Shigella and Shigellosis

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Shigella and Shigellosis

© 2009 Kenneth Todar, PhD

Shigella is a genus of gamma proteobacteria in the familyEnterobacteriaceae. Shigellae are Gram-negative, nonmotile, non-spore

forming, rod-shaped bacteria, very closely related to Escherichia coli.

Shigellosis

Shigellosis is an infectious disease caused by various species of Shigella.

People infected with Shigella develop diarrhea, fever and stomach cramps

starting a day or two after they are exposed to the bacterium. The

diarrhea is often bloody. Shigellosis usually resolves in 5 to 7 days, but in

some persons, especially young children and the elderly, the diarrhea can

be so severe that the patient needs to be hospitalized. A severe infection

with high fever may also be associated with seizures in children less than

2 years old. Some persons who are infected may have no symptoms at

all, but may still transmit the bacteria to others.

Shigella were discovered over 100 years ago by the Japanese

microbiologist, Shiga, for whom the genus is named. There are four

species of Shigella: S. boydii, S. dysenteriae, S. flexneri, and S.

sonnei. Shigella sonnei, also known as Group D Shigella, accounts for

over two-thirds of the shigellosis in the United States. Shigella flexneri,

or Group B Shigella, accounts for almost all of the rest. Other types of

Shigella are rare in this country, although they are important causes of

disease in the developing world. One type, Shigella dysenteriae type 1,

causes deadly epidemics in many developing regions and nations.

Diagnosis

Determining that Shigella is the cause of the illness depends on laboratory

tests that identify the bacteria in the stool of an infected person. Some of



the tests may not be performed routinely, so the bacteriology laboratory

should be instructed to look for the organism. The laboratory can also do

tests to determine which type of Shigella is involved, and which

antibiotics, if any, would be best for treatment.

Figure 1. Several media have been designed to selectively grow

enteric bacteria and allow differentiation of Salmonella and

Shigella f rom E. coli. The primary plating media shown here are

eosin methylene blue (EMB) agar, MacConkey agar, ENDO agar,

Hektoen enteric (HE) agar and Salmonella-Shigella (SS) agar.

Treatment

Shigellosis can usually be treated with antibiotics. The antibiotics

commonly used are ampicillin, trimethoprim/sulfamethoxazole (also

known as Bactrim or Septra), nalidixic acid and the fluoroquinolone,

ciprofloxacin. Appropriate treatment kills the bacteria present in the

gastrointestinal tract and shortens the course of the illness.

Some Shigella have become resistant to antibiotics and inappropriate use

of antibiotics to treat shigellosis can make the organisms more resistant

in the future. Persons with mild infections will usually recover quickly

without antibiotic treatment. Therefore, when many persons in a



community are affected by shigellosis, antibiotics are sometimes used

selectively to treat only the more severe cases. Antidiarrheal agents such

as loperamide (Imodium) or diphenoxylate with atropine (Lomotil) are

likely to make the illness worse and should be avoided.

R eiter's syndrome

Persons with diarrhea usually recover completely, although it may be

several months before their bowel habits are entirely normal. About 3%

of persons who are infected with Shigella flexneri may subsequently

develop pains in their joints, irritation of the eyes, and painful urination.

This condition is called R eiter's syndrome. It can last for months or

years, and can lead to chronic arthritis which is difficult to treat. Reiter's

syndrome is a late complication of S. flexneri infection, especially in

persons with a certain genetic predisposition, namely HLA-B27. [Human

Leukocyte Antigen B27 (HLA-B27) is a class I surface antigen in the major

histocompatibility complex (MHC) on chromosome 6 and presents

microbial antigens to T-cells. HLA-B27 has been strongly associated with

a certain set of autoimmune diseases referred to as the "seronegative

spondyloarthropathies".]

Hemolytic Uremic Syndrome (HUS)

Hemolytic uremic syndrome (HUS) can occur after S. dysenteriae type 1

infection. Convulsions may occur in children; the mechanism may be

related to a rapid rate of temperature elevation or metabolic alterations,

and is associated with the production of the Shiga toxin, which is

discussed below.

Transmission

Shigella is transmitted from an infected person to another usually by a

fecal-oral route. Shigella are present in the diarrheal stools of infected

persons while they are ill and for a week or two afterwards. Most Shigella

infections are the result of the bacterium passing from stools or soiled



fingers of one person to the mouth of another person. This happens when

basic hygiene and handwashing habits are inadequate. It is particularly

likely to occur among toddlers who are not fully toilet -trained. Family

members and playmates of such children are at high risk of becoming

infected. The spread of Shigella from an infected person to other persons

can be stopped by frequent and careful handwashing with soap, practiced

by all age groups.

Part of the reason for the efficiency of transmission is because a very

small inoculum (10 to 200 organisms) is sufficient to cause infection. As a

result, spread can occur easily by the fecal-oral route and readily occurs

in settings where hygiene is poor.

Epidemics may be foodborne or waterborne. Shigella infections may be

acquired from eating food that has become contaminated by infected food

handlers. Vegetables can become contaminated if they are harvested

from a field with contaminated sewage or wherein infected field workers

defecate. Shigella can also be transmitted by flies. Flies can breed in

infected feces and then contaminate food. Shigella infections can be

acquired by drinking or swimming in contaminated water. Water may

become contaminated if sewage runs into it, or even if someone with

shigellosis swims or bathes or, worse, defecates, in it.

Immunity and Vaccines

Once someone has had shigellosis, they are not likely to get infected with

that specific type again for at least several years. However, they can still

get infected with other types of Shigella. Presumably, this immunity is

due to secretory IgA. Circulating antibodies can also be detected in

immune individuals. Although CMI may not be ruled out, the cellular

immune response is ineffective against Shigella in animal models, and

Shigella-specific cytotoxic T lymphocytes have not been isolated from

convalescent individuals.



In addition, factors that permit the bacterium to optimize its lifestyle in

the human colon may also have been acquired by means of horizontal

gene transmission from other enteric bacteria in the colon after

acquisition of the prototypic virulence plasmid. An example of this is the

acquisition by horizontal transfer of O-antigen genes, such as those

present on the virulence plasmid of S. sonnei , and subsequent

inactivation of native O-antigen genes (30). Serotypic diversity due to the

variations in O antigen is seen among Shigella strains. Such diversity

likely facilitates evasion of the host humoral immune response.

Studies are underway around the world to develop a vaccine to prevent

shigellosis. Since the virulence of Shigella is well-understood, and

considering the present art of vaccine development, it seems that

vaccination should be feasible. The need of the vaccine is based on the

burden of disease globally: there are 160 million cases of shigellosis in

the world each year, resulting in about 1.5 million deaths. Three

approaches to shigella vaccine development that are under active

investigation are: 1) parenteral O-specific polysaccharide conjugate

vaccines; 2) nasal proteosomes delivering Shigella LPS; and 3) live,

attenuated invasive shigella deletion mutants that are administered

orally.

Several live attenuated Shigella vaccines of different serotypes have been

shown to be safe, immunogenic, and in one case, effective against

challenge with virulent strains. The ability to invade epithelial cells

remains critical for the success of these vaccine candidates. Live, orally

administered Shigella vaccine derivatives are also being evaluated as

multivalent mucosal vaccines able to deliver bacterial antigens to the gut

associated lymphoid tissues (GALT).

Incidence and R isk of Infection



In the United States, there are approximately 14,000 laboratory-

confirmed cases of shigellosis and an estimated 448,240 total cases (85%

due to S. sonnei ) that occur each year, according to CDC. Groups at

increased risk of shigellosis include children in child-care centers and

persons in custodial institutions, where personal hygiene is difficult to

maintain.

In the developing world, S. flexneri predominates. Epidemics of S.

dysenteriae type 1 have occurred in Africa and Central America with case

fatality rates of 5-15%.

Pathogenesis of Shigella flexneri

Shigella flexneri causes bacillary dysentery, the symptoms of which

include abdominal pain, diarrhea, fever, vomiting and blood or mucus in

the stool. The bacteria are transmitted by the fecal-oral route, and

through contaminated food and water. Once ingested, the bacteria

survive the gastric environment of the stomach and move on to the large

intestine. There, they attach to and penetrate the epithelial cells of the

intestinal mucosa. After invasion, they multiply intracellularly and spread

to neighboring epithelial cells, resulting in tissue destruction and

characteristic pathology of shigellosis.

Entry of Shigella flexneri into Epithelial Cells

In order for S. flexneri to enter an epithelial cell, the bacterium must first

adhere to its target cell. It is then internalized by a process which is

similar to the mechanism of phagocytosis. Generally, the bacterium

adheres to the membrane of the cell and is internalized via an endosome,

which it subsequently lyses to gain access to the cytoplasm where

multiplication occurs.

To aid its entry into the epithelial cell, the bacterial DNA encodes a

number of plasmid and chromosomal proteins. These proteins are the

invasion plasmid antigens (Ipa), surface presentation antigens



(Spa), membrane excretion proteins (Mxi), and virulence proteins

(Vir).

When the bacterium grows at 37oC, the virulence protein VirF induces the

expression of the VirB protein. The VirB protein then activates the ipa,

mxi, and spa promoters leading to expression of the spa and mxi operons.

This results in the synthesis and assembly of a protein complex called the

Mxi-Spa translocon. When the bacterium makes contact with the

epithelial cell membrane, the translocon becomes activated and secretes

the pre-synthesized Ipa proteins. IpaB, IpaC and IpaA associate to form a

complex which interacts with the host epithelial cell membrane to induce

a cascade of cellular signals which will lead to the internalization of the

bacterium via an endosome. The Ipa proteins are also required for escape

from the endosome.

Figure 2. Electron Micrograph of Shigella in a membrane-enclosed

endosome of an epithelial cell

Intracellular and Intercellular Spread

Extracellular S. flexneri cells are nonmotile, but intracellular bacteria

move to occupy the entire cytoplasm of the infected cell, and they areable to spread between cells. The genes necessary for intracellular and

intercellular spreading are virG (icsA) and icsB.

After entry into the cell, intracellular movement occurs if the bacterium

expresses both an Olm ("organelle-like movement") phenotype and an



alternative Ics phenotype. The expression the Olm phenotype allows the

bacteria to "slide" along actin stress cables inside the host cell, while the

expression of the Ics phenotype allows the bacteria to "spread" or infect

adjacent cells.

Specifically, movement of S. flexneri between adjacent cells is mediated

via the product of the virG (icsA) gene. The icsA gene elicits actin

polymerization at the poles of the bacteria and induces the formation of

protrusions. In some instances, these tightly packed actin filaments

appear to form a cylinder. The bacteria in the protrusions can move

through the host cell and penetrate into an adjacent cell w ithout coming

in contact with the extracellular medium where they would be rendered

nonmotile.

The mxiG gene is required for Ipa protein secretion, and is also essential

for entry. This gene and others in the Mxi-Spa translocon are also

required for intercellular dissemination.

Pathological Effects

Following host epithelial cell invasion and penetration of the colonic

mucosa, Shigella infection is characterized by degeneration of the

epithelium and inflammation of the lamina propria. This results in

desquamation and ulceration of the mucosa, and subsequent leakage of

blood, inflammatory elements and mucus into the intestinal lumen.

Patients suffering from Shigella infection will therefore pass frequent,

scanty, dysenteric stool mixed with blood and mucus, since, under these

conditions, the absorption of water by the colon is inhibited. This is in

opposition to the diarrheal symptoms seen in patients suffering from

extensive Shigella colitis, and the pathologic basis for this is unknown. It

is possible that prostaglandin interactions induced by the inflammatory

response to bacterial invasion contribute to diarrhea in patients with

Shigella colitis.



The Large Virulence Plasmid of Shigella flexneri

All virulent strains of Shigella flexneri possess a large 220kb plasmid that

mediates its virulence properties. This so-called the invasion plasmid

has been shown to encode the genes for several aspects of Shigella

virulence, including

- Adhesins that are involved in the adherence of bacteria onto the surface

of target epithelial cells

- The production of invasion plasmid antigens (Ipa) that have a direct role

in the Shigella invasion process

- Transport or processing functions that ensure the correct surface

expression of the Ipa proteins

- The induction of endocytic uptake of bacteria and disruption of endocytic

vacuoles

- The intra- and inter-cellular spreading phenotypes

- The regulation of plasmid-encoded vir genes

The presence of this plasmid was discovered in the 1980s, after the

observation that essentially the entire chromosome of S. flexneri could be

transferred to E. coli without reconstituting the virulence phenotype of the

donor. However, the ability to invade tissue culture cells was transferred

to E. coli by the conjugal mobilization of this plasmid from S. flexneri.

(see below)



Figure 3. Circular map of the large virulence plasmid of Shigella. Outer ring

depicts ORFs and their orientations, color coded according to functional

category: 1. identical or essentially identical to known virulence -associated

proteins (red); 2, homologous to known pathogenesis -associated proteins

(pink); 3. highly homologous to IS elements or transposases (blue); 4. weakly

homologous to IS elements or transposases (light blue); 5. homologous to

proteins involved in replication, plasmid maintenance, or other DNA metabolic

functions (yellow); 6. no significant similarity to any protein or ORF in the

database (brown); 7. homologous or identical to conserved hypothetical ORFs,

i.e., proteins of unknown function (orange); and 8. Tn501 insertion-associated

genes (green). The second ring shows complete IS elements. The third r ing

graphs G+C content, calculated for each ORF and plotted around the mean value

for all ORFs, with each value color coded for the corresponding ORF. Scale is in

base pairs. The figure was generated by Genescene (DNASTAR). Venkatesan,

M.M., et al. Complete DNA Sequence and Analysis of the Large Virulence Plasmid

of Shigella flexneri. Infect Immun. 2001 May; 69(5): 3271ï¿½3285.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=98286

The invasion locus on the virulence plasmid of Shigella is a pathogenicity

island-like cluster that consists of 38 ORFs of the ipa-mxi-spa operons

within a stretch of 32 kb of the plasmid. Genes within this locus are



critical for Shigella invasion of mammalian cells, although certain genes

outside this region are required for optimal invasion of tissue culture cells.

Table 1. Virulence-associated Genes and Functions Encoded by the Large Shigella

Virulence Plasmid

Gene

Protein

Product

MW

Regulatory or effector function

virF 30 kDa positive regulators of the virG and ipa-mxi-spa loci

invA(mxiB) 38 kDa Necessary for invasion (orients ipa gene products in outer

membrane

mxiA 76 kDA Same as above

ippI 18 kDa Same as above

ipaB 62 kDa Necessary for invasion: mediates endocytic uptake of shigellae

ipaC 43 kDa Same as above

ipaA 38 kDa Same as above

ipaD 78 kDa Not necessary for invasion (role unknown)

virB 33 kDa positive regulator of the virG and ipa-mxi-spa loci

virG (icsA) 120 kDaassembles actin tails that propel the bacteria through the cell

cytoplasm and into adjacent cells

ipaH 60 kDahas 5 alleles; IpaH7.8 facilitates the escape of Shigella from

phagocytic vacuoles

shET2 60kDa ShET2 enterotoxin

Evolution of the Shigella virulence plasmid

Recent genetic analyses suggest that shigellae do not constitute a distinct

genus as traditionally believed but rather are within the genus of E. coli ,

much like the enteric pathogenic E. coli . These analyses indicate that

Shigella emerged from E. coli seven or eight independent times during

evolution, leading to three clusters of Shigella, each of which contains



serotypes from multiple traditional species, and four or five additional

forms, each of which contains one traditional serotype. The three main

Shigella clusters are estimated to have evolved 35,000 to 270,000 years

ago, which predates the development of agriculture and makes shigellosis

one of the early infectious diseases of humans.

The defining event each time Shigella arose was almost certainly the

acquisition of an historical precursor of the current-day virulence plasmid.

The data also suggest that the loss of specific catabolic pathways

(inability to utilize lactose and mucate and to decarboxylate lysine), loss

of motility, and expansion of O-antigen diversity that are characteristic of

Shigella strains occurred more recently than the acquisition of the

plasmid.

Since the plasmid was acquired at distinct times, one would predict that

differences reflecting the evolution of the plasmid could be obtained by

genetic comparison of virulence plasmids of the seven different Shigella

evolutionary groups. Subsequent to the acquisition of the virulence

plasmid, divergence of Shigella clones from E. coli would involve clonal

divergence (accumulation of mutations by base substitution), horizontal

transfer of genetic material from other species, and loss of gene

sequences that interfere with pathogenicity.

Certain horizontal gene transfer events have been key to the evolution of

Shigella. A quintessential feature of Shigella is its ability to invade

mammalian cells and access the cell cytoplasm, defining a niche unique

among enteric Gram-negative bacteria, with the exception of

enteroinvasive E. coli . Thus, the acquisition and evolution of the ipa-mxi-

spa pathogenicity island, which encodes all of the genes required for cell

invasion and phagolysosomal lysis, permitted a major alteration in

pathogenesis. Likewise, the acquisition of virG (icsA), which mediates

actin assembly on Shigella, and virF and virB, the regulators of the virG

and ipa-mxi-spa loci, were key to the emergence of Shigella. Since all



Shigella serotypes contain these loci, they were probably all present on

the prototypic virulence plasmid.

The Shiga Toxin

The Shiga toxin, also called the verotoxin, is produced by Shigella

dysenteriae and

enterohemorrhagic Escherichia coli (EHEC), of which the strain O157:H7

has become the best known.

The syndromes associated with shiga toxin include dysentery,

hemorrhagic colitis, and hemolytic uremic syndrome. The name is

dependent upon the causative organism and the symptoms, which mayinclude severe diarrhea, abdominal pain, vomiting, and bloody urine (in

the case of hemolytic uremic syndrome).

The onset of symptoms is generally within a few hours, with higher doses

leading to more rapid onset. There is no antidote for the toxin. Supportive

care requires maintenance of fluid and electrolyte levels, and monitoring

and support of kidney function.

Immunoassays are available for rapid diagnosis of the toxin.

Inactivation of the toxin is achieved by steam treatment, oxidizing agents

such as bleach, and chemical sterilizing agents such as glutaraldehyde.

The toxicity of Shiga Toxin for the mouse (LD50) is <20 micrograms/kg by

intravenous or intraperitoneal administration. There is no published data

on the inhalation toxicity of Shiga toxin. However, there are often indirect

effects on the lungs when the toxin is taken in as a food contaminant.

Table 2. The toxin has been given several trivial names depending

on the bacterium that produces it and the gene that encodes it.



Source

Organism

Gene

Designation

Toxin

Name

Older

Names

Shigella

dy senteriae, type I

stxShiga toxin

(Stx)

Shiga toxin

Escherichia

coli stx1

Shiga toxin 1

(Stx1)

Shiga-like toxin I,

Verotoxin 1

stx2Shiga toxin 2

(Stx2)

Shiga-like toxin II,

Verotoxin 2

Structure of the Toxin

The toxin has a molecular weight of 68,000 da. It is a multi-subunit

protein made up one molecule of an A subunit (32,000 molecular weight)

responsible for the toxic action of the protein, and five molecules of the B

subunit (7,700 molecular weight) responsible for binding to a specific cell

type.

Mechanism of Action

The toxin acts on the lining of the blood vessels, the vascular

endothelium. The B subunits of the toxin bind to a component of the cellmembrane known as Gb3 and the complex enters the cell. When the

protein is inside the cell, the A subunit interacts with the ribosomes to

inactivate them. The A subunit of Shiga toxin is an N-glycosidase that

modifies the RNA component of the ribosome to inactivate it and so bring

a halt to protein synthesis leading to the death of the cell. The vascular

endothelium has to continually renew itself, so this killing of cells leads to

a breakdown of the lining and to hemorrhage. The first response is

commonly a bloody diarrhea. This is because Shiga toxin is usually taken

in with contaminated food or water.

The toxin is effective against small blood vessels, such as found in the

digestive tract, the kidney, and lungs, but not against large vessels such

as the arteries or major veins. A specific target for the toxin appears to



the vascular endothelium of the glomerulus. This is the filtering structure

that is a key to the function of the kidney. Destroying these structures

leads to kidney failure and the development of the often deadly and

frequently debilitating hemolytic uremic syndrome. Food poisoning with

Shiga toxin often also has effects on the lungs and the nervous system.

Shiga Toxin-Producing Escherichia coli (STEC)

Shiga toxin-producing Escherichia coli is a type of enterohemorrhagic E.

coli (EHEC) bacteria that can cause illness ranging from mild intestinal

disease to severe kidney complications. Enterohemorrhagic E. coli include

the relatively important serotype E. coli O157:H7, but other non-O157

strains, such as O111 and O26, have been associated with shiga toxin

production.

The incubation period for STEC ranges from 1 to 8 days, though typically

it is 3 to 5 days. Typical symptoms include severe abdominal cramping,

sudden onset of watery diarrhea, frequently bloody, and sometimes

vomiting and a low-grade fever. Most often the illness is mild and self-

limited generally lasting 1-3 days. However, serious complications such as

hemorrhagic colitis, Hemolytic Uremic Syndrome (HUS), or postdiarrheal

thrombotic thrombocytopenic purpura (TTP) can occur in up to 10% of

cases.

Cases and outbreaks of Shiga toxin-producing Escherichia coli have been

associated with the consumption of undercooked beef (especially ground

beef), raw milk, unpasteurized apple juice, contaminated water, red leaf

lettuce, alfalfa sprouts, and venison jerky. The bacteria have also been

isolated from poultry, pork and lamb. Person-to-person spread via fecal-

oral transmission may occur in high-risk settings like day care centers and

nursing homes.



Although anyone can get infected, the highest infection rates are in

children under age 5. Elderly patients also account for a large number of

cases. Outbreaks have occurred in child-care facilities and nursing homes.

For mild illness, antibiotics have not been shown to shorten the duration

of symptoms and may make the illness more severe in some people.

Severe complications, such as hemolytic uremic syndrome, require

hospitalization.

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Shigella and Shigellosis