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

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Final Processing

0019 Most blends are prepared a few months before ship-

ping, which allows the final blend to be stabilized.

Especially since such different wines are blended, it is

important to ascertain that no instabilities occur, and

stabilization and fining techniques are commonly

used to ensure a clear and stable wine in the bottle.

See also: Grapes; Wines: Dietary Importance; Yeasts

Further Reading

Gonzalez Gordon M (1972) Sherry, The Noble Wine.

London: Cassell.

Goswell RW (1986) Microbiology of fortified sherries. In:

Robinson RK (ed.) Developments in Food Microbiology,

2, pp. 1–19. London: Elsevier Applied Science.

Goswell RW and Kunkee RE (1977) Fortified sherries. In:

Rose AH (ed.) Economic Microbiology, vol. 1, Alcoholic

Beverages, pp. 477–535. London: Academic Press.

Martin B, Etievant PX and Henry RN (1990) The chemistry

of sotolon: a key parameter for the study of a key com-

ponent of flor sherry wines. In: Bessiere Y and Thomas

AF (eds) Flavour Science and Technology, pp. 53–56.

Chichester: John Wiley.

Reader HP and Dominguez M (1994) Fortified wines:

sherry, port and Madeira. In: Lea AGH and Piggott JR

(eds) Fermented Beverage Production, pp. 159–207.

Glasgow: Blackie.

Webb AD and Noble AC (1976) Aroma of sherry wines.

Biotechnology and Bioengineering XVIII: 939–1052.

Composition and Analysis

M Medina, J Moreno, J Merida, M Mayen, L Zea

and L Moyano, University of Cordoba, Campus de

Rabanales, Cordoba, Spain

Background

0001 Most sherry wines are produced in two southern

regions of Spain, Jerez and Montilla-Moriles, where

the grape varieties Palomino Fino and Pedro Xime-

nez, respectively, are preferentially grown. These

grapes are used to obtain so-called fino, oloroso,

and amontillado wines, which are three typical types

of these wines. These are obtained starting from the

same wine base but subjected to different aging con-

ditions. Fino wines have a very light yellow color

and an almond flavor with pungent notes. Like

some Italian- and French-like sherry wines, they are

obtained by biological aging under the action of flor

yeasts growing on the wine surface. This aging pro-

cedure involves a complex series of wine transfers

that leads to a mixture of different vintages in the

same cask. Oloroso wines are obtained by oxidative

aging, after fortification with ethanol, in order to

prevent growth of flor yeasts. They have a very dark

color that results from the oxidation of phenolic com-

pounds, and a flavor with distinct notes of oak and

walnut. Amontillado wines are produced by using the

previous two aging procedures in succession (bio-

logical aging first and then oxidative aging). These

wines are highly desirable, have a color in between

those of fine and oloroso wines, but closer to the

latter, and have a flavor with hazelnut notes that is

the most complex among the three.

Musts and Wines. General Composition

0002The prevailing climate of the sherry-making regions

of southern Spain determines to a great extent the

general composition of the musts and wines they

produce, and also some winemaking techniques. In

summer, temperatures above 35



C, or even above



C in areas far from the Mediterranean coast, are

usual during the grape-ripening period. A milder cli-

mate can be found in zones such as Sanlucar de Bar-

rameda, on the coast, which produces a wine similar

to the fino type (called Manzanilla) but with distinct-

ive sensorial characteristics. Under these climatic con-

ditions, grapes accumulate large amounts of reducing

sugars, resulting in concentrations above 200 g l

1

in the must (specific gravity 1.094 g ml

1

) or even

more than 270 g l

1

(specific gravity 1.113 g ml

1

)in

warmer areas. As a result, the base wines obtained

after alcoholic fermentation have a high ethanol con-

tent and are classified according to their suitability for

producing the different types of sherry wines. Wines

to be aged biologically are fortified up to 15% v/v

ethanol content, if necessary, whereas those destined

for the production of oloroso wines are fortified to

17–18% v/v.

0003The climatic conditions also result in musts with a

low acidity (3–4.5 g l

1

as tartaric acid) that is usually

increased by addition of tartaric acid, although wine-

makers in some areas continue to use gypsum for this

purpose. The musts are also supplied with SO

concentrations of around 100 mg l

1

0004The special features of the process by which flor

yeasts produce fino wine have aroused much interest

among researchers. The yeast types most frequently

used for this purpose are Saccharomyces cerevisiae

varieties beticus, cheresiensis, and montuliensis in

the Jerez region, and races capensis and bayanus in

SHERRY/Composition and Analysis 5257

the Montilla-Moriles region. These yeasts develop an

aerobic metabolism, consuming ethanol (1–1.5% v/v)

and glycerol (decreasing from 7–8 g to less than

0.5 g l

1

), in addition to some acids such as acetic

(the concentration varying as the volatile acidity

falls from 0.4 to 0.04 g l

1

) and lactic acid, which

are converted to acetaldehyde and other byproducts.

Quantitatively, changes during the biological aging

process depend on the distribution of the yeast popu-

lation and on the stability of the film they form; this

results in some differences among fino wines pro-

duced in different zones. However, the development

of flor yeasts is essentially influenced by the ethanol

content in the wine (around 15% v/v) and the

temperature during aging (below 22.5



C). Any devi-

ation from these conditions (e.g., higher tempera-

tures) deteriorates the flor film and retards the aging

process.

0005 In the absence of flor yeasts, oloroso wines con-

sume no glycerol, which remains at levels of 6.5–

8gl

1

during oxidative aging. However, their volatile

acidity increases during aging (up to 0.68 g l

1

acetic acid), because of the oxidation of ethanol and

acetaldehyde. Amontillado wines exhibit an inter-

mediate behavior between those of the previous two

as a result of the mixed aging procedure used.

0006 The three types of quality sherry wines require very

long aging times: at least 4–5 years for fino wines and

more than 10 years for amontillado wines. In order to

shorten the process, some authors recommend the use

of various procedures to accelerate biological aging of

the wine. Such procedures are based on an increase in

the population densities of flor yeasts, which can be

obtained by using containers with an increased sur-

face-to-volume ratio or controlled aerations of the

wine during aging. Oloroso wines have also been

subjected to accelerated aging procedures involving

higher temperatures or maceration with oak wood

shavings.

Volatile Compounds and Flavor

Musts and Wines

0007 Palomino Fino and Pedro Ximenez, the two grape

varieties preferentially used to obtain sherry wines

in the Jerez and Montilla-Moriles regions, are occa-

sionally aromatic with an overall free terpene content

in the musts of 0.27–0.37 mg l

1

for the latter variety.

In order to improve the sensory profile of the must,

cold maceration with grape skin at 10



C for 24 h has

been found to increase the terpene content by a factor

of 4.5, but these results probably do not offset the

cost of the operations involved.

0008Traditionally, the musts are fermented with wild

yeasts, the population consisting primarily of various

Saccharomyces cerevisiae varieties. The content in

higher alcohols (mainly isobutanol, isoamyl alcohols,

and phenethyl alcohol) at the end of fermentation

ranges from 207 to 405 mg l

1

. Compared with

other types of white wines, the ester fraction is less

concentrated, and so the wine has a stronger sensory

profile prior to aging. This is also a result of a high

fermentation temperature (frequently above 25



C),

which favors the formation of higher alcohols. Pure

and mixed cultures of the yeasts have been tested with

a view to softening the sensory profile of the base

wine. In this sense, some authors have studied the

enzymatic activity of acetyl transferase and esterase

in pure cultures of the fermentation yeasts in order to

determine the contribution of specific yeast varieties

involved in the alcoholic fermentation process to the

flavor of the base wine. However, the need to ferment

large amounts of sugars, as a result of a high tolerance

to ethanol, makes replacing the wild yeasts very

difficult.

Wine Aging

0009Most of the analytical and sensory differences among

sherry wines result from differences in the aging

procedures to which the base wines are subjected.

On the one hand, the biological aging process that

yields fino wine is affected by flor yeasts, which use

some compounds in the base wine as carbon sources,

thereby altering its composition to a variable extent

depending on the duration of the process. On the

other hand, the oxidative aging process that produces

oloroso wine allows for no biological transformation,

so the changes involved should be preferentially

ascribed to evaporation, chemical oxidation, and

extraction from constituents of the wood casks.

Because amontillado wine is obtained by a com-

bination of both types of aging procedure, its ana-

lytical properties are inherited to a variable extent,

even though its sensory profile is unique to this wine

type.

0010More than 300 different compounds have been

identified in sherry wine-type fino. Only a small frac-

tion, however, can be specific to them, with most of

the analytical differences observed being ascribed to

changes in the concentrations of compounds also

present in other types of white wines. At this point,

the contents of acetaldehyde and derivatives such as

1,1-diethoxyethane and acetoin are noteworthy. The

concentrations of these compounds increase during

biological aging as a result of the aerobic metabolism

of flor yeasts consuming the ethanol of the wine.

Their concentrations in commercially available

5258 SHERRY/Composition and Analysis

wines are in the range of 400–500 mg l

1

for acetalde-

hyde, 40–75 mg l

1

for 1,1-diethoxyethane, and

8–19 mg l

1

for acetoin, depending on the duration

of the aging process. Acetaldehyde is thus used as a

measure of wine maturity, being largely responsible

for the typical pungent flavor of fino wines. Some

lactones and their oxygen derivatives also impart

different notes to the flavor of wines obtained by

biological aging. In fact, despite their low concentra-

tions, some lactones such as sotolone (0.036–

0.143 mg l

1

), g-butyrolactone (27–54 mg l

1

) and

pantolactone (3–8.5 mg l

1

) impart a caramel-like

odor to these wines and increase their concentration

during biological aging. In particular, the former is

considered an important contributor to the flavor of

fino wines, introducing nut-curry notes. One other

distinctive feature of the biological aging process is

the decrease in volatile acidity resulting largely from

the consumption of acetic acid by flor yeasts, which

reduces its typical odor and flavor.

0011 Changes in other aroma compounds are not as

clear as those in the aforementioned compounds.

Although some authors have reported a decrease in

the contents of major higher alcohols, others have

found slightly increased or constant contents. These

contradictory results are probably a consequence of

differences in the experimental conditions used, par-

ticularly as regards the distribution of the flor yeast

population and the aging temperature. In relation to

the ester, changes during aging can also be observed

giving contradictory results, although increased con-

tents in ethyl lactate and diethyl succinate, and de-

creased contents in ethyl, isoamyl, isobutyl, and

phenethyl acetates, appear to be the norm. Some

authors have found that flor yeasts synthesize small

amounts of terpenes such as linalool, a-terpineol and

z-nerolidol.

0012 The aroma fraction of oloroso and amontillado

wines is mainly influenced by a concentrating effect

during their lengthy aging; as a result, the contents

in major higher alcohols and esters increase with

increasing wine age. Also, chemical esterification

is favored by a high ethanol content in the wine, so

the concentrations of esters such as ethyl acetate,

ethyl lactate, and diethyl succinate increase during

aging.

Phenolic Compounds and Color

0013 Color is the most obvious distinctive feature of sherry

wines for the consumer. Thus, fino wines, which are

obtained by biological aging, exhibit a very pale

yellow color, whereas amontillado and, especially,

oloroso wines, have a dark color.

Musts and Wines

0014More than 20 phenolic compounds in monomeric

and simple oligomeric forms have been identified in

base wines prior to aging. Although their concentra-

tions depend markedly on the particular climatic con-

ditions of each year and on the grape ripening status,

flavan-3-ol derivatives invariably exhibit the highest

concentrations of all (100–120 mg l

1

, most of which

is contributed by catechin and epicatechin). The con-

centrations of hydroxycinnamic acids range from

1 to 5 mg l

1

, and those of their esters from 45 to

75 mg l

1

, in which t-caftaric acid is the best repre-

sented. Gallic acid exhibits the highest contents

among hydroxybenzoic acids (the overall concentra-

tion ranging from 15 to 25 mg l

1

). Flavonols and

coumarins are also present, at concentrations of

10–20 and about 50 mg l

1

, respectively.

0015Fino wine is a delicate product and it tends to get

browner after bottling. This alters not only its color

but also its sensory properties. Thus, fresh fino wine

has an absorbance at 420 nm of 0.100–0.120 A.U. At

0.170–180 A.U., it is normally discarded. Depending

on the particular production technique used, this

absorbance threshold is reached within about a year

after bottling. This has promoted the development of

improved production techniques to balance supply

and demand better in order to avoid overstocking.

Because brown pigments in the wine are formed by

oxidation of phenolic compounds, winemaking tech-

niques such as must hyperoxidation have been used

to decrease the contents in these compounds and

increase the resistance of the resulting wines to

browning. However, one should bear in mind the

complexity of the aging process, by which a given

cask contains mixed wines from different vintages.

Reliable conclusions on this point can only be reached

from a ‘solera and criadera system’ exclusively using

hyperoxidized wines from various vintages, requiring

a long-term research. Also in order to improve the

resistance to browning, traditional practices such as

the addition of SO

to grapes during the harvest

period have been suppressed, because these result

in an increased degree of extraction of phenolic

compounds from the grape skin.

Wine Aging

0016Because of their differential aging processes, the

three types of sherry wine differ in their contents in

phenolic compounds. Thus, flavan-3-ol derivatives,

predominate in fino wines (with concentrations of

60–80 mg l

1

), followed by hydroxycinnamic esters

(15–25 mg l

1

), hydroxybenzoic acids (15–30 mg l

1

flavonols (5–10 mg l

1

), and hydroxycinnamic acids

SHERRY/Composition and Analysis 5259

(1.5–2.5 mg l

1

). The contents in vanillic and ferulic

acid, catechin, epicatechin, and procyanidin B2 and

B4, have been found to decrease during biological

aging, whereas those of syringic acid and procyanidin

B1 appear to increase during the process. It should be

noted that this type of wine does not brown under flor

yeasts, thus preserving its pale color with slight vari-

ations for years. This has traditionally been ascribed

to the flor yeasts that grow on its surface, making the

diffusion of atmospheric oxygen across the flor film

difficult and thus protecting wine from this. In add-

ition, flor yeasts consume dissolved oxygen in the

wine to maintain their aerobic metabolism, thereby

preventing the oxidation of phenolic compounds.

However, the only contribution of this protective

effect to color stability in the wine subjected to bio-

logical aging has been partly questioned by some

authors who believe that the wine is partially oxygen-

ated over the years as a result of two actions. On the

one hand, older wine is mixed with younger wine

three or four times a year, which introduces some

oxygen during the transfer operations. On the other

hand, the flor film fails in some seasons because of its

strong dependence on temperature. The lack of the

flor film facilitates the access to oxygen. In fact, the

absorbance values at 420 nm measured at the end

of summer are normally higher than those recorded

in late spring and autumn. Thereby, in addition to

the aforementioned protective effect of the flor

film, some authors think that yeasts may be able to

retain brown polymers formed by oxidation of

phenols.

0017 Oloroso wines are considerably darker than fino

wines as a result of their oxidative aging, showing

absorbance values at 420 nm, ranging from 1.30 to

1.60 A.U.

0018 The phenolic contents of these wines exceed

those in fino wines, with levels of 35–45 mg l

1

for hydroxybenzoic acids, 5–20 mg l

1

for hydroxy-

cinnamic acids, 20–25 mg l

1

for hydroxycinnamic

esters, and 100–130 mg l

1

for flavanes. Oxidative

aging usually takes place at a higher temperature

than does biological aging, which results in volume

losses in the aging wine, with a subsequent concen-

trating effect on the soluble compounds.

0019 Amontillado wines show intermediate levels in

between the previous two because of the combined

aging process used in their production, so their cor-

rected absorbances at 420 nm range from 0.950 to

1.100 A.U. However, in general, their phenol con-

tents are similar to those of oloroso wines, with

values of 45–75 mg l

1

for hydroxybenzoic acids,

5–15 mg l

1

for hydroxycinnamic acids, and 125–

160 mg l

1

for flavanes.

Other Compounds

0020Amino acids are important compounds because they

are used by the flor yeast as a nitrogen source.

l-Proline is the predominant compound of this

fraction in the wine base, accounting for about 60%

of total nitrogen, with a concentration of 4.5–

7.5 mmol l

1

. Other abundant amino acids include

l-tryptophan (8%), l-phenylalanine (7.5%), l-cyst-

eine (6%), and l-threonine (3%). l-Proline is the

most heavily used nitrogen-containing compound

and the principal source of nitrogen for the flor

yeast, and so this compound is consumed most

during biological aging. In the deficiency of l-proline,

flor yeasts use other amino acids such as l-glutamic

acid, l-alanine, l-arginine, and l-tryptophan. Olor-

oso wines show slightly increased amounts of

amino acids as a result of the aforementioned

effect of volume concentration during oxidative

aging.

0021The most abundant polyalcohols quantified in

sherry wines are inositol and erythrol, with variable

levels depending on the method of wine-making. No

significant changes have been observed during aging,

because the initial variability of the wine base is

greater than the contribution of the maturation pro-

cesses. However, there is a general trend towards

higher contents in these compounds for the oloroso

and amontillado wines.

See also: Amino Acids: Properties and Occurrence;

Flavor (Flavour) Compounds: Structures and

Characteristics; Oxidation of Food Components;

Phenolic Compounds; Sherry: The Product and its

Manufacture; Yeasts

Further Reading

Baron R, Mayen M, Merida J and Medina M (1997)

Changes in phenolic compounds and browning during

biological aging of Sherry type wine. Journal of Agricul-

tural and Food Chemistry 45: 1682–1685.

Cortes MB, Moreno JJ, Zea L, Moyano L and Medina M

(1999) Response of the aroma fraction in sherry wines

subjected to accelerated biological aging. Journal of

Agricultural and Food Chemistry 47: 3297–3302.

Domecq P (1989) Sherry: state of art on a very special

fermentation product. In: Proceedings of the Inter-

national Symposium on Yeasts (Issy XIII), pp. 15–35.

Leuven: Leuven Press.

Fabios M, Lopez-Toledano A, Mayen M, Merida J and

Medina M (2000) Phenolic compounds and browning

in sherry wines subjected to oxidative and biological

aging. Journal of Agricultural and Food Chemistry 48:

2155–2159.

Garcı

a-Barroso C, Lo

pez Sa

nchez L, Otero JC, Cela-Torri-

jos R and Pe

rez-Bustamante JA (1989) Studies on the

5260 SHERRY/Composition and Analysis

browning of the fino sherry and its relation to

polyphenolic compounds. Zeitschrift fu

r Lebensmittel-

unterschung und -forschung 189: 322–325.

Guichard E, Etievant P, Henry R and Mosandl A (1992)

Enantiomeric ratios of pantolactone, solerone, 4-carbo-

ethoxy-4-hydroxy-butyrolactone and of sotolon, a

flavor impact compound of flor-sherry and botrytized

wines. Zeitschrift fu

r Lebensmittelunterschung und -for-

schung 195: 540–544.

Ibeas JI, Lozano I, Perdigones F and Jimenez J (1997)

Effects of ethanol and temperature on the biological

aging of sherry wines. American Journal of Enology

and Viticulture 48: 71–74.

Martin B, Etievant PX, Lequere JL and Schlich P (1992)

More clues about sensory impact of sotolon in some

flor sherry wines. Journal of Agricultural and Food

Chemistry 40: 475–478.

Martinez P, Rodriguez LP and Benitez T (1997a) Evolution

of flor yeast population during the biological aging of

fino sherry wine. American Journal of Enology and

Viticulture 48: 160–168.

Martinez P, Rodriguez LP and Benitez T (1997b) Velum

formation by flor yeasts isolated from sherry wine.

American Journal of Enology and Viticulture 48: 55–62.

Mauricio JC, Moreno J and Ortega JM (1997) In vitro

specific activities of alcohol and aldehyde dehydro-

genases from 2 flor yeasts during controlled wine

aging. Journal of Agricultural and Food Chemistry 45:

1967–1971.

Mesa JJ, Infante JJ, Rebordinos L and Cantoral JM (1999)

Characterization of yeasts involved in the biological

aging of sherry wines. Lebensmittel-Wissenschaft und

Technologie 32: 114–120.

Moyano L, Moreno J, Millan C and Medina M (1994)

Flavour in Pedro Xime

nez grape musts subjected to

maceration processes. Vitis 33: 87–91.

Webb AD and Noble AC (1976) Aroma of sherry wines.

Biotechnology and Bioengineering XVIII: 939–1052.

SHIGELLA

K A Lampel, US Food and Drug Administration, Laurel,

MD, USA

R Sandlin, Towson University, Towson, MD, USA

Characteristics

0001 Shigella spp. are the causative agents of shigellosis

(bacillary dysentery). These organisms are endemic

worldwide, resulting in high morbidity and mortality

in third world countries. Shigella are small, slender,

Gram-negative rod-shaped bacteria that are nonmo-

tile and nonspore-forming. The genus is a member of

the family Enterobacteriaceae and is closely related to

Escherichia coli. Shigella spp. are divided into four

major groups based on biochemical reactions and the

O antigen type (Table 1). The four species are

S. dysenteriae (A), S. flexneri (B), S. boydii (C), and

S. sonnei (D), and are further subdivided into sero-

types (except S. sonnei), based on differences in the O

antigen. The severity of disease varies, depending on

the particular species, from mild diarrhea to bacillary

dysentery (stools containing blood, mucous, and in-

flammatory cells). S. sonnei produces the mildest

form of disease, whereas S. dysenteriae produces the

most severe. S. flexneri and S. boydii cause intermedi-

ate forms of the disease. Additionally, the geographic

distribution and epidemiology produced by the

different species are different (Table 1). For example,

S. dysenteriae is responsible for epidemics of the dis-

ease, whereas shigellosis outbreaks in industrialized

nations are usually caused by S. sonnei. In addition,

S. flexneri is more frequently found in developing

countries, and S. boydii is usually limited to the

Indian subcontinent.

0002Shigella spp. are facultative anaerobes with rela-

tively simple nutritional requirements. Typically, Shi-

gella spp. are lysine decarboxylase negative and

lactose nonfermenting (except for a slow reaction

with S. sonnei), characteristics that separate them

from closely related commensal E. coli. However,

enteroinvasive E. coli (EIEC) share many properties

with shigellae producing clinical symptoms indistin-

guishable from shigellosis. Moreover, EIEC has

similar biochemical traits (e.g., nonmotile, lactose

negative) and possess immunologically cross-reactive

somatic antigens with several Shigella serotypes.

Thus, similarities between EIEC and Shigella can

complicate the accurate diagnosis of shigellosis.

0003The World Health Organization has estimated that

worldwide, diarrheal diseases account for approxi-

mately 5 million deaths each year among children

with nearly 10% caused by Shigella spp. S. dysenter-

iae type I is the only Shigella spp. known to produce

shiga toxins, a common etiological agent of epidemic

dysentery. S. dysenteriae has also been shown to

persist in the poorest regions of the world, notably

in parts of Africa, Central America and Asia, where

malnutrition is common. In the USA, of the estimated

76 million people that contract foodborne illness

SHIGELLA

5261

annually, an estimated 300 000 cases are caused by

Shigella. In a recent report from the US Center for

Disease Control and Prevention, Emerging Infections

Program Foodborne Diseases Active Surveillance

Network (FoodNet; http://www.cdc.gov/foodnet),

2324 of the 12 631 confirmed cases of known food-

borne illnesses reported (only nine diseases were

followed), were caused by Shigella spp., which

ranked Shigella as the third leading cause of diseases

of bacterial origin. As expected, most of the reported

Shigella cases were caused by S. sonnei (85%) and the

remainder by S. flexneri (15%).

0004 Shigella is principally a human disease, although

outbreaks have been described in subhuman pri-

mates. One distinctive characteristic of the disease

is its high communicability attributed to the low in-

fectious dose required to cause illness. For example,

ingestion of fewer than 100 organisms has been

shown to result in clinical disease in volunteer studies.

Thus, the low infectious dose is the primary reason

cited for ease of Shigella spread in populations living

under crowded and unsanitary conditions. The in-

cubation period for Shigella is 1–7 days, with the

onset of symptoms usually appearing in 3 days. The

clinical presentation of shigellosis can vary from

mild diarrhea to severe dysentery with symptoms in-

cluding fever, cramps, and frequent bowel movements

containing blood, pus, and mucus. Infections are as-

sociated with mucosal ulceration, rectal bleeding, and

dehydration. Polymorphonuclear leukocytes in the

stools is one characteristic feature of the inflamma-

tory nature of dysentery. Mortality has been reported

as high as 10–15% in some strains, but the infection is

usually self-limiting and resolves without treatment

within 1–2 weeks. Complications of shigellosis

include reactive arthritis, and hemolytic uremic

syndrome.

0005 Asymptomatic carriers of shigellae may be partially

responsible for the perpetuation of shigellosis out-

breaks. Owing to socioeconomic factors that affect

both living and sanitary conditions, adults, particu-

larly those that have resided in one area for a long

period of time, are thought to exacerbate the spread

and maintenance of this pathogen. Furthermore, shi-

gellosis appears to have the highest incidence in

summer months, presumably because of increased

contact of asymptomatic carriers with uninfected

populations. Therefore, asymptomatic carriers may

play a significant role in incubating this pathogen

through the colder months.

0006Unlike most pathogens, Shigella spp. are not

indigenous to any food. Most cases of illness are a

result of close contact with infected individuals, with

transmission most frequently occurring via the fecal-

to-oral route. The organism is commonly found in

water contaminated with human feces and can be

spread via food, flies, fingers, and feces. In highly

endemic areas, shigellosis is associated with children

who frequently lack adequate personal hygiene

habits. Children, ages 1–6, are most susceptible to

infection by Shigella spp. The carrier state usually

lasts 1–4 weeks, but long-term carriers have been

described and are thought to be reservoirs of infec-

tion. It has been estimated that during the acute phase

of the disease, 10

–10

colony-forming units (CFU)

per gram of stool are shed, whereas in convalescing

patients, 10

–10

CFU per gram of stool are shed. To

control the spread of shigellae, infected individuals

are monitored for the presence of the organism in

stool until cleared.

Virulence Factors

0007Members of the genus Shigella are facultative intra-

cellular pathogens. Following ingestion, the organ-

isms are capable of surviving the acidic barrier of

the stomach in order to reach the large intestine.

Shigellosis is caused when virulent Shigella organisms

induce their uptake into M cells and epithelial cells of

the large intestine. After invasion, the bacteria are

released from the phagosome into the cytoplasm

and multiply intracellularly utilizing the host cell

cytoplasm for nutrients. The infection is perpetuated

by the spread of the organism to neighboring epithe-

lial cells through projections from the originally

infected cell. This cycle results in damage and de-

struction of the intestinal epithelial cell, resulting in

tbl0001 Table 1 Characteristics of Shigella spp.

Species Serogroup Serotypes Geographic distribution Distinguishing characteristics

S. dysenteriae A 15 Indian subcontinent, Africa, Asia, Central

America

Produce Shiga toxin, causes most severe

dysentery, high mortality rate if untreated

S. flexneri B 6 Most common isolate in developing

countries

Less severe dysentery

S. boydii C 19 Indian subcontinent, rarely isolated in

developed countries

Biochemically identical to S. flexneri,

distinguished by serology

S. sonnei D1

Most common isolate in developed countries Mildest form of shigellosis

Forms I (smooth) and II (rough) are serotypically distinguishable.

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SHIGELLA

dysentery. Shigella spp. are also able to induce apop-

tosis or programmed cell death of macrophages. Viru-

lence factors that contribute to these processes are

both chromosomally encoded and present on a large

220-kb virulence plasmid (Table 2). The nucleotide

sequence of the entire plasmid was recently reported

(GenBank Accession No. AL391753). The virulence

plasmid is essential for many virulence phenotypes,

and in the absence of the plasmid, the organism is

avirulent. A related virulence plasmid is also present

in EIEC and is essential for this organism’s ability to

cause disease. The genetic factors involved in the

virulence phenotype can be loosely grouped into

genes that encode proteins involved in epithelial cell

uptake, genes that encode proteins involved in the

intercellular spreading phenotype, and genes involved

in the regulation of these processes.

0008 The expression of the virulence genes is regulated

by a variety of factors, most notably temperature.

Temperature is an important factor in the regulation

of virulence genes in bacterial pathogens, including

Salmonella typhimurium, Bordetella pertussis, Yersi-

nia spp., and Listeria monocytogenes, as well as

Shigella. Virulence gene expression in Shigella is

repressed at 30



C and induced at 37



C by the action

of three gene products. The virulence plasmid en-

coded VirF is a transcriptional activator that is a

member of the AraC family of activators. VirF func-

tions to upregulate expression of VirB. VirB is en-

coded on the virulence plasmid and is thought to

induce the expression of a variety of essential viru-

lence genes including the ipa, spa, and mxi genes.

VirR (H-NS) is a chromosomally encoded global

negative regulator that is involved in a variety of

regulatory pathways. H-NS acts to block VirF activity

at the VirB promoter. The exact mechanism is un-

known, but several factors, including temperature,

affect regulation at both the transcriptional and trans-

lational level. Mutations in H-NS result in an absence

of temperature regulation and a constitutive virulent

phenotype, whereas mutations in VirF or VirB result

in an inability to produce the Ipa, Spa, and Mxi

proteins and an avirulent phenotype.

0009Upon contact with a target epithelial cell, Shigella

spp. are induced to secrete up to 25 proteins, includ-

ing the Ipa B, C, D, and A proteins. Some of these

proteins, including IpaC and IpaA, are translocated

directly into the host cell cytoplasm. The Ipa proteins

tbl0002 Table 2 Virulence loci of Shigella

Locus Protein Role in virulence

Plasmid-encoded genes

ipaA 70-kDa protein Invasion; associates with vinculin

ipaB 62-kDa protein Invasion; lysis of vacuole; induction of apoptosis

ipaC 43-kDa protein Invasion; induces formation of filopodia and lamellipodial extensions

ipaD 38-kDa protein complex with IpaB Invasion; forms antisecretion

ipgC 17-kDa protein Chaperon for IpaB and IpaC

ipgD 59.8-kDa protein Modulates invasion of bacteria into epithelial cells

ipaH Family of proteins Present in plasmid and chromosome

mxi/spa 20 proteins Secretion of Ipa and other virulence proteins

icsA (virG) 120-kDa protein Actin polymerization for intracellular motility and intercellular

spread

sen ShET2 Enterotoxin

virB Transcriptional activator Temperature regulation of virulence genes

virF Transcriptional activator Temperature regulation of virulence genes

osp Outer Shigella proteins Encoded proteins secreted by type III secretory system

Chromosomal-encoded virulence-associated genes

virR (hns) Histone-like protein Repressor of virulence gene expression

rfa; rfb Enzymes for LPS core and

O-antigen biosynthesis

Unipolar localization of IcsA

stx

Shiga toxin Destruction of vascular tissue

vacB (rnr) Exoribonuclease RnaseR Posttranscriptional regulation of virulence gene expression

cpxR Response regulator of virF CpxA–CpxR two-component system

Iuc Aerobactin and receptor Acquisition of iron in the host

SodB Superoxide dismutase Defense against oxygen-dependent killing in host

set

ShET1 Enterotoxin

dsbA Disulfide bond catalyst Facilitates secretion of IpaB and IpaC

sigA 139.6-kDa exported cytopathic

protease

Intestinal fluid accumulation

The stx locus and production of Shiga toxin are observed only in S. dysenteriae 1.

The set locus and production of ShET1 is observed almost exclusively in S. flexneri.

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are encoded on the virulence plasmid by the

ipaBCDA operon and are recognized by convalescent

patient sera. Ipa B, C, and D are essential for invasion

and are thought to be involved in signaling the uptake

of the bacteria by the host cell. Additionally, IpaB is

hypothesized to be responsible for release from the

phagocytic uptake vacuole and for induction of apop-

tosis in infected macrophages. The Ipas are secreted

by a dedicated virulence protein secretion apparatus

known as a Type III secretory system. Related secre-

tion systems have been described for a variety of plant

and animal pathogens and share both functional and

sequence homologs. The group of proteins that com-

pose this secretion apparatus are encoded by the mxi/

spa loci present on the virulence plasmid. These pro-

teins have been shown to comprise a needle complex

with both cytoplasmic and transmembrane domains

that span the inner and outer membrane of the bac-

terial cell to allow the release of the Ipa proteins as

well as other secreted proteins. Mutation in any of

these structural components of the secretory appar-

atus results in a loss of virulence, owing to the inabil-

ity to secrete the Ipa proteins. The icsA (virG) locus

on the virulence plasmid is another essential virulence

factor. This outer membrane protein is asymmetric-

ally localized at the old pole of the bacterial cell,

where it catalyzes the polymerization of host cell

actin. This results in the formation of an actin tail at

one end of the bacterium that propels the organism

through the host cell and allows the bacterium to

enter the neighboring cell without leaving the initially

infected host cell and encountering the exterior en-

vironment. Functional homologs of IcsA have been

described in L. monocytogenes, Rickettsia,and

Vaccinia.

0010 In addition to the mucosal damage that results

from the process of epithelial cell invasion and

intracellular multiplication, shigellae also produce

enterotoxins that are presumably responsible for the

diarrheal component of the disease. One serotype,

S. dysenteriae, elaborates a potent toxin (Shiga

toxin) that inhibits protein synthesis and exhibits

both cytotoxic and enterotoxic activity. Some E. coli

strains also produce Shiga-like toxins and have been

associated with outbreaks spread by contaminated

meats and sprouts. The Shiga toxin is produced only

in S. dysenteriae, which causes the most severe form

of dysentery, and may contribute significantly to the

severity of the disease.

0011 Because the virulence plasmid-encoded genes are

essential to the disease process, the ability of the

pathogen to maintain the presence of the plasmid is

critical for pathogenesis. It has been shown that

the expression of the plasmid-encoded virulence

genes destabilizes the maintenance of the plasmid,

presumably owing to the high metabolic demand of

synthesizing the virulence proteins. The virulence

plasmid is lost from the bacterial cell at a higher

frequency at 37



C when virulence protein expression

is upregulated than when the bacteria are grown

under repressing conditions at 30



C. This observa-

tion could affect the detection of the organisms from

clinical, food, and environmental samples using

DNA-based or serological assays.

Shigella in Foods

Regulatory Issues

0012Unlike other bacterial foodborne pathogens, such as

Salmonella spp., Shigella spp. are not associated with

any specific food. The contamination of foods with

Shigella usually results from an infected food handler

or from contaminated water, either from irrigation or

after processing. In the former case, the poor personal

hygiene of one or more individuals can lead to diar-

rheal diseases. Each year, there are a significant

number of outbreaks of shigellosis from consumption

of contaminated foods. Many of these outbreaks are

confined to single countries, but international travel

and global commerce have been influential factors in

the spread of the disease. For example, in 1994, con-

taminated lettuce from one country was the source of

an outbreak of shigellosis that occurred in several

European countries.

0013Many shigellosis outbreaks have been traced to the

consumption of raw or fresh vegetables, particularly

in salads, seafood, bakery products, chicken, and

hamburger. In response to the increasing number of

foodborne outbreaks resulting from consumption of

contaminated fresh produce, the US Food and

Drug Administration published a document entitled

‘Guidance for Industry-Guide to Minimize Micro-

bial Food Safety Hazards for Fresh Fruits and Vege-

tables’ (http://www.foodsafety.gov/ dms/

prodguid.html). These voluntary guidelines address

concerns in the following areas: agricultural and

packinghouse water use, manure management,

worker hygiene, field and packinghouse sanitation,

and transportation. The aim of these recommenda-

tions is to reduce transmission of shigellae and other

enteric pathogens via fresh vegetables and fruits.

0014Another factor in the spread of Shigella sp. is im-

proper storage temperature of contaminated foods.

For the consumer, steps can be taken to reduce risk in

contracting shigellosis: purchase food at reliable

sources, wash food adequately, store food at suitable

(refrigerated) temperatures, cook food adequately,

refrigerate left-overs promptly, and wash cooking

utensils and equipment adequately. The transmission

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SHIGELLA

of Shigella spp. via foods is very efficient, and because

of its low infectious dose, action to remove contamin-

ated food from commerce should be swift.

0015 Probably the most effective means to reduce the

spread of many bacterial and parasitic pathogens is

proper handwashing. Many agencies, including the

World Health Organization, have emphasized ad-

equate hand washing before and during meal prepar-

ation by those that work in food establishments and

the consumer as an effective means in reducing food-

borne outbreaks.

0016 To address the concerns of the introduction of

pathogens during food processing, a program, Hazard

Analysis and Critical Control Point (HACCP), was

established to identify and control specific food-

related practices. This analysis determines where tests

should be implemented to assay for the presence of

foodborne pathogens at critical stages of food produc-

tion. HACCP is an alternative approach to the trad-

itional method of analyzing the final food product.

Although HACCP may be a good monitoring system

for other pathogens commonly known to be associ-

ated with foods, such as Salmonella spp. and E. coli

O157:H7, testing for the presence of shigellae in

foods at this point may not be useful. For example,

contamination of foods with Shigella spp. commonly

occurs between the processing plant and the con-

sumer. However, proper adherence to good manufac-

turing process, such as hand washing and temporary

removal of employees with diarrheal illness, can

reduce outbreaks of shigellosis as well as other

forms of foodborne diseases. As an example, at a

mass gathering, a large outbreak occurred with nearly

half of the attendees (3000 people) developing shigel-

losis from consuming meals prepared where one food

handler was found to be ill.

Survival

0017 Since shigellae are not indigenous to any particular

food, this pathogen may be found in any food matrix.

In the past, foods containing contaminated raw vege-

tables have been implicated in many shigellosis out-

breaks. Other foods that are common vehicles for the

spread of shigellae include tossed salads, potato

salads, chicken, and shellfish. In a recent study, the

survivability of Shigella in packaged vegetables (ster-

ile and unsterile) held at different temperatures (5



C, and room temperature) was reported. The

greatest reduction in number of shigellae recovered

was from the first 24 h. However, the data indicate

that shigellae do survive in the vegetables tested,

albeit after a 3 to 7 log reduction, for at least 10

days (although the initial inoculum was high (10

the final numbers (CFU g

1

) were between 10

and 10

0018In laboratory conditions, Shigella spp. can grow at

temperatures between 6



Cand48



C and at a pH of

between 4.8 and 9.3. In addition, Shigella can survive

at room temperature for up to 50 days in foods such

as milk, flour, eggs, clams, shrimp, and oysters, and

only 5–10 days in acidic foods (e.g., orange juice,

tomato juice, carbonated soft drinks) and 1–2 weeks

in refrigerated, fermented milk. It should be noted

that although shigellae do not survive well in acidic

foods, these organisms are able to survive the acidic

conditions of the stomach.

Detection of Shigella from Foods

0019Foods are not routinely examined for the presence of

shigellae. An outbreak of shigellosis is usually identi-

fied initially from clinical findings and epidemi-

ological investigations. Therefore, 7–10 days may

have passed before the first individual becomes ill.

During this time, the fate and condition of the con-

taminated food is unknown, presenting a challenge to

the laboratory responsible for isolating the etiological

agent. Moreover, the need to rapidly isolate and iden-

tify shigellae in foods is necessary to reduce the spread

of this pathogen.

0020Bacteriological scheme The Bacteriological Analyt-

ical Manual describes one protocol to isolate Shigella

spp. from foods. Twenty-five-gram sample portions

are added to 225 ml of Shigella broth supplemented

with novobiocin (0.5 mgml

1

for S. sonnei;3mgml

1

for other shigellae; this antibiotic is added to suppress

the natural bacterial flora of the food) and held for at

least 10 minutes at room temperature. The broth is

contained in a 500-ml flask and incubated overnight

at 37



C with shaking. Alternative protocols use other

enrichment broths, such as Gram-negative broth,

where food samples are blended in a sterile stomacher

bag, the contents poured into a flask and incubated at



C with shaking for 16–20 h.

0021Recovery of injured cells owing to a lengthy time

delay on foods and/or storage conditions before pro-

cessing a sample for analysis is problematic. Bile salts

and desoxycholate have an adverse affect on growth

of impaired cells; therefore, selective media without

these compounds are recommended. Food samples

are added to 100 ml of tryptic soy broth, the pH

adjusted to 7.0, and then blended. After 8 h of incu-

bation at 37



C, 125 ml of enrichment broth are

added and the incubation extended for an additional

16–20 h.

0022After enrichment in broth, cultures can be plated

on selective (low to high) agar medium. To optimize

the recovery of shigellae from foods, two or three

different ranges of selective agar plates should be used

(see Table 3). MacConkey agar is a low-selectivity

SHIGELLA

5265

medium. Shigella produce colonies that are translu-

cent and slightly pink (Shigella are lactose-negative)

with or without rough edges. Eosin methylene

blue (EMB) and tergitol-7 agar are alternative low-

selectivity agars containing lactose. Colorless col-

onies on eosin methylene blue plates or bluish

colonies on the yellow–green tergitol-7 agar are indi-

cative of Shigella. The preferred media for Shigella

isolation are desoxycholate and xylose–lysine–deso-

xycholate (XLD) agars, intermediate selective media.

Shigella colonies on XLD agar medium are translu-

cent and red (alkaline), whereas on desoxycholate

medium, Shigella produce reddish colonies. Although

most Shigella spp. do not ferment xylose, some

species, e.g., S. boydii (variable) may be missed, and

therefore, plating on XLD and MacConkey agar

plates is recommended. Highly selective media in-

clude Salmonella–Shigella and Hektoen agars, media

designed to differentiate and isolate Salmonella and

Shigella spp. from other enteric bacteria. However,

some Shigella spp., such as S. dysenteriae type I,

are unable to grow on highly selective Salmonella–

Shigella medium. Shigella produce colorless, translu-

cent colonies on this agar. Colonies of Shigella on

Hektoen agar appear to be green and moist, as do

colonies from Salmonella spp. (some are blue green

with or without black centers); E. coli strains, and

other coliforms, such as Klebsiella and Enterobacter,

form yellow, yellow–orange or salmon colonies. All

presumptive colonies are inoculated into semisolid

(motility) test agar for further testing; Shigella spp.

are nonmotile.

0023 Biochemical tests are used to further identify Shi-

gella spp. Suspected colonies that are Gram-negative,

nonmotile rods are inoculated on to lysine iron, Kli-

ger iron, or triple sugar iron agar. On triple sugar iron

agar slants, Shigella typically produce acid from glu-

cose utilization (yellow butt) with no gas production,

and because they do not utilize lactose or sucrose, the

slant retains its color (red); no black color is produced

in the butt, indicating the absence of H

S(Table 4).

Further biochemical characterizations show that Shi-

gella spp. are negative for phenylalanine deaminase,

sucrose, and lactose fermentation, do not utilize cit-

rate, acetate, KCN, malonate, inositol, adonitol, and

salicin, and lack lysine decarboxylase. Shigellae are

negative for the Voges–Proskauer test (S. sonnei and

S. boydii serotype 13 are positive). However, all shi-

gellae are methyl red-positive and are unable to pro-

duce acid from glucose and other carbohydrates (acid

and gas production occur with S. flexneri serotype 6,

S. boydii serotypes 13 and 14, and S. dysenteriae 3).

S. dysenteriae type I is catalase-negative, and S. son-

nei has ornithine decarboxylase activity.

0024Table 4 and 5 show key biochemical reactions to

differentiate Shigella from E. coli and also to distin-

guish each Shigella sp. from each other. Growth on

Christensen citrate, sodium mucate, or acetate agar is

one characteristic that discriminates between E. coli

and Shigella; shigellae are unable to utilize citrate,

acetate, or mucate as sole carbon source.

0025Other biochemical tests are used to identify the

serotypes of Shigella. The ability to utilize mannitol,

dulcitol, xylose, rhamnose, raffinose, glycerol, and

indole and the presence of ornithine decarboxylase

tbl0004Table 4 Tests to differentiate Shigella spp. from E. c oli

Test Reaction

Shigella E. coli

Motility þ

Gas from glucose 

Lysine decarboxylase þ

Christensen’s citrate þ

Acetate þ

Mucate þ

Most positive, some negative (enteroinvasive E. c oli are nonmotile).

Some strains of S. flexneri 6 produce small amounts of gas from glucose.

Some exceptions.

Enteroinvasive E. coli are also negative.

tbl0003 Table 3 Growth characteristics of Shigella, E. coli, and Salmonella on selective media

Organism Medium Colonyappearance

Shigella MacConkey Colorless (lactose nonfermentor); S. sonnei colonies are colorless to pink

, translucent, flat with

jagged edges

Hektoen Green and moist

XLD Colorless

E. coli MacConkey Lactose fermentor; flat, pink colonies surrounded by darker pink region (indicates sorbitol

fermentors, nonsorbitol fermentors form colorless colonies)

Hektoen Yellow/yellow orange, salmon

XLD Yellow

Salmonella MacConkey Colorless (lactose nonfermentors)

Hektoen Green

XLD Red with black center

S. sonnei may ferment lactose slowly (> 40 h). Form I vs. Form II; smooth to rough look as a result of the loss of a 120-MDa virulence plasmid.

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