flagel-

lum, but on the surface of solid media young cultures

may also show unsheathed peritrichous flagella.

0003V. parahaemolyticus is a halophilic organism. It

can grow on/in ordinary media containing 1–8%

NaCl, but the most abundant growth occurs in the

presence of 2–4% salt. It fails to grow on/in media

lacking salt. It grows between 10



C and 44



C, but

the optimal temperature for growth is between 30



and 37



C. It fails to grow at 4



C and lower tempera-

tures not only arrest multiplication of the organism

but also cause a rapid decrease in viable cell numbers.

The vibrio can grow at a pH range from 5.6 to 9.6 but

grows best at pH 7.6–8.6. Under optimal conditions,

the generation time of the vibrio in the exponential

phase of growth is estimated at 9–13 min.

0004Cultures of V. parahaemolyticus produce moist,

circular colonies, opaque in appearance, attaining

a size of 2–3 mm on ordinary agar media containing

2–3% salt after incubation at 37



C for 24 h. Some-

times mucoid and viscid colonies may be seen.

After several subcultures, however, dissociated

translucent or rugose colony variants may also

occur. Cultures of this vibrio, especially those isolated

5988 VIBRIOS/

Vibrio parahaemolyticus

from environmental sources, often show swarming

on the surface of nutrient agar. In broth containing

2–4% salt, most strains produce heavy homologous

turbidity, but rugose variants form a pellicle on the

surface of the broth.

0005 With few exceptions, V. parahaemolyticus pro-

duces both lysine and ornithine decarboxylases as

well as indole. Arginine dihydrolase is not produced.

It gives a negative reaction in the Voges–Proskauer

test, and a positive reaction in the oxidase test. It

ferments glucose, maltose, mannose, trehalose, and

mannitol without gas production but fails to attack

lactose, sucrose, xylose, and sorbitol. The majority of

strains ferment arabinose, but occasional strains fail

to do so. Urea is not usually decomposed, but some

strains are able to hydrolyze it. It has been suggested

that the ability to hydrolyze urea may be a marker

for those Kanagawa-negative strains which are

associated with illness but which nevertheless pro-

duce a thermolabile TDH-related hemolysin (TRH)

related to, though different from, the direct thermo-

stable direct hemolysin (TDH) of Kanagawa-positive

strains.

0006 The vibrio is susceptible to most antibiotics except

penicillin, ampicillin, and polymyxin B, but resistant

mutants do occur. It is also susceptible to the vibrio-

static agent O/129 (2,4-diamino-6,7-diisopropyl pter-

idine) at a concentration of 150 mgml

1

but resistant

at 10 mgml

1

0007 Strains of V. parahaemolyticus may be typed with a

combination of both somatic O and capsular K anti-

gens, based on a serological scheme in which 13 O

and 75 K antigens were established. The number of K

antigens has since been extended to 84. Most strains

of the vibrio are inagglutinable with O antisera in the

living state, but after heating suspensions for 30 min

at 100



C they become O-agglutinable. H antigens are

not included in the antigenic scheme, since in all

strains they are regarded as serologically identical,

although antigenically the polar flagellum and the

peritrichous flagella are different.

Incidence in the Aquatic Environment

0008 V. parahaemolyticus occurs in estuarine environ-

ments throughout the world. It also occurs occasion-

ally in fresh-water sites, especially in brackish water

during the summer, but only in small numbers. The

halophilic nature of the vibrios is related to their

ability to survive in the aquatic environment. They

survive in estuarine water for more than 1 week at

15–20



C, whereas they rapidly become extinct in

fresh water under the same conditions as those of

estuarine waters. The fate of the vibrios in fresh

water is also partly concerned with osmotic pressure

which causes autolysis of the cells. In brackish water,

however, they may survive as long as in estuarine

water in the summer. They are not found in the estu-

arine water in winter, although the vibrio can usually

be isolated from sediment even when the water tem-

perature is less than 10



C. Indeed, it is likely that it

survives in sediment in estuarine areas during the

winter. The vibrio produces chitinase and the action

of this enzyme enables it to adhere to the chitin of

marine life. It thus colonizes zoo-plankton and the

surface of shellfish. It therefore occurs in the digestive

tracts of shellfish as a result of their ingestion of zoo-

plankton. V. parahaemolyticus probably survives in a

viable but nonculturable state in a cold environment,

as reported for V. cholerae and V. vulnificus.

Reservoirs

0009Gastroenteritis due to V. parahaemolyticus is usually

associated with seafoods. Coastal fish and shellfish

are usually contaminated with V. parahaemolyticus in

the summer. On the surface of market marine fish, the

vibrios do not proliferate if kept below 10



C, but at



C their numbers increase rapidly. About 10–100

cells of V. parahaemolyticus have been found on the

surface of coastal fish just after landing. If the fish

were kept at atmospheric temperature, especially in

summer, the number of vibrios increases to more

than 10

cells within a few hours. It has not been

found on market fish in winter.

0010Seafoods responsible for illness vary with local

eating habits in different countries. In Japan, the

high incidence of infection is undoubtedly due to

the national custom of eating the meat of raw fish,

shellfish, and other fish products. Any kind of seafish

served as sushi and sashimi can act as the vehicle

for the transmission of gastroenteritis. Although

the vibrio may be killed within several minutes in

0.5% acetic acid, raw fish or shellfish with vinegar,

which are widely eaten in Japan, nevertheless often

transmit this infection. Also, infection is sometimes

associated with consumption of cured vegetables

cross-contaminated with the vibrio in the kitchen.

The incidence of V. parahaemolyticus infection may

be less in other countries where fish are not usually

consumed uncooked. Nevertheless, cases of gastroen-

teritis caused by this organism have been reported in

East European countries, the UK, Africa, and the

USA. In the USA, for example, 40 outbreaks of infec-

tion were reported to the Centers for Disease Control

and Prevention between 1973 and 1998. In countries

other than Japan, although fish are usually cooked

shortly before consumption, crab meat and shrimp,

which are the seafoods most often implicated with

this infection, are usually extracted either by hand or

VIBRIOS/

Vibrio parahaemolyticus

5989

mechanically after cooking, becoming contaminated

from other sources. Oysters and clams which are

consumed raw are also significantly associated with

gastroenteritis caused by this vibrio. It is possible that

V. parahaemolyticus may cause diarrheal diseases in

developing countries more often than is currently

recognized. In such countries, waterborne infection

with this vibrio should also be considered.

Clinical Infection with

V. parahaemolyticus

0011 Although V. parahaemolyticus is associated with oc-

casional skin and soft-tissue infections as well as with

otitis media in fish handlers and bathers, gastroenter-

itis is the essential clinical manifestation of foodborne

infection. Symptoms usually occur about 12 h after

consumption of contaminated food. The outstanding

features are severe abdominal cramps and diarrhea,

together with nausea, vomiting, and fever. These

symptoms range from mild with a few loose stools

to very severe. In addition, dysentery-like stools with

blood and mucus may occur in some cases. Recovery

of patients and disappearance of the vibrio from

stools are usually complete within a few days. The

mortality rate is very low. Person-to-person transmis-

sion has not been recognized.

0012 Although the size of the infectious dose necessary

to produce clinical symptoms may vary with the strain

and individual, according to the data from human

volunteer experiments it, probably lies between

about 10

and 10

viable cells. However, based on

the low numbers of Kanagawa-positive cells usually

found in samples of the food implicated or in water-

borne infections encountered in developing countries,

it appears that the numbers of viable vibrios needed

to cause illness may be much smaller.

Virulence Factors

0013 It has been found that strains of V. parahaemolyticus

isolated from patients with gastroenteritis were

hemolytic, on a modified brain–heart infusion agar

containing human blood, whereas those from sea fish

and the marine environment were nonhemolytic. This

hemolysis is referred to as the Kanagawa reaction. It

was found that 96% of 2720 strains from patients

with gastroenteritis were Kanagawa-positive com-

pared with only 1% of 650 environmental strains.

Feeding tests with Kanagawa-negative strains in 15

adult volunteers failed to induce any clinical signs of

illness. The hemolysin responsible for the Kanagawa

reaction is known as TDH. Although V. parahaemo-

lyrticus is known to produce at least four hemolytic

substances – TDH, a thermolabile hemolysin, phos-

pholipase A, and lysophospholipase – it is probable

that only TDH plays a significant role in the patho-

genesis of gastroenteritis.

0014Occasional outbreaks of gastroenteritis may be

associated with Kanagawa-negative vibrios. These

strains produce a thermolabile TRH but not the

thermostable TDH.

0015TDH is a 21-kDa protein not affected by heating at

100



C for 15 min at pH 6.0. It is a pore-forming

toxin which expresses hemolytic activity, cytotoxi-

city, and cardiotoxicity. In hemolytic tests, TDH is

strongly active against erythrocytes of dogs, rats,

mice, and humans, weakly active against those of

rabbits and sheep, and inactive against horse erythro-

cytes. Although it is clear that TDH is associated with

the pathogenesis of infection, it is not clear how this

toxin causes diarrhea. TDH, when inoculated at

doses of 100 mg into ligated ileal loops in rabbits,

failed to produce fluid accumulation but caused ero-

sive lesions and necrosis of the intestinal mucosa.

Such histological changes do not occur when whole

bacteria are tested in ileal loops.

0016It has been reported that a TDH-positive parent

vibrio strain caused fluid accumulation in ligated

ileal loops in rabbits, whereas a TDH-negative

mutant did not cause fluid accumulation. Similar

results were obtained with culture supernatants of

TDH-positive strains tested on rabbit ileal tissues

mounted in an Ussing chamber, which represents a

more sensitive measure of secretory activity. In these

assays, the ability of TDH to alter iron transport in

the intestinal tract was demonstrated at nanogram

levels, with no histological changes. TDH induces

intestinal chloride ion secretion and trisialoganglio-

side G

T1b

appears to be the cellular receptor. How-

ever, recent work suggests that other unknown

receptor(s) may be present. TDH uses Ca

2þ

as an

intracellular second messenger, and is thus the first

bacterial enterotoxin for which a linkage between

changes in intracellular calcium and secretory activity

has been established.

0017TDH is encoded by two copies, tdh1 and tdh2,of

the tdh gene in V. parahaemolyticus. The two gene

copies are not identical and the predicted protein

products differ in seven amino acid residues, although

the proteins themselves are immunologically indistin-

guishable. The level of TDH production may be

under the control of a regulator similar to that of

ToxR in V. cholerae.

0018Although TRH and TDH are similar in their

biological, immunological, and physicochemical prop-

erties, TRH is thermolabile and differs from TDH in its

activity on erythrocytes. TRH is encoded by the trh

gene which shares 69% identitiy with the tdh2 gene.

TRH is linked epidemiologically to gastroenteritis, but

the secretory mechanism of this toxin is still uncertain.

5990 VIBRIOS/

Vibrio parahaemolyticus

0019 Several adhesive factors, including fimbriae, peri-

trichous flagella, outer membrane proteins, and a

mannose-resistant, cell-associated hemagglutinin,

have also been proposed, but no substantial studies

have yet implicated any of the candidate adhesins in

the pathogenicity of the organism.

Isolation and Identification

0020 Several selective agar media have been devised, but

thiosulfate citrate bile salts sucrose (TCBS) agar is

recommended for the isolation of V. parahaemolyti-

cus. In clinical microbiology laboratories, MacCon-

key agar containing 0.5% additional NaCl is also

convenient for routine culture of diarrheal stools.

0021 Enrichment culture is used for the detection of the

vibrio from food and marine samples. Polymyxin salt

broth, containing 2% NaCl and 50 mgml

1

of poly-

myxin B (pH 7.6) may be used for the selective

growth of V. parahaemolyticus. It should be noted

that some factor(s) in shellfish may inhibit the growth

of vibrios. It is therefore recommended that shellfish

are cut into small pieces, but not homogenized. After

shaking the enrichment broth vigorously, the pieces of

shellfish are removed with forceps. However, enrich-

ment culture of seafoods incriminated in outbreaks of

infection may be unrewarding, because most yield

Kanagawa-negative isolates, in contrast to those

from patients.

0022 The colonial appearance of V. parahaemolyticus on

TCBS agar is so typical that provisional identification

of the isolates from stool specimens may be made

directly from the plates. However, isolates from

marine sources must be further examined in order to

differentiate them from related organisms. The add-

ition of 1% NaCl to medium for biochemical tests is

essential to obtain valid reactions.

0023 The Kanagawa reaction is a reliable test for the

recognition of virulent strains. For determination,

isolates should if possible be tested using Wagatsuma

agar, which contains 0.5% yeast extract, 1% pep-

tone, 0.5% mannitol, 0.05% K

HPO

, 7% NaCl,

0.0001% crystal violet, 1.5% agar, and washed

human red blood cells. However, as adequate sources

of human blood, or of blood from other suitable ani-

mals, are not always readily available, other suitable

media may be considered. Thus, an enzyme-linked

immunosorbent assay has been described for the

detection of TDH-producing vibrios.

0024 Several molecular approaches for the detection

of Kanagawa-positive vibrios have also been de-

veloped. DNA and oligonucleotide probes specific

for the genes tdh and trh have been described. How-

ever, the probes also hybridize with tdh genes in some

strains of non-O1 V. cholerae, V. hollisae, and

V. mimicus. A polymerase chain reaction technique

has been reported using oligonucleotide primers

derived from the nucleotide sequence of the tdh gene.

0025Serotyping of isolates of V. parahaemolyticus may

be performed by slide agglutination tests using O and

K antisera. In outbreaks of V. parahaemolyticus infec-

tion, however, the same serovar as that identified in

patients is seldom detected in incriminated seafoods.

Serotyping of isolates from seafoods and marine

sources is thus not usually significant unless they are

Kanagawa-positive.

See also: Fish: Spoilage of Seafood; Shellfish:

Contamination and Spoilage of Molluscs and

Crustaceans; Vibrios: Vibrio cholerae

Further Reading

Asakawa Y, Akahane S and Noguchi M (1974) Quantita-

tive studies on pollution with Vibrio parahaemolyticus

during distribution of fish. In: Fujino T et al. (eds) Inter-

national Symposium on Vibrio parahaemolyticus pp.

97–103. Tokyo: Saikon Publishing Co.

Daniels NA, MacKinnon L, Bishop R et al. (2000) Vibrio

parahaemolyticus infections in the United tates, 1973–

1998. J Infect Dis 181: 1661–1666.

Honda T, Ni Y, Yoh M et al. (1989) Production of mono-

clonal antibodies against thermostable direct hemolysin

of Vibrio parahaemolyticus and application of the anti-

bodies for enzyme-linked immunosorbent assay. Med

Microbiol Immunol 178: 245–253.

Kato T, Obara Y, Ichinoe H et al. (1965) Grouping of Vibrio

parahaemolyticus (biotype 1) by hemolytic reaction.

Shokuhin Eisei Kenkyu 15: 83–86 (in Japanese).

Lee C and Pan S-F (1993) Rapid and specific detection of

the thermostable direct hemolysin gene in Vibrio para-

haemolyticus by polymerase chain reaction. J Gen

Microbiol 139: 3225–3231.

Nishibuchi M, Ishibashi M, Takeda Y et al. (1985) Detec-

tion of the thermostable direct hemolysin gene and

related DNA sequences in DNA colony hybrid-ization

test. Infect. Immun 49: 481–486.

Nishibuchi M, Hill WE, Zon G et al. (1986) Synthetic

oligodeoxyribonucleotide probes to detect Kanagawa

phenomenon-positive Vibrio parahaemo-lyticus. J Clin

Microbiol 23: 1091–1093.

Nishibuchi M, Taniguchi T, Misawa T et al. (1989) Cloning

and nucleotide sequence of the gene (trh) encoding the

hemolysin related to the thermostable direct hemolysin

of Vibrio parahaemolyticus. Infect. Immun. 57: 2691–

2697.

Osawa R, Okitsu T, Morozumi H et al. (1996) Occurrence of

urease-positive Vibrio parahaemo-lyticus in Kanagawa,

Japan, with special reference to presence of thermostable

direct hemolysin (TDH) and the TDH-related hemolysin

genes. Appl Environ Microbiol 62: 725–727.

Sakazaki R, Iwanami S and Fukumi H (1963) Studies on

the enteropathogenic, facultatively halophilic bacteria,

VIBRIOS/

Vibrio parahaemolyticus

5991

Vibrio parahaemolyticus I. Morphological, cultural and

biochemical properties and its taxonomical position. Jpn

J Med Sci Biol 16: 161–188.

Sakazaki R, Iwanami S and Tamura K (1968a) Studies on

the enteropathogenic, facultatively halophilic bacteria,

Vibrio parahaemolyticus. II. Serological characteristics.

Jpn J Med Sci Biol 21: 313–324.

Shinoda S, Miwatani T, Honda T et al. (1974) Antigenicity

of flagella of Vibrio parahaemolyticus. In: Fujino T et al.

(ed.) International Symposium on Vibrio parahaemo-

lyticus pp. 193–197. Tokyo: Saikon Publishing Co.,

Tokeo.

Sanyal SC and Sen PC (1974) Human volunteer study on

the pathogenicity of Vibrio parahaemolyticus In: Fujino

T et al. (eds) International Symposium of Vibrio para-

haemo-lyticus pp. 227–235. Tokyo: Saikon Publishing

Co.

Takikawa I (1958) Studies on pathogenic halophilic bac-

teria. Yokohama Med Bull 2: 313–322.

Yamamoto K, Honda T and Miwatani T (1992) Enzyme-

labeled oligonucleotide probes for detection of the genes

for thermostable direct hemolysin (TDH) and TDH-

related hemolysin (TRH) of Vibrio parahaemolyticus.

Canad J Microbiol 38: 410–416.

Vibrio vulnificus

A C Wright, University of Florida, Gainesville, FL, USA

J G Morris, Jr., University of Maryland School of

Medicine, Baltimore, MD, USA

Background

0001 Vibrio vulnificus, first recognized as a distinct species

in the late 1970s, is a Gram-negative bacterium that is

indigenous to the estuarine environment. Virtually all

oysters harvested in the USA during warmer, summer

months harbor this organism, and it has been

reported in association with shellfish and fish in a

number of locations in South America, Asia, and

Europe. For persons in ‘high risk’ groups (i.e., persons

with liver disease, iron overload states, or immuno-

suppression), ingestion of V. vulnificus in raw or

undercooked seafood can result in a syndrome of

‘primary septicemia,’ characterized by intractable

shock and a mortality rate of greater than 50%.

These infections are the leading cause of death asso-

ciated with consumption of shellfish in the USA. The

microorganism can also cause serious wound infec-

tions and may be a cause of gastroenteritis. In popu-

lation-based studies in US coastal areas, the incidence

of V. vulnificus infections is approximately 0.5/

100 000 persons per year. Case series from Korea

and Taiwan suggest that V. vulnificus infections are

even more common in these latter areas.

0002Virulence of V. vulnificus is most closely linked

with the presence of a polysaccharide capsule and is

greatly influenced by availability of iron in the host

during infection. V. vulnificus is also known to pro-

duce a variety of extracellular factors, including a

cytolysin, siderophores, pili, lipopolysaccharide, pro-

teases, and other exoenzymes; however, the role of

these factors in virulence remains unclear. Animal

studies of the lethality of this organism have indicated

that mutants deficient in cytolysin and protease do

not exhibit reduced virulence. Mutants deficient in

siderophores or pili expression are somewhat less

virulent than wild-type strains, but these results are

complicated by the pleiotropic nature of these muta-

tions. However, excess iron in the host and expression

of capsular polysaccharide (CPS) by V. vulnificus

clearly increase the lethality of this disease. For mice

with normal iron status, the LD50 ranges from 10

, depending on the strain and the ability to pro-

duce CPS. The LD50 for unencapsulated mutants in

iron-overloaded mice is also about 10

; however, en-

capsulated strains in iron-overloaded mice may be

lethal at a dose of one bacterium.

0003There is tremendous diversity in capsular types

among strains, and no one type (or group of types)

predominates among clinical or environmental isol-

ates. Three biotypes (or two biotypes and one distinct

serovar) of V. vulnificus are currently recognized. The

majority of clinical and environmental V. vulnificus

isolates reported to date are in biotype 1. Strains

initially classified as biotype 2 are responsible for

sepsis in eels; they do not cause human disease.

More recently, it has been recognized that the biotyp-

ing characteristics described for these strains lacked

specificity; however, the eel pathogens are homoge-

neous in their lipopolysaccharide-based serogroup,

leading to their reclassification as serovar E. Biotype

3 strains have been described in association with

wound infections related to handling of live fish

(tilapia) from fish farms in Israel.

Occurrence in the Environment

0004As noted above, V. vulnificus is commonly isolated

from water and organisms, particularly shellfish, col-

lected from estuarine environments. The highest

numbers are found in areas with intermediate sali-

nities (5–25 p.p.t.) and warmer temperatures (opti-

mally, > 20



C). Isolation has been reported from

the US Atlantic, Gulf, and Pacific coasts, as well

as the Atlantic coast of Europe, the Mediterranean,

the Indian Ocean, Malaysia, and the Pacific Coast

of Asia. It is likely that the organism is present in

5992 VIBRIOS/

Vibrio vulnificus

virtually all estuarine areas with appropriate salinity

and temperature ranges. Using a DNA probe, V. vul-

nificus has been identified in 80% of Chesapeake Bay

water samples collected during months in which

water temperatures exceeded 8



C: concentrations

ranged from 3.0 10

to 2.1 10

CFU

1

, rep-

resenting approximately 8% of the total culturable

heterotrophic bacteria. However, none of the samples

collected during February and March, when tem-

peratures were less than 8



C, were positive for this

organism.

0005 V. vulnificus is generally not recovered from the

environment during colder months, and several lines

of evidence suggest that it may enter a viable but

nonculturable state, where recovery on typical solid

media is not possible. Other studies have suggested

that the majority of V. vulnificus cells die off with the

stress of cold temperature or starvation, and only a

small resistant subpopulation remains viable. How-

ever, both experimental and epidemiological data

strongly indicate that nonrecoverable forms probably

are not virulent, as little or no disease is observed in

colder months. Thus, the relevance of this issue to the

seafood industry may be negligible. However, further

study may shed light on how the species is maintained

in the environment and help elucidate survival mech-

anisms that could be targets for control measures.

Entry to the Food Chain

0006 Epidemiologically, there is a very strong association

between V. vulnificus infection and eating raw

oysters, particularly those harvested from warmer

waters. It should be noted that most, if not all, serious

disease is associated with the raw, freshly shucked

product; proper cooking eliminates the disease risk.

In Chesapeake Bay studies, V. vulnificus was found in

virtually all oysters collected during warmer months

(water temperature 7.6



C or above), with the

numbers of this species approximately two orders of

magnitude above those found in the surrounding

water. In studies conducted by the US Food and

Drug Administration, V. vulnificus was detected in

virtually all oysters from the US Gulf Coast. The

numbers varied based on temperature and salinity;

representative data from this study are shown in

Figure 1. The prevalence of this organism in the en-

vironment correlates well with disease incidence, as

approximately 90% of the infections occur between

April and October.

0007 Unlike other pathogens associated with seafood

consumption, several studies have shown no correl-

ation between the presence of V. vulnificus and

fecal coliforms. V. vulnificus is clearly an indigenous

estuarine species with an environmental reservoir;

however, many aspects of this reservoir are still un-

clear. For example, does this organism exist and rep-

licate as a free-living species or in association with

algae, shellfish, or fish? While it may be assumed that

concentrations of V. vulnificus in shellfish, in part,

reflect filtering of the organism from surrounding

water, there are data suggesting that V. vulnificus

can assume a symbiotic relationship with oysters. At

a practical level, this relationship implies specific at-

tachment or internalization, which may account for

the difficulties in clearing the organism from oysters

by using traditional depuration techniques.

0008The condition and overall health of the oyster may

also influence the numbers of V. vulnificus in oyster

tissue. Numerous variables such as heavy metal con-

centrations, water turbidity, availability and types of

food sources, spawning status, etc. may influence the

ability of oysters to clear or control endogenous bac-

terial populations. Bacterial growth also may be

enhanced by coinfection with parasites such as Per-

kinsus marinus, the presence of which may depress

innate defense mechanisms.

Fate on Processing/Storage and Control

Measures

0009V. vulnificus can persist in oyster shellstock for

extended periods of time. In oysters held at 18



for 30 h, or at ambient temperatures for 12 h, there

are significant increases in V. vulnificus counts,

highlighting the importance of refrigeration after

harvest and during storage and transport. Currently

in the USA, Hazard Analysis Critical Control Point

0.1

1994 1995Months

..A....S...O...N....D...J....F...M...A...M....J...J...A...S....

100

V. vulnificus (MPN per g)

1000

10 000

100 000

fig0001Figure 1 Weekly densities of V. vulnificus in oysters from Gulf

Coast sites in Alabama (+), Florida (), and Louisiana (

) during

a 14-month period. Each point represents the geometric mean

(n ¼2) of the MPN of bacteria per gram of oyster meat. From

Motes ML, DePaola A, Cook DW et al. (1998) Influence of water

temperature and salinity on Vibrio vulnificus in northern Gulf and

Atlantic Coast oysters. Applied and Environmental Microbiology 64:

1459–1465, with permission.

VIBRIOS/

Vibrio vulnificus

5993

recommendations are being implemented that pro-

vide more rapid postharvest refrigeration of shellfish

to limit increases in the numbers of V. vulnificus

resulting from temperature abuse.

0010 A variety of approaches have been proposed to

reduce or eliminate V. vulnificus from oyster shell-

stock. Traditionally, depuration or the incubation of

oysters in recirculating, sanitized fresh water has been

used to reduce bacterial contamination of fecal origin

in shellstock; however, several studies found this treat-

ment to be ineffective for V. vulnificus control. In one

study, V. vulnificus was cleared from the digestive

organs but persisted in other tissues following depur-

ation treatment. Recently, alternative treatments such

as freezing, cold or hot water immersion, relaying to

high salinity waters, high pressure, and irradiation

have shown promising results for the reduction and

possible elimination of this species from oysters.

Unfortunately, many of these treatments are lethal to

oysters as well, and their application to the ‘oyster on

the half shell’ market may be limited. Some suppliers

are currently using these methods to produce a prod-

uct that greatly resembles a freshly shucked oyster but

will not be sold as live product.

Detection in Food, Water, and

Physiological Samples

0011 V. vulnificus has traditionally been evaluated in food

and water samples using most probable number

(MPN) calculations and standard microbiologic tech-

niques. In this procedure, oysters are homogenized in

buffer and serially diluted to MPN enrichment tubes,

which are subsequently screened for vibrios by

growth on selective/differential media. Unfortunately,

owing to the diversity and abundance of vibrios in the

environment, this method lacks reliability, and fur-

ther confirmatory methods such as species-specific

monoclonal antibody are generally required for

identification.

0012 More recently, there has been increasing use of

molecular approaches, including DNA probes and

PCR techniques. Several of the probes and PCR

primers are based upon the DNA sequence for the

V. vulnificus cytolysin gene, vvh. This gene appears to

be common to most, if not all, strains and is highly

species-specific. Applications include a colony hy-

bridization protocol that can be combined with total

viable counts on nonselective medium to provide

more direct enumeration. Multiplex PCR analyses

have also been described that simultaneously detect

V. vulnificus and other Vibrio spp. Unfortunately,

these probes, as well as the standard MPN methods

described, do not discriminate between potentially

virulent and avirulent strains.

0013For patient samples, V. vulnificus will grow with-

out difficulty in standard blood culture media or on

nonselective media (such as blood agar) routinely

used for wound cultures; identification and speciation

of the organism are possible through any of the stand-

ard, commercially available microbiology identifica-

tion systems. As with all Vibrio species, isolation of

the organism from stool generally requires the use of

a specific selective culture media (thiosulfate citrate

bile-salts sucrose).

Symptoms and Characteristics

0014V. vulnificus has been linked with three different clin-

ical syndromes: (1) ‘primary septicemia’ (presence of

the organism in the blood, without an obvious source

such as a wound infection), (2) wound infections,

and, possibly, (3) gastroenteritis. Serious V. vulnificus

infections occur almost exclusively in persons who

have underlying liver disease (including alcoholic

liver disease), are immunosuppressed, or have chronic

diseases such as diabetes or renal failure. Risk of

infection is also substantively increased in persons

with increased iron stores, as found in patients with

hemochromatosis.

Primary Septicemia

0015Approximately one-third of patients with primary

V. vulnificus septicemia present in shock or become

hypotensive within 12 h of hospital admission. Three-

fourths of patients have distinctive bullous skin

lesions. Thrombocytopenia is common, and there is

often evidence of disseminated intravascular coagula-

tion. Complications such as gastrointestinal bleeding

are not infrequent. The mortality rate for persons

with primary V. vulnificus septicemia is more than

50% overall and more than 90% in persons who are

hypotensive. Although the actual infection may be

cleared rapidly, patients often require prolonged

hospitalization in intensive care units because of the

associated shock syndrome and resultant multiorgan

system failure.

Wound Infections

0016V. vulnificus may contaminate wounds exposed to

estuarine waters or shellfish. In persons in the risk

groups noted above, the infection may spread rapidly,

producing severe myositis and fasciitis reminiscent of

gas gangrene and requiring amputation in extreme

cases.

Gastroenteritis

0017V. vulnificus has also been associated with gastro-

enteritis. However, an etiologic role is difficult to

5994 VIBRIOS/

Vibrio vulnificus

establish, as isolation from stool samples is of uncer-

tain significance, given the ubiquitous presence of the

organism in both water and shellfish.

Treatment and Prevention

0018 In V. vulnificus septicemia, the sooner antibiotic ther-

apy is initiated, the greater the chance that the patient

will survive. Based upon clinical observations and

in vitro susceptibilities, tetracycline and quinolone

antibiotics have been recommended for management

of V. vulnificus infections. Recent in vitro and animal

studies from Taiwan indicate that there is synergism

between minocycline and cefotaxime in treatment of

serious V. vulnificus infections, leading to the recom-

mendation that these latter two drugs be used as ‘first

line’ therapy. Patients with serious infections need to

be aggressively managed in an intensive care unit

setting to minimize the possible consequences of

hypotension, septic shock, and multiorgan system

failure.

0019 Given the high mortality associated with V. vulni-

ficus infections, persons in the high-risk groups noted

above should avoid eating raw or undercooked shell-

fish, particularly oysters. By law, restaurants in many

states are now required to post warnings about this

risk. Persons at high risk for infection should also

avoid situations in which estuarine-associated

wounds are likely to occur.

See also: Fish: Spoilage of Seafood; Shellfish:

Contamination and Spoilage of Molluscs and Crustaceans

Further Reading

Bisharat N, Agmon V, Finkelstein R et al. (1999) Clinical,

epidemiological, and microbiological features of Vibrio

vulnificus biogroup 3 causing outbreaks of wound infec-

tion and bacteremia in Israel. Lancet 354: 1421–1424.

Blake PA, Merson MH, Weaver RE, Hollis DG and

Heublein PC (1979) Disease caused by a marine Vibrio:

clinical characteristics and epidemiology. New England

Journal of Medicine 300: 1–5.

Brasher CS, Depaola A, Jones DD and Bej AK (1998)

Detection of microbial pathogens in shellfish with multi-

plex PCR. Current Microbiology 37: 101–107.

Bush CA, Patel P, Gunawardena S et al. (1997) Classifica-

tion of Vibrio vulnificus strains by the carbohydrate

composition of their capsular polysaccharides. Analyt-

ical Biochemistry 250: 186–195.

Chuang YC, Ko WC, Wang ST et al. (1998) Minocycline

and cefotaxime in the treatment of experimental murine

Vibrio vulnificus infection. Antimicrobial Agents and

Chemotherapy 42: 1319–1322.

Cook DW (1997) Refrigeration of oyster shellstock: condi-

tions which minimize the outgrowth of Vibrio vulnifi-

cus. Journal of Food Protection 60: 349–352.

Dixon DW and Rodrick GE (1998) Effect of gamma radi-

ation on shellstock oysters: extension of shelf-life and

reduction in bacterial numbers, with particular reference

to Vibrio vulnificus. In: Combination Processes for Food

Irradiation, pp. 97–110. Vienna: IAEA.

Elliot EL, Kaysner CA, Jackson L and Tamplin ML (1998)

Vibrio cholerae, V. parahaemolyticus, V. vulnificus and

other Vibrio spp. In: U.S. Food and Drug Administra-

tion Bacteriological Analytical Manual, 8th edn.

Gaithersburg, MD: AOAC International.

ISSC (2000) Gulf Oyster Industry Council, FL. Dept of

Agriculture, and FDA Office of Seafood Processing con-

trols for vibrios in raw oysters. Online abstracts of the

25th Annual Meeting of Seafood Science and Technol-

ogy Society, Longboat Key, FL http://www.seafood.uc-

davis.edu/organize/SSTSA.htm

Motes ML and DePaola A (1996) Offshore suspension

relaying to reduce levels of Vibrio vulnificus in oysters

(Crassostrea virginica). Applied and Environmental

Microbiology 62: 3875–3877.

Motes ML, DePaola A, Cook DW et al. (1998) Influence of

water temperature and salinity on Vibrio vulnificus in

northern Gulf and Atlantic Coast oysters. Applied and

Environmental Microbiology 64: 1459–1465.

Park SD, Shon HS and Joh NJ (1991) Vibrio vulnificus

septicemia in Korea: Clincal and epidemiologic findings

in seventy patients. Journal of the American Academy of

Dermatology 24: 397–403.

Strom MS and Paranjpye RN (2000) Epidemiology and

pathogenesis of Vibrio vulnificus. Microbes and Infec-

tion 2: 177–188.

Tamplin ML and Capers GM (1991) Persistence of Vibrio

vulnificus in tissues of Gulf coast oysters (Crassostrea

virginica) exposed to seawater disinfected with UV

light. Applied and Environmental Microbiology 58:

1506–1510.

Wright AC, Hill RT, Johnson JA, Roghman M-C, Colwell

RR and Morris JG Jr. (1996) Distribution of Vibrio

vulnificus in the Chesapeake Bay. Applied and Environ-

mental Microbiology 62: 717–724.

VIBRIOS/

Vibrio vulnificus

5995

VINEGAR

M Plessi, Dipartimento di Scienze Farmaceutiche,

Universita

degli Studi di Modena, Modena, Italy

Summary and Background

0001 Vinegar is traditionally the product of the acetous

fermentation of dilute alcoholic solutions. At the pre-

sent time it is produced microbiologically from nat-

ural alcoholic solutions or by dilution of acetic acid.

Vinegar and its characterization are presented in this

article.

0002 Vinegar has been known since ancient times, for

more than 10 000 years, and has been used for its

typically acid flavor. It uses were many and various

for centuries, not only for food but also for medicinal

and ritual uses. It is traditionally the product of the

acetic fermentation of dilute alcoholic liquors and

it was wine that was the first alcoholic liquid used,

thus the derivation of its name (from the French vin

aigre ¼ sour wine).

History

0003 During Babylonian times vinegar was used as a con-

diment and for food preservation. Hippocrates recog-

nized its medicinal properties and it is also mentioned

in the Bible as a remedy on account of its sedative

and curative properties. Pliny reported that vinegar,

diluted with water (known as posca), was given to

the Roman legionnaires during their long marches.

0004 The medicinal use of vinegar was widespread

during the Middle Ages and the Renaissance, for

both internal and topical use. It was in fact used as a

digestive, a prophylactic against liver disorders, an

anthelmintic, for sore throats, and to rub on the

wrists against fever, but also against hair loss and

tinea.

0005 Vinegar production as an industry began during the

period of the Communes; those who swore not to

reveal the secret of its fabrication were admitted to

the corporation of manufacturers. Although widely

used, little was known about the real nature of vin-

egar and its fermentation until the eighteenth century.

In 1723, Stahl obtained acetic acid from vinegar, but

nothing was known of the causes which determined

the formation of acetic acid. The first breakthrough

came with Berzelius, who, at the beginning of the

nineteenth century, explained how acetic acid was

formed from alcohol by a process of oxidation.

Persoon (1822) and Ku

tzing (1837) were the first

to suggest the presence of microorganisms in the

process. Finally, in 1864, Pasteur brilliantly demon-

strated the biological origin of the acetic fermenta-

tion, indicating Mycoderma aceti as the fermentation

agent. Later studies clarified the nature of the micro-

organisms, which turned out to belong to several

species.

0006Vinegar is now used above all to give foodstuffs a

pleasant acidic flavor; it is also used as a preserver of

foodstuffs and is recognized to have certain pharma-

cological qualities.

Production

0007At the present time, vinegar is still produced micro-

biologically from a mostly natural alcoholic solution,

but can also be produced by dilution of acetic acid.

Microbiological Production

0008Vinegar is the product of the acetic fermentation

of slightly alcoholic liquids (less than 10–12% by

volume of ethyl alcohol); transformation of alcoholic

liquids into vinegar is not really fermentation, but

oxidation.

0009Raw materials may be wine, cider, beer, and other

liquors deriving from the alcoholic fermentation of

cereals, fruit and potatoes, sugar solutions, such as

molasses, honey, and whey, and also diluted pure

ethanol with the addition of nutrients. These are the

so-called brewed vinegars, derived from the oxidation

activity of aerobic microorganisms. The microorgan-

isms belong to the genus Acetobacter (initially called

Mycoderma), and the most widespread species are A.

aceti, A. pasteurianus,andA. hansenii. These are the

ones that oxidize ethyl alcohol into acetic acid. The

oxygen used for the bioxidation is that of air.

0010These microorganisms, in the shape of aspori-

genous rods or cocci, form membranes of different

consistencies (vinegar mother). The optimum tem-

perature for their multiplication is between 18 and



C, depending on the species.

0011Ethanol is dehydrogenated to acetic acid and the

reduced cosubstrates are oxidized via the respiratory

chain (Figure 1). Similarly, but less abundantly and in

an anaerobic atmosphere, acetic acid is formed by

dismutation of 2 molecules of acetaldehyde (derived

in their turn from alcohol by oxidation). In theory, 1 g

of alcohol yields 1.3 g of acetic acid; in practice,

however, the yield is 15–20%, lower mainly because

alcohol, acetaldehyde, and acetic acid tend to volatil-

ize. The theoretical amount of air required for 1 l of

vinegar containing 6% of acetic acid is about 120 l,

5996 VINEGAR

whereas, in practice, given the slow rate of liquid–gas

exchange, the amount required is much greater.

0012 Acetous fermentation is accompanied by secondary

fermentation which combines to produce the flavor

and typical aroma; small quantities of volatile sub-

stances are formed (e.g., ethane, acetaldehyde, ethyl

formate, ethyl acetate, isopentyl acetate, butanol,

methylbutanol, 3-hydroxi-2 butanone or acetyl-

methylcarbinol), which vary from vinegar to vinegar

depending on the starting material and which,

because of their individual characteristics, produce

vinegars with a variety of odor, taste, color, and

other properties. The fermentation is usually stopped

at a minimum residual ethanol level to avoid

overoxidation, as oxidation of acetic acid to

water and CO

.(See Fermented Foods: Origins and

Applications.)

0013 Methods of manufacture The manufacture of

vinegar is an ancient craft, known and practiced all

over the world from time immemorial. A certain

amount of vinegar is still manufactured following

the centuries-old empirical methods of the small

producer, but since the last century a flourishing

vinegar-manufacturing industry has developed.

0014Industrial vinegar-manufacturing processes fall

into three main categories. The slow processes,

which are the oldest commercial procedure and re-

semble home-brewing techniques, are no longer used.

The Orleans process is still in use for the production

of high-quality vinegar; the procedure has remained

unchanged through the years but is very slow and

requires a great deal of space (Figure 2). The starting

liquor is placed in a large cask, containing wood

shavings or grape stalks, where the acetification pro-

cess gets underway. After about 8 days, the liquid is

withdrawn and transferred into barrels, which are left

only one-half or two-thirds full and where the liquid

remains until acidity reaches its peak (about 3

months). From now on, two-thirds or three-quarters

of the vinegar is withdrawn from the bottom of the

barrel every week and an equal volume of liquid from

the generator cask is added from the top. (See Barrels:

Wines, Spirits, and Other Beverages.)

0015The processes of the second and third categories

aim at a closer contact, with maximum possible

CHOOH

Alcohol-DH

NAD(P) NAD(P)H + H

Aldehyde-DH

COOH

NAD(P)NAD(P) + H

Respiratory chain

2 H

O + 6 ATP

fig0001 Figure 1 Ethanol oxidation to acetic acid by Acetobacter spp.

fig0002 Figure 2 Vinegar making Orleans method: A, starting vat; B, acetifier casks; C, vats of clarification.

VINEGAR 5997