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© 2009 Kenneth Todar, PhD

c
 is a genus of gamma proteobacteria in the family



 Shigellae are Gram-negative, nonmotile, non-spore
forming, rod-shaped bacteria, very closely related to ×


 
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Shigellosis is an infectious disease caused by various species of c 

People infected with c 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 diarrhe a 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. ?

c  were discovered over 100 years ago by the Japanese

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


c 
c 
c 


c


 also known as Gr ? ?c  accounts for
over two-thirds of the shigellosis in the United States. c


or Gr ? c  accounts for almost all of the rest. Other types of
c are rare in this country, although they are important causes of
disease in the developing world. One type, c

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causes deadly epidemics in many developing regions and nations. ?

 



Determining that c  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 c  is involved, and which
antibiotics, if any, would be best for treatment. ?

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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 c  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. ?

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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 withc   may subsequently
develop pains in their joints, irritation of the eyes, and painful urination.
This condition is called m¬r'
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r . 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 c
  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".] ?

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Hemolytic uremic syndrome (HUS) can occur after c
 type 1
infection. Convulsions may occ ur 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. ?

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

fecal-oral route. c  are present in the diarrheal stools of infected
persons while they are ill and for a week or two afterwards. Most c 
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 a re at high risk of becoming
infected. The spread of c 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 becau se 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.c  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. c  can also be transmitted by flies. Flies can breed in
infected feces and then contaminate food. c  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. ?

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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 c 
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 c  in animal models, and
c -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 c
 , and subsequent
inactivation of native O-antigen genes (30). Serotypic diversity due to the
variations in O antigen is seen among c  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 c  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   vaccine development that are under active
investigation are: 1) parenteral O-specific polysaccharide conjugate
vaccines; 2) nasal proteosomes delivering c  LPS; and 3) live,
attenuated invasive   deletion mutants that are administered
orally. ?

Several live attenuated c  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 c  vaccine derivatives are also being evaluated as
multivalent mucosal vaccines able to deliver bacterial antigens to the gut
associated lymphoid tissues (GALT). ?

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In the United States, there are approximately 14,000 laboratory-
confirmed cases of shigellosis and an estimated 448,240 total cases (85%
due to c
 ) 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, c

  predominates. Epidemics of c

  type 1 have occurred in Africa and Central America with case
fatality rates of 5-15%. ?

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c   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.


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In order for c
  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



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(Vir).

When the bacterium grows at 37 oC, the virulence protein VirF induces the
expression of the VirB protein. The VirB protein then activates the 
and  promoters leading to expression of the  and operons.
This results in the synthesis and assembly of a protein complex called the
$! ?¬r

 
. When the bacterium makes contact with the
epithelial cell membrane, the translocon becomes activate d 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.

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Extracellular c
  cells are nonmotile, but intracellular bacteria
move to occupy the entire cytoplasm of the infected cell, and they are
able to spread between cells. The genes necessary for intracellular and
intercellular spreading are 
 and 


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 c
  between adjacent cells is mediated
via the product of the 
 gene. The 
 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  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.

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Following host epithelial cell invasion and penetration of the colonic

mucosa, c  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 c  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 c  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
c  colitis.
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All virulent strains of c   possess a large 220kb plasmid that
mediates its virulence properties. This so -called the 


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has been shown to encode the genes for several aspects of c 
virulence, including

- Adhesins that are involved in the adherence of bacteria on to the surface

of target epithelial cells

- The production of invasion plasmid antigens (Ipa) that have a direct role
in the c  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  genes

The presence of this plasmid was discovered in the 1980s, after the
observation that essentially the entire chromosome of c
  could be
transferred to ×

  without reconstituting the virulence phenotype of the
donor. However, the ability to invade tissue culture cells was transferred
to ×

  by the conjugal mobilization of this plasmid from c
 

(see below)
Figure 3. Circular map of the large virulence plasmid of c . 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. Tn  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 c  
Infect Immun. 2001 May; 69(5): 3271�3285.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=98286

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

island-like cluster that consists of 38 ORFs of the   operons
within a stretch of 32 kb of the plasmid. Genes within this locus are
critical for c  invasion of mammalian cells, although certain genes
outside this region are required for optimal invasion of tissue culture cells.

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Recent genetic analyses suggest that shigellae do not constitute a distinct
genus as traditionally believed but rather are within the genus of ×

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much like the enteric pathogenic ×

 . These analyses indicate that
c  emerged from ×

  seven or eight independent times during
evolution, leading to three clusters of c , each of which contains
serotypes from multiple traditional species, and four or five additional
forms, each of which contains one traditional serotyp e. The three main
c  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 c  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
c  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 t he plasmid could be obtained by
genetic comparison of virulence plasmids of the seven different c 
evolutionary groups. Subsequent to the acquisition of the virulence
plasmid, divergence of c  clones from ×

  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
c . A quintessential feature of c  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 ×

 . Thus, the acquisition and evolution of the 
 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  (
), which mediates
actin assembly on c , and  and , the regulators of the 
and   loci, were key to the emergence of c . Since all
c  serotypes contain these loci, they were probably all present on
the prototypic virulence plasmid.

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The  ?¬ $
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  and
enterohemorrhagic ×


  (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 may
include 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 dia gnosis 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 (LD 50) 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.

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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.



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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 cell
membrane known as Gb3 and the complex enters the cell. When the
protein is inside the cell, the A s ubunit 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 vascula r
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.

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Shiga toxin-producing ×


  is a type of enterohemorrhagic ×


  (EHEC) bacteria that can cause illness ranging from mild intestinal
disease to severe kidney complications. Enterohemorrhagic ×

  include
the relatively important serotype ×

  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 ×


  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.