Bacteriology at UW- Madison

The Microbial World

University of Wisconsin - Madison

Shigella and shigellosis

Shigella is a genus of the bacterial family Enterobacteriaceae. Shigellae are Gram-negative, nonmotile, non-spore forming, rod-shaped bacteria, very closely related to Escherichia coli.

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 teansmit the Shigella bacteria to others.

Shigella were discovered over 100 years ago by a Japanese microbiologist named Shiga, for whom the genus are named. There are four species of Shigella: boydii, dysenteriae, flexneri, and 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 from 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 actually 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.

Reiter'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 Reiter'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.

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  are transmitted from an infected person to another who become infected. Shigella are present in the diarrheal stools of infected persons while they are sick 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, a practice that is important among 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 easily occur by the fecal-oral route and occurs in areas where hygiene is poor. Epidemics may be foodborne or waterborne. Shigella can also be transmitted by flies.

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. Flies can breed in infected feces and then contaminate food. Shigella infections can also 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, much less, 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, although CMI may not be ruled out. Circulating antiboies can also be detected in immune individuals.

Currently, there is no vaccine to prevent shigellosis. However, since the virulence of Shigella is well-understood, and considering the present art of vaccine development, it seems that vaccination should be feasable.

OFF THE WALL
On the topic of vaccines, the following information is from the Walter Reed Army Institute of Research, Walter Reed Army Institute of Research, Vaccines: The Best Revenge

"So far, the institute has four vaccines in the works. 'Ideally, the goal would be to have one vaccine that will protect against multiple pathogens that can easily be given to deploying soldiers,' said Maj. David Katz, a senior clinical investigator at WRAIR. 'So soldiers can take it before they deploy to an area, and they'll be protected.'

A vaccine to combat Shigella flexneri, called SC602, was developed along with The Institut Pasteur. Since 1992 it has undergone clinical trials in the States and Bangladesh. 'The wonderful  thing about the shigella vaccines is... the bacteria (used in them) are alive but weakened to diminish the amount of symptoms,' Katz said. 'The body thinks it's infected and gives an immune response, but you don't get infected like a natural infection because the bacteria don't spread from cell to cell.'

Receiving the oral vaccine before deploying is key, Katz said. 'Most of the soldiers will get hit right when they arrive in a new area, either because they're eating on the economy or they're in a new area and their system has not been primed.'

'Another reason to give the vaccine ahead of time is because of potential side effects,' said Dr. Thomas Hale, Chief of the Department of Enteric Infections at WRAIR. 'The vaccine can cause some short-term fevers and mild diarrhea in 20 percent of the people who receive it, so soldiers need to take it well before they get on a plane.'

A vaccine for Shigella sonnei, which often attacks travelers and stateside daycare centers, is a possible stand alone product, Hale said. 'This one vaccine could make a significant difference in the health of soldiers deployed to the Middle East (where 90 percent of outbreaks occur) and the developing world.'

 Drs. Malabi Venkatesan and Antoinette Hartman from WRAIR developed the oral vaccine, called WRSS1, that is currently in clinical trials in conjunction with the University of Maryland Medical School and the National Institute of Allergy and Infectious Diseases.

The Department of Enteric Infections at WRAIR has teamed up with the Israel Defense Force for a vaccine trial evaluating WRSS1 this winter. 'Israel has cities that are very Westernized, but almost everyone has a compulsory military obligation, so they go from cities to field posts and the incidence of diarrheal disease is significant,' Katz said.

To combat the deadly form of diarrhea, dysentery--also called bloody diarrhea--WRAIR researchers are working with the Bloomberg School of Public Health at Johns Hopkins University to test the oral Walter Reed Shigella-Dysentery-1 vaccine, WRSD1.

The other diarrhea-causing bacteria WRAIR and the Navy Medical Research Unit researchers are trying to disable is E. coli. Whereas shigella bacteria invades a cell's wall and moves from cell to cell to spread the disease, E. coli prefers to stick to the intestine's lining, homestead and crank out toxins that cause diarrhea. To outsmart the unwanted tenant, researchers are trying to make antibodies that will prevent squatters from colonizing because they can't stick to the intestine. The vaccine's been tested in a time-release capsule form as well as a transdermal patch,

'It should be easy for the soldier to use: Just pop the patch on and that's it,' Katz said. Though having one vaccine to combat all major forms of infectious diarrhea is a ways off, the quest to prevent soldiers from needing to run, trot and quick step will be WRAIR researchers' best revenge on Montezuma."

Incidence and Risk of Infection

Meanwhile, back in the USA, 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. 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%.

In the United States, groups at increased risk of shigellosis include children in child-care centers and persons in custodial institutions, where personal hygiene is difficult to maintain; Native Americans; orthodox Jews; international travelers; men who have sex with men; and those in homes with inadequate water for handwashing.

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 37^oC, 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 Shigellain 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 are able 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 "organelle-like movement" (Olm) 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 ellicits 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 without 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 contributes to diarrhea in patients with Shigella colitis.

The Large Virulence Plasmid of Shigella flexneri

All virulent strains of Shigella flexneri possess a large 230kb 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

- Ligands 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 intercellular 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.The 230kb plasmid has been subjected to SalI endonuclease digestion and 23 fragments labeled A through F have been identified and mapped.

Figure 3.The Circular SalI restriction map of Shigella flexneri 2a plasmid pMYSH6000. Adapted from Hale (1991). The large Shigella plasmid encodes many of the virulence-associated genes which are summarized in the table below.
 
 

The Shiga Toxin

The Shiga toxin, also called the verotoxin, is produced by the bacteria 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 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 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.

Structure of the Toxin
The toxin has a molecular weight of 68,000 da. It is a multisubunit 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 cell membrane 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.