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

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See also: Escherichia coli: Occurrence; Occurrence and

Epidemiology of Species other than Escherichia coli;

Immunoassays: Radioimmunoassay and Enzyme

Immunoassay

Further Reading

Banwart GJ (1989) Basic Food Microbiology, 2nd edn.

New York: Van Nostrand Reinhold.

Doyle MP and Padhye VV (1989) In: Doyle MP (ed.) Food-

borne Bacterial Pathogens, pp. 235–281. New York:

Marcel Dekker.

Hill WE (1996) The polymerase chain reaction: applica-

tions for the detection of foodborne pathogens. Critical

Reviews in Food Science and Nutrition 36: 123–173.

Hill WE, Ferreira JL, Payne WL and Jones VM (1985)

Probability of recovering pathogenic Escherichia coli

from foods. Applied and Environmental Microbiology

49: 1374–1378.

International Commission on Microbiological Specifica-

tions for Foods (1988) Microorganisms in Foods. 1.

Their Significance and Methods of Enumeration, 2nd

edn. Toronto: University of Toronto Press.

Jay JM (2000) Modern Food Microbiology, 6th edn.

Guithersburg, MD: Aspen.

Mehiman IJ (1984) In: Speck ML (ed.) Compendium of

Methods for the Microbiological Examination of

Foods, 2nd edn, pp. 265–285. Washington, DC: Ameri-

can Public Health Association.

Meng J, Doyle MP, Zhao T and Zhao S (2001) Escherichia

coli O157:H7 (1997). In Doyle MP, Beuchat LR and

Montville TJ (eds) Food Microbiology: Fundamentals

and Frontiers, 2nd edn. pp. 193–213. Washington,

DC:ASM Press.

Nataro JP and Kaper JB (1998) Diarrheagenic Escherichia

coli. Clinical Microbiology Reviews 11: 142–201.

Padhye NV and Doyle MP (1992) Escherichia coli O157:

H7: Epidemiology, pathogenesis, and methods for detec-

tion in food. Journal of Food Protection 55: 555–565.

Food Poisoning

K S Venkitanarayanan, University of Connecticut,

Storrs, CT, USA

M P Doyle, University of Georgia, Griffin, GA, USA

Introduction

0001 Pathogenic strains of E. coli cause more types of

infections in humans than any other Gram-negative

bacterium. They are a frequent cause of urinary tract

infections and meningitis, and also have been associ-

ated with wound infections and pneumonia. As early

as 1923, E. coli was suspected to be associated with

infantile diarrhea. The first documented outbreak of

foodborne E. coli infection in the United States oc-

curred in 1971. The implicated food was imported

French Camembert and Brie cheeses. E. coli strains

that cause gastrointestinal disease have been classified

into five groups: the enterotoxigenic E. coli (ETEC),

the enteroinvasive E. coli (EIEC), the enteropatho-

genic E. coli (EPEC), the enterohemorrhagic E. coli

(EHEC), and the enteroaggregative E. coli (EAEC).

Each of the five groups of gastrointestinal pathogenic

E. coli consists of certain serotypes that are com-

monly associated with that group (Table 1).

Enterotoxigenic

E. coli

(ETEC)

0002ETEC produce at least one of two types of enterotox-

ins, namely heat-stable enterotoxin (ST) or heat-labile

enterotoxin (LT). Originally, ETEC were recognized

as the etiological agents of diarrhea in piglets. Subse-

quently, ETEC were isolated from diarrheal stools of

children and adults. ETEC cause a watery diarrhea

similar to that produced by Vibrio cholerae, although

usually not as severe in intensity. Diarrhea is usually

accompanied by a low-grade fever, abdominal

cramps, malaise and nausea. The mechanism of

ETEC infection is similar to that of Vibrio cholerae.

Following ingestion, ETEC cells using fimbriae attach

to epithelial cells lining the ileum. The fimbriae are

proteinaceous filaments that specifically recognize

carbohydrate receptors on the intestinal cells. Adher-

ence allows the bacteria to resist removal by the

peristaltic movement of intestinal contents.

0003After adhering to the intestinal wall, ETEC pro-

duce one or more toxins (i.e., LT and ST) that induce a

diarrheal response. LT is a heat-labile protein with a

native molecular weight of about 86 000. LT consists

of an enzymatically active 28-kDa A subunit and five

identical 11.5-kDa subunits B. The A subunit, con-

sisting of A

and A

peptides linked by a disulfide

bond, is responsible for the enzymatic activity of the

toxin. The B units are arranged in a ring form, and

bind strongly to the ganglioside GM

and weakly to

the ganglioside GD

. Following binding to intestinal

mucosal cells, LT is endocysted and translocated

through the cell by a process involving trans-golgi

vesicular transport. The A

peptide transfers ADP-

ribosyl moiety from NAD to the a-subunit of the

GTP-binding protein, G

, which in turn stimulates

adenyl cyclase activity. LT ADP ribosylates the ade-

nylate cyclase regulatory subunit, causing the accu-

mulation of cyclic AMP (cAMP) in the intestinal cell.

This leads to an increased secretion by crypt cells and

decreased absorption by villous tip cells. There are

two distinct classes of LT: LT-I is immunologically

related to cholera toxin; LT-II is similar to cholera

ESCHERICHIA COLI

/Food Poisoning 2157

toxin in function but is not serologically cross-

neutralizable. LT-I is produced by E. coli strains that

are pathogenic for both humans and animals,

whereas LT-II is mainly produced by strains of animal

origin. Two antigenic variants of LT-II, namely LT-IIa

and LT-IIb have been reported. Similar to LT-I, LT-II

also increases intracellular cAMP level; however, the

receptor for LT-II is GD

, instead of GM

0004 ST is a heat-stable protein that is much smaller than

LT, with a molecular weight of less than 5000. There

are two forms of ST, i.e., STa and STb, but only STa is

involved in human infection. STb-producing strains

are usually of animal origin and do not infect

humans. STa consists of 18–19 amino acids with a

molecular weight of 2 kDa. Two types of STa have

been reported, which include STp (ST porcine or

STIa) and STh (ST human or STIb). Each of these

two variants contains 13 identical amino acids. STa is

produced from its precursor containing 72 amino

acids, as a result of cleavage by a signal peptidase.

The primary receptor of STa is guanyl cyclase C

(GC-C). Stimulation of GC-C occurs once STa binds

to it, resulting in the production of increased levels

of intracellular cyclic guanosine monophosphate

(cGMP). This ultimately results in the inhibition of

sodium chloride transport and increased secretion of

water from intestinal cells.

0005 Both LT-I and ST are plasmid-encoded, whereas

LT-II is chromosomally encoded. The large plasmids,

approximately 50–70 MDa in size, that encode LT-I

and ST usually encode the fimbriae used for adher-

ence as well. ST genes are located inside a transposon-

like element. The presence of these genes on plasmids

has contributed to their spread throughout homo-

conjugative strains.

0006 In developed countries with good hygiene, ETEC is

not an important cause of disease. Patients typically

have a mild, uncomplicated, watery diarrhea that

requires only symptomatic treatment. In these coun-

tries, ETEC is most often associated with ‘traveler’s

diarrhea’, contracted while travelling in another

country with lower hygienic standards. Occasionally,

when food or water becomes contaminated, extensive

outbreaks of ETEC diarrhea can occur. In 1975, over

2000 staff and visitors at a national park in the USA

developed an infection after the park’s water supply

became contaminated with an ETEC strain that pro-

duced both LT and ST. Outbreaks of ETEC have been

mainly associated with drinking contaminated water,

but examples of foods that have been implicated in

outbreaks include crab meat, curried turkey mayon-

naise, raw vegetable salad, and Brie and Camembert

cheeses. A recent study published by the Centers for

Disease Control and Prevention, USA, reported an

estimated 79 000 cases of ETEC diarrhea in the United

States, with about 70% of the cases foodborne.

0007In developing countries, ETEC can be a major

cause of dehydration and death, particularly among

children under 5 years of age. This higher incidence of

ETEC infection with higher mortality rates is associ-

ated with lower hygienic standards and poor nutri-

tion. In many countries, children are believed to have

two to three ETEC infections per year during the first

3 years of life. The disease caused by ETEC in these

situations can be severe, and is often indistinguishable

from cholera.

Enteroinvasive

E. coli

(EIEC)

0008EIEC strains are biochemically, genetically, and

pathogenically related to Shigella spp. Similar to Shi-

gella spp., EIEC are nonmotile, lactose negative, and

lysine decarboxylase-negative. Further, serologically,

EIEC and Shigella spp. demonstrate numerous cross-

reactions. The mechanisms by which they cause

disease are also very similar.

0009The pathogenesis of EIEC involves cellular inva-

sion and spread, which are mediated by chromosomal

and plasmid-borne virulence genes. Like Shigella

spp., EIEC strains infect the colon by invading epithe-

lial cells, a phenotype mediated by both plasmid and

chromosomal loci. Moreover, EIEC elaborate endo-

toxins, which are believed to play a role in the devel-

opment of diarrhea. The events that occur in the

tbl0001 Table 1 Characteristics of groups of gastrointestinal E. c oli pathogens

Group Virulence factors Serogroups

ETEC Enterotoxins (LT, ST) O6, O8, O15, O20, O25, O27, O63, O78, O80, O85, O115, O218ac, O139, O148, O153, O167

Fimbriae

EIEC Colonic invasiveness O28ac, O29, O124, O136, O143, O144, O152, O164, O167

EPEC Enteroadhesiveness factor (?) O18ab, O18ac, O26, O44, O86, O111, O114, O119, O125, O126, O127, O128ab, O142, O158

EHEC Shiga toxins O26, O111, O113, O121, O145, O157

Locus of enterocyte effacement

Fimbriae (?)

EAEC Fimbriae (?) O3, O4, O6, O7, O17, O44, O51, O68, O73, O75, O77, O78, O85, O111, O127, O142, O162

ETEC, enterotoxigenic E. coli; EPEC, enteropathogenic E. c oli; EIEC, enteroinvasive E. coli; EHEC, enterohemorrhagic E. c oli ; EAEC, enteroaggregative

E. coli .

2158

ESCHERICHIA COLI

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pathogenesis of EIEC involve epithelial cell penetra-

tion, lysis of the endocytic vacuole, intracellular

multiplication, movement through the cytoplasm,

and extension into adjacent epithelial cells. Severe

infections result in a strong inflammatory reaction

leading to ulceration of the mucosa. The resulting

symptoms are typical for bacillary dysentery: fever

and diarrhea, with some blood and mucus in the

stools. Some patients will also demonstrate chills,

headache, muscle pain, and abdominal cramps.

0010 The genes mediating invasion are carried on a 140-

MDa plasmid. Another plasmid-borne gene (desig-

nated as sen) in EIEC, which codes for a novel

63-kDa protein (enterotoxin) has been cloned and

sequenced. A mutation in the sen gene has been

reported to cause a significant loss in the enterotoxic

activity of the EIEC. Although the role of entero-

toxins in EIEC pathogenesis is unclear, toxins are

believed to play a critical role in the development of

watery diarrhea.

0011 EIEC strains have caused outbreaks of disease

world-wide, with most reported outbreaks taking

place in countries with high hygienic standards, such

as the USA, the UK, France, and Australia. Despite

these outbreaks, EIEC is not a common cause of

gastrointestinal disease. Outbreaks have resulted

from the consumption of contaminated Brie and

Camembert cheese and potato salad, but water

remains the most commonly implicated vehicle of

infection.

Enteropathogenic

E. coli

(EPEC)

0012 The EPEC group has historically been a collection of

serogroups associated with infantile and neonatal

diarrhea. Until the mechanism by which EPEC cause

disease was elucidated, the group was defined princi-

pally on the basis of O and H serotypes. However,

with a better understanding of the mechanism of

EPEC’s pathogenicity, this group of pathogens is cur-

rently defined on the basis of pathogenesis character-

istics. EPEC strains do not produce LT or ST

enterotoxins that are associated with ETEC. They

are also noninvasive when evaluated by the Sereny

test, but some strains are capable of invading tissue

culture cells. Some strains do produce Shiga toxin,

but these strains have since been reclassified as Shiga

toxin-producing E. coli (STEC).

0013 EPEC are capable of producing attaching and

effacing lesions on host cells. EPEC strains harbor a

characteristic large plasmid that confers adherence on

the surface of cells. A three-stage model has been

proposed in the pathogenesis of EPEC. The first

stage involves localized adherence of EPEC to host

cells, which is mediated by a type IV fimbria known

as bundle-forming pilus (BFP) encoded by fourteen

bfp genes present on the large plasmid. Studies have

shown that mutations in bfp genes prevent expression

of BFP, resulting in a failure of localized adherence of

EPEC to cells. The second stage involves infliction

of attaching and effacing lesions on cells, which are

mediated by a 35 637-bp pathogenicity island known

as the locus of enterocyte effacement (LEE). The

attaching and effacing lesions consist of localized

destruction of microvilli and the formation of a cup-

like pedestal composed of cytoskeletal proteins at the

site of EPEC binding to host cells. In the third stage,

EPEC attaches intimately to host cells via a receptor

followed by cellular alterations in the cytoskeleton.

The eae gene located in the locus of enterocyte efface-

ment encodes an outermembrane protein intimin,

which confers strong attachment of EPEC to cells.

Studies of mutations induced in the eae gene have

revealed that EPEC mutants not only lack the ability

to attach intimately to host cells, but also have a

diminished ability to cause diarrhea in experimental

human infections.

0014EPEC infections are most commonly associated

with infantile diarrhea, which can last for as long as

14 days. Other symptoms can include fever, vomiting,

and abdominal pain. When adults are affected by

EPEC, symptoms are usually diarrhea, nausea, ab-

dominal cramps, headache, and chills. EPEC infantile

diarrhea most commonly occurs in tropical countries

with poor hygiene. In Brazil, EPEC is the most

common cause of diarrhea in children under 6

months of age. In temperate countries, EPEC are not

an important cause of diarrhea, although occasional

outbreaks do occur. Foods implicated in outbreaks

include pork, meat pies and water.

Enterohemorrhagic

E. coli

(EHEC)

0015The observation that some strains of E. coli produce

toxins that kill Vero (African green monkey kidney)

tissue culture cells by Shiga toxin provides a basis for

the STEC group of E. coli. Shiga toxins (also known

as Vero toxins) inhibit protein synthesis by cleaving

rRNA at a specific nucleotide, in a manner identical

to the action of Shiga toxin produced by Shigella

dysenteriae. Two major Shiga toxins have been

characterized. Shiga toxin I (Stx1, VT1) is structur-

ally practically identical to Shiga toxin, and can be

neutralized by antiserum raised against Shiga toxin.

Shiga toxin II (Stx2, VT2) is structurally similar to

Stx1, having at least 74% nucleotide homology with

Stx1, but cannot be neutralized by antiserum raised

against Shiga toxin or Stx1.

0016The STEC group as a whole has not been associated

with foodborne illnesses. However, a subdivision of

ESCHERICHIA COLI

/Food Poisoning 2159

the STEC, known as enterohemorrhagic E. coli

(EHEC), has been associated with outbreaks and spor-

adic cases of hemorrhagic colitis following the con-

sumption of contaminated foods. EHEC is a more

prevalently recognized cause of illness in developed

countries than developing countries. Interestingly, a

principal reservoir of EHEC is cattle and possibly

other ruminants, whereas humans are the principal

reservoir of EIEC and ETEC strains involved in

human illness. For this reason, EHEC infections in

humans are more often associated with foods of

animal origin, or those contaminated with feces of

animals than the other types of pathogenic E. coli.

Additionally, the illnesses caused by EHEC strains are

far more likely to lead to death or long-term disabil-

ities than those caused by other E. coli gastrointest-

inal pathogens.

0017 The two major virulence factors in the patho-

genesis of EHEC infections include the production

of Shiga toxins, and attaching and effacing lesions

which are similar to those incurred by EPEC. The

Shiga toxins are composed of a single catalytic sub-

unit A, and five receptor-binding B subunits. The B

subunits form a pentamer with a center pore through

which the carboxyl terminus of the A subunits

extends. The B pentamer of Shiga toxins binds to a

specific glycolipid receptor known as globotiosyl cer-

amide (Gb

), which is present on the cell surface. This

is followed by entry of the A subunit into the cell by

endocytosis and transport into the Golgi apparatus,

and then to the endoplasmic reticulum. The A subunit

translocates into the cytoplasm, where it catalyses the

N-glycosidation of adenine 4324 of 28S ribosomal

RNA, leading to a failure of peptide chain elongation

and inhibition of protein synthesis. The block in pro-

tein synthesis results in the death of renal endothelial

cells and intestinal epithelial cells, or any cells con-

taining Gb

receptor. Both Stx1 and Stx2 are encoded

by a bacteriophage related to the lambda phage.

0018 Similar to EPEC, EHEC possess the entire LEE,

encoded by a 35-kb pathogenicity island. Although

the classical attaching and effacing histopathology

has been observed in gnotobiotic piglets, infant

rabbits, and cultured epithelial cells infected with

E. coli O157:H7, such lesions have not been reported

in clinical biopsy specimens from patients with EHEC

infection. The cellular responses leading to attaching

and effacing histopathology in EHEC infections in-

clude high concentrations of polymerized actin on

mucosal lesions, increased levels of IP

and intracel-

lular calcium. In addition to Shiga toxins, and attach-

ing and effacing lesions, a plasmid-encoded fimbria

has been identified among E. coli O157:H7 strains,

but its importance in adherence and pathogenesis has

not been demonstrated.

0019Most EHEC isolates belong to the serotype

O157:H7, although other serotypes, such as

O26:H11, and O111:NM, also have been reported in

human illnesses. Hemorrhagic colitis is characterized

by a watery diarrhea that progresses to grossly bloody

diarrhea, indicative of significant amounts of gastro-

intestinal bleeding. Severe abdominal pain also results,

although fever is usually not present. The illness typic-

ally lasts from 2 to 9 days. It is rarely fatal, except

among the elderly and young children. However, hem-

orrhagic colitis and other gastrointestinal infections

caused by EHEC strains are often antecedants to

more serious conditions, presumably caused by action

of Shiga toxins on kidneys and other organs. Hemo-

lytic uremic syndrome (HUS) and thrombotic throm-

bocytopenic purpura (TTP) occasionally result from a

few days to a few weeks after acquiring a gastrointest-

inal infection with EHEC, especially among the very

young and the elderly. Both are manifestations of

damage to kidneys, which can lead to kidney failure

and death. The clinical features of TTP are similar to

HUS, but TTP also involves damage to the central

nervous system. Patients often develop blood clots in

the brain, frequently leading to death.

0020EHEC was first implicated as a cause of human

illness following two outbreaks of hemorrhagic colitis

in the USA in 1982. Forty-seven people became ill after

eating contaminated ground-beef sandwiches. Several

outbreaks have since been reported from the USA,

Canada, and UK. A chain of outbreaks involving

more than 40 E. coli O157:H7 cases was reported

from Scotland in 1996. In the UK, the number of

laboratory isolations of EHEC O157 from human

infections increased from 76 in 1986 to 1087 in

1997. A massive outbreak of E. coli O157:H7 involv-

ing more than 9500 cases and nine deaths occurred in

Japan in 1996. A study published by the Centers

for Disease Control and Prevention, USA, in 1999

reported an estimated 62 500 foodborne cases of

E. coli O157:H7 annually in the United States. Out-

breaks of hemorrhagic colitis have been associated

frequently with inadequately cooked ground beef.

Outbreaks have also been associated with consump-

tion of a variety of other foods such as salami, roast

beef, venison jerky, unpasteurized milk, apple juice,

coleslaw, and contaminated municipal water. More-

over, vegetables such as lettuce, alfalfa and radish

sprouts, and potatoes have also been associated with

outbreaks of E. coli O157:H7 outbreaks.

Enteroaggregative

E. coli

(EAEC)

0021First described in 1987, EAEC are now recognized as

a cause of infantile diarrhea, especially in cases that

persist for more than 14 days. EAEC are especially a

2160

ESCHERICHIA COLI

/Food Poisoning

problem in developing countries; however, an increas-

ing number of cases have been reported in industrial-

ized countries. EAEC are characterized by their

ability to adhere to cultured cell monolayers with an

aggregative or ‘stacked brick’ pattern.

0022 The pathogenesis of EAEC has not been fully elu-

cidated; however, a characteristic histopathologic

pattern and a number of virulence factors have been

identified. A surface fimbrial structure designated as

Aggregative Adherence Fimbriae I (AAF/I) that medi-

ates adherence to Hep-2 cells and erythrocyte hemag-

glutination has been identified in EAEC. A 60-MDa

plasmid containing two separate gene clusters en-

codes AAF/I. A second fimbriae, AAF/II, that is mor-

phologically and genetically distinct from AAF/I has

also been reported in EAEC. Although EAEC is

reported to produce a 4100-Da homolog of the

heat-stable enterotoxin (EAST1), its role in vivo in

pathogenicity is not clear. EAEC also produces a 108-

kDa cytotoxin that incurs destructive lesions in a rat

ileal loop, and is believed to be responsible for muco-

sal damage in patients. Lesions such as shortening of

villi, hemorrhagic necrosis of villous tips, edema, and

mononuclear infiltration of submucosa are observed

in EAEC infection.

0023 Typical clinical manifestations of EAEC infection

include watery, mucoid diarrhea, and low-grade fever

with no vomiting. In some patients, grossly bloody

stools are observed. In studies with volunteers, the

mean incubation period of EAEC infection was

14 h, with a range of 7–22 h. The duration of diarrhea

is typically longer than 14 days. Although outbreaks

of EAEC were commonly reported from developing

countries such as Brazil, Chile, Mexico, India, and

Bangladesh, some of the recently reported outbreaks

occurred in industrialized countries, including Japan

and the UK. A massive outbreak of EAEC involving

2697 schoolchildren was reported from Japan in

1993. Four outbreaks of EAEC occurred in the UK

in 1994. Although foods have not been implicated as

vehicles in any EAEC outbreaks, the role of food and

water in the transmission of EAEC merits investiga-

tion. Diagnosis of EAEC infection involves isolation

of E. coli from stool specimens and a demonstration

of aggregative adherence pattern in HEp-2 cells.

A DNA probe and a PCR assay specific for EAEC

have also been reported.

Treatment

0024 In developed countries, medical intervention in cases

of gastrointestinal E. coli infections is rarely required.

The illness typically is of short duration and resolves

without any significant incident. Antibiotic therapy is

usually not necessary, with the exception of newborn

children with severe EPEC infection. In most other

cases, treatment with antimicrobial agents does not

appear to have a substantial effect on the duration or

intensity of disease. A principal concern is to prevent

dehydration, especially among infants. Diarrheal

episodes in developing countries are generally con-

siderably more severe than in developed countries.

Oral rehydration with aqueous solutions containing

sodium, potassium, chloride, bicarbonate, and glu-

cose has substantially decreased mortality. The

World Health Organization recommends a rehydra-

tion solution containing 90 mM sodium, 20 mM po-

tassium, 80 mM chloride, 30 mM bicarbonate and

111 mM glucose.

0025Infection by E. coli O157:H7 is usually self-limited

as well, although patients with severely bloody diar-

rhea, HUS, or TTP are hospitalized. Care must be

taken to prevent dehydration and excessive blood

loss. Patients who develop HUS or TTP will often

require extensive treatment, including dialysis and

blood transfusions.

Control and Prevention

0026Strains of ETEC, EIEC, and EPEC that cause human

disease are usually not associated with animals. The

principal source of these bacteria is believed to be feces

of human carriers. Therefore, the primary step in pre-

venting these infections is avoiding fecal contamin-

ation of foods and water. Water used for cleaning of

food contact surfaces should be chlorinated to ensure

absence of viable E. coli cells. Untreated human

sewage should not be used to fertilize vegetables or

crops intended for human consumption. Food hand-

lers should be educated in proper personal hygiene and

in techniques of handling food safely. Foods should be

heated thoroughly to kill E. coli and stored under

refrigeration conditions to prevent bacterial multipli-

cation. E. coli O157:H7 and other EHEC serotypes

differ from other E. coli gastrointestinal pathogens in

that the strains that cause human illness are also found

in the intestinal tract of domestic animals used in food

production. Consumers should be certain that all

foods of animal origin are thoroughly cooked or pas-

teurized before consumption, and care should be

taken to prevent contamination of ready-to-eat

foods with uncooked material. Food producers and

processors should avoid contamination of animal

products with feces during slaughter or milking.

0027Vaccination of cattle is one potential method for

reducing carriage of E. coli O157:H7. Although a

number of studies have attempted to develop a

vaccine targeting various colonization factors such

as intimin, an effective vaccine against E. coli

O157:H7 in cattle has not been reported. However,

ESCHERICHIA COLI

/Food Poisoning 2161

an alternative strategy involving administration of

probiotic bacteria to cattle that reduce colonization

of E. coli O157:H7 was recently reported. Protection

against ETEC infection has been demonstrated in

animals immunized with fimbrial antigens used by

these organisms to adhere to intestinal epithelium,

and in the litters of pigs immunized with these fim-

briae while pregnant. Several studies aimed at induc-

ing immunogenicity against ETEC in humans have

been reported. A recent study revealed that peroral

administration to children of inactivated, whole cells

ETEC coupled with the B subunit of cholera toxin

was effective in eliciting an IgA response against

ETEC colonization factor antigen I.

See also: Diarrheal (Diarrhoeal) Diseases; Shigella;

Vibrios: Vibrio cholerae

Further Reading

Bertschinger HU, Neif V and Tschape H (2000) Active oral

immunization of suckling piglets to prevent colonization

after weaning by enterotoxigenic Escherichia coli with

fimbriae F18. Veterinary Microbiology 71: 255–267.

Donnenberg MS and Nataro JP (2000) In: Cary JW, Linz JE

and Bhatnagar D (eds) Mechanism of Pathogenesis and

Toxin Synthesis, pp. 87–130. Lancaster, PA: Technomic

Publishing.

Doyle MP (1990a) Foodborne illness: pathogenic Escheri-

chia coli, Yersinia enterocolitica and Vibrio parahaemo-

lyticus. Lancet 336: 1111–1115.

Doyle MP (1990b) In: Cliver DO (ed.) Foodborne Diseases,

pp. 209–215. San Diego, CA: Academic Press.

Doyle MP (1991) Escherichia coli O157:H7 and its signifi-

cance in foods. International Journal of Food Micro-

biology 12: 289–302.

Doyle MP and Padhye VV (1989) In: Doyle MP (ed.) Food-

borne Bacterial Pathogens, pp. 235–281. New York:

Marcel Dekker.

Gorbach SL (1987) Bacterial diarrhea and its treatment.

Lancet ii: 1378–1382.

Gross RJ and Rowe B (1985) Escherichia coli diarrhea.

Journal of Hygiene 95: 531–550.

Gyles CL (1998) Vaccines and Shiga toxin-producing

Escherichia coli O157:H7 in animals, pp. 434–444. In:

Kaper JB and O’Brien AD (eds) Escherichia coli and

other Shiga toxin-producing E. coli strains. Washington,

DC: ASM Press.

Mead PS, Slutsker L and Dietz V et al. (1999) Food-related

illness and death in the United States. Emerging Infec-

tious Diseases 5: 607–625.

Meng J, Doyle MP, Zhao T and Zhao S (2001) Escherichia

coli O157: H7 (1997) In: Doyle MP, Beuchat LR and

Montville TJ (eds) Food Microbiology: Fundamentals

and Frontiers, 2nd edn, pp. 193–213. Washington, DC:

ASM Press.

Nataro JP and Kaper JB (1998) Diarrheagenic Escherichia

coli. Clinical Microbiology Reviews 11: 142–201.

Nataro JP, Steiner T and Guerrant RL (1998) Enteroag-

gregative Escherichia coli. Emerging Infectious Dis-

eases 4: 251–261.

Savarino SJ, Hall ER, Bassily S et al. (1999) Oral, inacti-

vated, whole cell enterotoxigenic Escherichia coli plus

cholera toxin B subunit vaccine: results of the initial

evaluation in children. Journal of Infectious Diseases

179: 107–114.

Zhao T, Doyle MP, Harmon BG et al. (1998) Reduction of

carriage of enterohemorrhagic Escherichia coli

O157:H7 in cattle by inoculated with probiotic bacteria.

Journal of Clinical Microbiology 36: 641–647.

Occurrence and Epidemiology

of Species other than

Escherichia coli

P Guglielmetti, Universita

di Siena, Siena, Italy

A Bartoloni, Universita

di Firenze, Firenze, Italy

Background

0001The Enterobacteriaceae are a large family of micro-

organisms, widely distributed in nature, that may

have either saprophytic or pathogenic interactions

with the host. In this article, some members of the

Enterobacteriaceae, other than Escherichia coli, Sal-

monella spp., Shigella spp., and Yersinia enterocoli-

tica, are reviewed, with particular reference to their

occurrence, isolation, and identification procedure

and their role in foodborne poisoning. (See Shigella;

Yersinia enterocolitica: Properties and Occurrence.)

Characteristics and Occurrence

0002The very large group of Gram-negative bacteria in

the Enterobacteriaceae family are among the most

important organisms in the field of human health.

The bacteria included in this family share properties.

They are Gram-negative, nonsporing, straight rods

(0.3–1.0 mm by 1.0–6.0 mm), motile by peritrichous

flagella, or nonmotile. They grow well on peptone,

meat extract, and usually MacConkey’s media, both

aerobically and anaerobically, often with gas produc-

tion by fermentation, rather than oxidation. They are

catalase-positive and oxidase-negative, reduce nitrate

to nitrite, and have a 39–59% guanine and cytosine

(G þC) content of deoxyribonucleic acid (DNA).

0003The family of Enterobacteriaceae includes 26

genera and more than 60 species. Enterobacteriaceae

are widely distributed on plants, fruits, vegetables in

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/Occurrence and Epidemiology of Species other than

Escherichia coli

the soil, and water, and in the intestine of humans

and animals. Some strains occupy a limited ecolo-

gical niche (e.g., Salmonella typhi); others (e.g.,

Klebsiella and Proteus) are widely distributed in the

environment.

0004 Only organisms in four genera – Escherichia, Sal-

monella, Shigella, and Yersinia – have been clearly

documented as enteric pathogens. The occurrence

and characteristics of genera potentially implicated

in intestinal disease, but for which a definitive role

has to be confirmed, are discussed here with special

reference to food poisoning.

Citrobacter

0005 There are three species in the Citrobacter genus that

may have some importance. Citrobacter freundii is

isolated from human and animal feces, as well as

from extraintestinal specimens. It is also commonly

found in soil water and food. Citrobacter diversus is a

rare but important agent of neonatal meningitis and

infections in compromised hosts. Citrobacter amalo-

naticus, widely distributed in the environment, is

found in a variety of human clinical specimens as an

opportunistic pathogen.

Edwardsiella

0006 The most frequently identified species in the Edward-

siella genus is E. tarda, which is rarely isolated but a

well-documented cause of extraintestinal infections.

It is suspected, but not firmly established, to be an

enteric pathogen. Other species in the Edwardsiella

genus are extremely rare.

Enterobacter

0007 All Enterobacter species are found in water, sewage,

soil, and vegetables. Enterobacter cloacae is the most

frequently isolated Enterobacter species from humans

and animals. Its role as an enteric pathogen has not

been demonstrated. However, it may be an opportun-

istic pathogen and has an increasing importance in

hospital-acquired infections (intensive care units,

emergency units, urology). Enterobacter cloacae is

less susceptible to chlorination than Escherichia coli.

It may also be isolated in meat, hospital environ-

ments, and on the skin of man as a commensal.

0008 Enterobacter sakazakii has no importance as an

enteric pathogen, but it is a rare cause of neonatal

meningitis and sepsis; it occurs in the environment

and in food.

0009 Enterobacter agglomerans is occasionally isolated

from clinical specimens and has no conclusive im-

portance as an enteric pathogen. Enterobacter aero-

genes, phenotypically very similar to E. gergoviae,

has been identified in dairy products and water.

Hafnia

0010The only species in the Hafnia genus is H. alvei,

previously known as Enterobacter alvei. It is fre-

quently found in clinical specimens, especially in

feces from healthy individuals. This organism has

been reported by some authors as a potential causa-

tive agent of intestinal disease. It has also been

isolated from water and dairy products.

Moellerella

0011The genus Moellerella has been recently described

(1984), and it includes only one species, M. wiscon-

sensis, which has been reported by some authors as a

potential agent of gastroenteritis. Moellerella wiscon-

sensis has been mainly isolated from human feces.

Morganella

0012Morganella morganii is the only species in the genus

Morganella. This organism was once assumed to be a

cause of diarrhea, but confirmation about its role in

intestinal disease has been lacking in recent years. It

is now considered an opportunistic pathogen, and

has been isolated from blood, sputa, and pus in

patients with bacteremias, respiratory tract, and

wound infections. Morganella morganii is widely

distributed in nature and is commonly identified

from the intestines of humans, dogs, other mammals,

and reptiles.

Proteus

0013Among the species in Proteus genus, P. mirabilis and

P. vulgaris may cause infections in humans. The

former is more frequently identified in clinical speci-

mens than P. vulgaris, mainly in human urinary tract

infections. Proteus urinary tract infections are more

commonly found in hospitalized and compromised

hosts. Approximately 25% of the healthy population

are intestinal carriers. The patients may infect them-

selves by acquiring the environmental strains or with

their own flora. Proteus urinary tract infections may

give rise to bacteremias that are often fatal.

0014Proteus mirabilis has been suggested as a possible

causative agent of outbreaks of gastroenteritis,

resulting from the consumption of contaminated

food. Its role as a causative agent of acute gastroenter-

itis is very difficult to assess because of the high

carrier rate in healthy individuals.

Providencia

0015Among the species in the Providencia genus, P. alca-

lifaciens may be isolated from human and animal

feces. The role of P. alcalifaciens in disease produc-

tion is not known.

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Serratia

0016 Serratia marcescens is an important opportunistic

pathogen, is responsible for many nosocomial infec-

tions, and is not an intestinal pathogen. Many other

Serratia species have been described, all of which are

widely distributed in the environment (soil, water,

plants, intestine of different species).

Klebsiella

0017 Klebsiellae are opportunistic pathogens that may give

rise to several extraintestinal infections. An increase

in Klebsiella species infections has been reported,

particularly in those hospitals where the strains isol-

ated were multiple-antibiotic-resistant. The main res-

ervoir of these strains is considered to be the human

intestinal tract, whereas hands are the main vehicles

for transmission. Enterotoxin-producing strains have

been described.

0018 Klebsiellae are widely distributed in nature, occur-

ring in botanical, aquatic, and soil environments. The

two species K. terricola and K. planticola are thought

to be pure environmental strains. Klebsiella pneumo-

niae and K. oxytoca are normally found in human

and animal intestinal tracts, but in lower numbers, if

compared with E. coli. Klebsiella oxytoca may also

be isolated from plants and aquatic environment.

Klebsiellae are not considered enteric or foodborne

pathogens, but their role in determining intestinal

disease is not excluded.

0019 Many other genera and species are included in the

large family of Enterobacteriaceae, but only those

genera that may be important in food contamination

and as opportunistic pathogens are described here.

Growth Factors, Isolation, and

Identification

0020 In common with all Enterobacteriaceae, the bacteria

listed above grow very well on a broad variety of

culture media. The isolation depends on the origin

of the specimen. In clinical microbiology laborator-

ies, specimens from a normally sterile body site are

cultured on blood or chocolate agar. These media are

very rich in nutrient elements and allow the growth of

many strains. All the Enterobacteriaceae grow well

on such media. Specimens that are likely to be a

mixture of microorganisms (feces and vomitus) are

generally cultured on selective media to enhance the

recovery of the Enterobacteriaceae. A preliminary

grouping of enteric bacteria and other Gram-nega-

tive organisms may be carried out using media such

as MacConkey agar or eosin methylene blue agar.

The characteristic appearance of the most common

organisms described on blood agar or MacConkey

agar allows a preliminary identification of these bac-

teria. (See Food Poisoning: Tracing Origins and

Testing.)

0021When a small number of Enterobacteriaceae are

thought to be present in the specimen, enrichment

techniques in liquid media may be of value to isolate

them. This is generally the case for the bacteria pre-

viously reviewed, which are present in lower

numbers when compared with E. coli. Enrichment

techniques are associated with subcultures in appro-

priate selective media in which the presence of anti-

biotics and other substances makes the isolation of

the suspected microorganism easier. For example, the

selectivity of the media for Klebsiella isolation is

based upon the high resistence of this genus to car-

benicillin, in contrast to other Enterobacteriaceae.

Selective media are routinely used in clinical micro-

biology laboratories to isolate Enterobacteriaceae

from feces. Commonly used selective media for

Enterobacteriaceae are MacConkey agar, xylose–

lysine–deoxycholate agar, or Hektoen enteric agar.

Most of these media inhibit the growth of Gram-

positive and commensal organisms and differentiate

between lactose fermenters and nonlactose ferment-

ers. Special enrichment and selective media are often

available according to the species investigated. These

kinds of media are very efficient for isolating Mor-

ganella, Serratia, Proteus, and Providencia. For

example, when feces are examined for Proteus, the

rate of isolation is increased from 8.2 to 23.6% for

P. mirabilis, when primary plating is preceded by

enrichment with tetrathionate or selenite broth. Se-

lective or enrichment methods are not available for

Citrobacter, Enterobacter, and Hafnia, and for

Edwardsiella, little information exists on selective

isolation. Although klebsiellae are usually present in

the intestinal tract in low numbers, their elevated and

mucoid appearance in agar media is very character-

istic and easy to identify. However, the isolation of

this microorganism from polymicrobic sources can

be facilitated by using selective media.

0022The identification of Enterobacteriaceae at the

genus and species level is based on several conven-

tional biochemical tests. The suspected colony is in-

oculated, from a pure culture in nonselective media,

into a series of tubes with different media for each

biochemical test; the tests are read after 24 and 48 h.

At present, the procedure is simplified by using

a series of miniaturized and standardized tests

that are available commercially. These identification

procedures are utilized in the large majority of clinical

microbiology laboratories.

0023To isolate and identify these bacteria from food,

enrichment procedures are normally performed.

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Role in Foodborne Poisoning

0024 In general, the microorganisms reviewed are part of

the normal intestinal flora. Mainly for this reason, it

is very difficult to establish their role in intestinal

disease. More than the isolation and identification

of a single species in feces of patients with diarrhea

and/or in a suspected food, it is the epidemiological

analysis of the episode that is important, especially in

the case of a foodborne outbreak. Moreover, some of

these organisms, mainly Enterobacter, Proteus, Serra-

tia, and Klebsiella, may be isolated from food without

any evidence of a concomitant intestinal pathology in

consumers. The high frequency of recovery and count

of the Klebsiella–Enterobacter–Serratia group indi-

cates that these bacteria are natural flora of some

foods, such as vegetables, and are not necessarily

contaminants from humans.

0025 In general, proof of their role in causing illness and

their mode of transmission is lacking. For this reason,

the reports on foodborne poisoning caused by these

Enterobacteriaceae are sporadic and lack definitive

confirmation.

0026 Nevertheless, some authors have reported the asso-

ciation of these bacteria with human intestinal dis-

ease. Furthermore, the evidence of the production of

an enterotoxin by some of these bacteria suggests

their hypothetical role as agents of intestinal disease.

0027 Reports are available for Citrobacter, Edward-

siella, Enterobacter, Hafnia, Moellerella, Proteus,

Providencia, and Klebsiella.

0028 Citrobacter has been isolated from feces of patients

with diarrhea and from apparently normal persons.

The same observations were obtained in animals. In

adults with diarrhea, the microorganism was more

often found than in asymptomatic controls; Citrobac-

ter in symptomatic patients was above all isolated in

pure culture. Unfortunately, the lack of data concern-

ing serotypes and biological activity of the isolated

strains did not allow the formation of a consensus

opinion concerning their pathogenicity. Citrobacter

has also been isolated from feces of patients and

from implicated food in a few foodborne disease

outbreaks. Patients had mild to serious diarrhea;

symptoms were abdominal cramps, fever, chills,

headache, tenesmus, vomiting, nausea, dizziness,

and fainting. The documented incubation period in

one of these outbreaks was 10–15 h, and the duration

of the illness was 12 h. Strains potentially implicated

in these outbreaks were isolated from corn pudding,

spoiled milk, raw milk, macaroni, meat, liver sausage

and smoked meat.

0029 After an outbreak in a nursery, Proteus mirabilis,

serotype 09:H2, was isolated from spaghetti with

cheese, and from the feces of symptomatic children

who had eaten the implicated food. The role of Pro-

teus species has been suspected in other foodborne

outbreaks, but the lack of information about other

intestinal pathogens, particularly staphylococci and

Clostridium perfringens, or in some cases the associ-

ation of well-known agents of gastroenteritis, does

not permit a final conclusion.

0030A well-documented role of Proteus spp. and

Klebsiella spp. in the genesis of foodborne infections

is that of histamine fish (scombroid) poisoning.

Scombroids, in common with many other Atlantic

and Pacific fish, contain high levels of histidine in

their flesh. When the fish are inadequately cooled,

Proteus and Klebsiella may multiply rapidly, decar-

boxylating histidine into histamine and other related

products. These substances are responsible for an

allergy-type illness, which starts within an hour of

ingestion of the implicated fish. (See Fish: Spoilage

of Seafood.)

0031Organisms belonging to the Providencia genus

have been isolated more frequently in children and

adults with diarrhea, compared with asymptomatic

subjects. In a foodborne disease outbreak, Providen-

cia, associated with Proteus mirabilis and Providen-

cia rettgeri, has been isolated from patients with

watery diarrhea and from the suspected food (chicken

fricassee).

0032Klebsiellae are considered normal inhabitants of

the human intestinal tract. Nevertheless, occasional

reports suggest their potential role as agents of gas-

troenteritis. Several strains of K. pneumoniae gave

positive responses in rabbit ileal loop, and a heat-

stable enterotoxin, similar to the E. coli toxin, has

been purified and characterized. A foodborne disease

outbreak that involved 190 people has been associ-

ated with Klebsiella contamination of food, but

no definitive evidence was produced. Klebsiella is a

well-established cause of opportunistic infections,

particularly in hospitalized patients. Food prepared

and supplied by hospital kitchens is at risk of colon-

ization by Enterobacteriaceae. Klebsiella was identi-

fied in high numbers from 12 foods in a hospital

kitchen and has been isolated from vegetables and

seeds, sugar, and milk. The most probable origin of

these contaminants is the incoming food, but spread

of these microorganisms in the hospital environment

may also occur by means of colonized subjects. In

addition, other Enterobacteriaceae, such as Entero-

bacter, Serratia, Citrobacter,andProteus, are com-

monly found in vegetables.

0033Like K. pneumoniae, Enterobacter cloacae in the

rabbit ileal loop is strongly positive in certain strains,

and a heat-stable toxin, resembling the heat-stable

enterotoxin of E. coli, has been partially purified

and described. The presence of such toxins may be

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2165

the in-vitro evidence of the potential pathogenicity

in vivo. Nevertheless, there are few reports of food-

borne outbreaks associated with Enterobacter.

0034 Hafnia alvei has been associated with an outbreak

of mild gastroenteritis among hospitalized individ-

uals and staff members. It has been isolated from

the feces of patients with diarrhea and from milk

that was implicated as the vehicle of the outbreak.

0035 Edwardsiella has been identified in the feces of

both patients with diarrhea and asymptomatic sub-

jects. The role of this genus in intestinal disease is

unknown.

See also: Fish: Spoilage of Seafood; Food Poisoning:

Tracing Origins and Testing; Shigella; Yersinia

enterocolitica: Properties and Occurrence