Shigellae: Classification, Structure, Virulence factor, Pathogenesis and Laboratory Diagnosis
The History of Discovery
- The agents of bacterial dysentery were first discovered by Chantemesse in 1888. In 1898 they were thoroughly studied by K. Shiga. A novel causative agent of dysentery was described later by S. Flexner and co-workers in 1900.
- At the borderline of 1900s some other agents of bacterial dysentery were isolated. К. Duval in 1904, as well as V. Kruse and co-workers in 1907, and К. Sonne in 1915 revealed the species, able to ferment lactose unlike the previously described isolates.
- Finally all these microbial representatives were placed into separate genus called Shigella in honor of K. Shiga.
- Shigellae cause bacterial dysentery or shigellosis. This infection circulates predominantly among the population of developing countries, affecting near to 150 million people annually with more than 1 million death cases. About 70% of patients are 1-5-year-old children.
- Shigella genus pertains to the family Enterobacteriaceae. The genus contains 4 species: S. dysenteriaе of group A, S. flexneri (group B), S. boydii (group C), and S. sonnei (group D).
- Different shigella species comprise numerous biotypes and serovars.
- Genetic analysis revealed that all shigellae share more than 90% of genomic DNA sequence with escherichiae.
- Therefore, they can be accounted as single genomospecies. However, the evident phenotypic dissimilarities of these bacteria, largely dependent on acquisition of various mobile genetic elements, result in striking differences in their virulence for humans. Hence, they remain to exist as separate genera and species.
Structure and Properties of Shigellae
All shigella closely resemble other Enterobacteriaceae members: small gram-negative non-motile rods without spores, possessing multiple pili. Certain strains can form thin capsule.
- These bacteria readily grow on basic nutrient media with optimal temperature about 37°C, pH 7.2.
- The growth reveals round small convex transparent colonies. In meat peptone broth shigellae produce homogenous turbidity.
- After cultivation in lactose-containing media (McConkey agar, or Ploskirev’s medium) most of shigellae form lactose-negative transparent colonies. S. sonnei can slowly ferment lactose.
- Shigellae are facultatively anaerobic bacteria. They utilize various carbohydrates with acid formation. Individual biovars (Newcastle subsp.) can produce small amounts of gas. All shigellae ferment glucose.
- Most of bacteria, except S. dysenteriaе, ferment mannitol. S. sonnei can metabolize lactose and sucrose within several days.
- The bacteria can’t produce hydrogen sulfide, but certain strains display proteolytic activity with indole formation.
- Shigellae are classified into 4 groups according to their antigenic properties. These groups comprise more than 40 serotypes.
- All of these bacteria contain group-specific O-antigen, some isolates produce capsular K antigen.
- Somatic lipopolysaccharide O-antigen possesses endotoxin activity.
- Pathogenic shigellae produce large number of virulence factors, responsible for microbial adherence, invasiveness, intercellular spread, apoptosis of host immune cells, and intestinal epithelium death that results in severe inflammation of large intestine with hemocolitis.
- Bacterial invasion is controlled by special structures of type III secretion system (so-called secreton III), which includes bacterial injectisome, or needle complex. Once attached to the host cells via needle complex, bacteria inject the number of invasion proteins into target cell.
- These effector proteins re-build cytoskeleton of affected cell, thus promoting intercellular microbial spread and further invasion.
- All virulent Shigellae contain a large 220-kb plasmid harboring pathogenicity island that determines the “invasive phenotype” of bacteria. Invasive proteins are encoded predominantly by ipa/spa (invasive plasmid antigen) genetic locus.
- Deep damage of bowel epithelium is promoted by cytotoxic action of bacterial Shiga toxin (STX toxin), which is encoded by chromosomal stx gene. Maximal toxin production is essential for S. dysenteriaе type 1.Toxin action pattern is very similar with enterohemorrhagic E. coli verotoxins.
- As in EHEC, STX toxin is composed of A and B subunits. Several receptor B-subunits bind to cellular receptor glycolipid Gb3.
- Exotoxin internalization is followed by subunit A cleavage. Toxic A1 fragment possesses RNA N-glycosidase activity and thereby cleaves N-glycosidic bond within 28S ribosomal RNA. Termination of protein synthesis causes the death of host cells.
- LPS-containing endotoxin of shigellae activates phagocytes and other immune cells that is followed by exuberant cytokine release and tissue inflammation.
- Shigellae are not the highly resistant bacteria, but they can survive in the environment within 5-10 days. The most resistant is S. sonnei that keeps viability for months in water and different foodstuffs, e.g. dairy products.
- Bacteria are killed by heating at 56°C within 10-15 minutes and inactivated readily by standard medical disinfectants (chloramine, hypochlorite, phenol, etc.).
Pathogenesis and Clinical Findings in Shigellosis
- Different clinical forms of bacterial dysentery or shigellosis are caused by enteroinvasive shigellae.
- The disease is transmitted by fecal-oral route and direct contact. It is “food, fingers, feces, and flies”-transmitted disorder.
- Water outbreaks of shigellosis are related with S. flexneri, while foodborne disease cases ensue from S. sonnei infection. The disease caused by S. dysenteriae is particularly severe.
- The main sources of infection are the patients with dysentery and bacterial carriers. The disease affects only humans.
- Incubation period lasts from 1 to several days.
- Infectious dose of 10-100 microbial cells is enough to cause the disease in adults (e.g., for S. dysenteriae infection).
- Some shigellae are killed, passing through the stomach. The rest of bacteria comes to the bowel and invade the colon mucosa. Bacteria are specific to the rectal and large intestine mucous membranes.
- The main intestinal entry site for shigellae is the follicle-associated epithelium that covers the mucosa-associated lymph nodes. Special epithelial M cells (microfold cells) are the primary targets for microbial invasion.
- After cell contact with bacterial needle complex, IpaB and IpaC proteins create a pore in eukaryotic cell membrane, and invasive proteins are injected inside the target cells.
- They trigger intracellular actin polymerization that results in membrane pocket formation. This way M-cells engulf and translocate shigellae into the cytoplasm. Bacterial VirG protein activates cell actin attachment to the pole of microbial cell with formation of actin comet.
- Comet bacterial cell is able to move within the infected cells and can readily achieve the neighboring enterocytes (“lateral spread” of shigellae) thus promoting further microbial invasion.
- Intestinal macrophages become invaded in a similar manner. Invasive IpaB protein induces macrophage release of most potent proinflammatory cytokines IL-1 and IL-18 and at the same time triggers phagocyte death via caspase 1-mediated apoptosis, thereby preventing shigellae from the death within macrophages.
- Inflammatory cytokines cause the injury of intestinal wall. But at the same time they activate immune inflammation, attracting neutrophils to the invaded bacteria. Efficient leukocyte reaction restricts the infection up to its termination.
- Massive cytolysis of intestinal epithelium is promoted also by Shiga cytotoxin action.
- All these events lead to severe colon destruction resulting in hemorrhagic colitis. General intoxication is followed by abdominal pain, fever, and hemorrhagic diarrhea with water loss. Intermittent painful rectal spasm (or tenesmus) is characteristic for developed shigellosis.
- The disease can be self-restricted within several days, but profound dehydration and acidosis require urgent therapy and even can cause lethal outcome especially in children.
- The immunity acquired after the dysentery is group- and type-specific but relatively weak and of a short duration. For this reason the disease may recur many times and in some cases may become chronic.
- Shiga toxin as a potent antigen elicits the synthesis of neutralizing antibodies.
Laboratory Diagnosis of Shigellosis
- Reliable results of laboratory examination depend on correct sampling of stool specimen and its immediate inoculation onto a selective and differential medium at the patient’s bedside. The inoculated material should be rapidly delivered to the laboratory.
- As an example, the clinical specimen (feces) should be best collected directly from patient’s rectum by rectal swab and planted immediately after the collection onto McConkey agar, EMB or Ploskirev’s medium.
- Ploskirev’s medium contains meat-peptone agar, lactose, indicator neutral red, and bile salts with brilliant green dye to suppress concomitant microflora. The similar composition is of McConkey agar.
- The plates are incubated at 37°C for 24 hours. When growing, shigellae produce lactose-negative transparent colonies.
- The culture is further isolated in butt-slant double sugar agar (Russel’s medium). It contains meat-peptone agar, 1% lactose, 0.1% glucose, and indicator dye. Inoculation of bacteria is performed both in aerobic and by stab in anaerobic conditions.
- As the result, the color change will appear only in the butt of medium due to glucose fermentation in anaerobic conditions. The slant part of agar would be intact because the most of shigellae are lactose-negative.
- The pure culture obtained is further identified according to its biochemical and serological properties. In the latter case the culture is tested by agglutination reaction with specific sera.
- For rapid identification of DNA of shigellae species in clinical specimens sensitive and reliable molecular genetic tests are applied (e.g., PCR).
- Serological examination has no value in diagnosis of shigellosis.
Treatment and Prophylaxis of Shigellosis
- Taking into account the increased resistance of shigellae to the long list of antimicrobial agents (e.g., ampicillin, trimethoprim-sulfamethoxazole, doxycycline, or chloramphenicol) fluoroquinolones (norfloxacin) and third generation cephalosporins (e.g., cefotaxime or ceftazidime) are most commonly used now for treatment of shigellosis.
- Important measures of supportive symptomatic treatment include urgent infusion therapy to compensate water and electrolyte loss. The treatment of shigellosis with probiotics restores the normal composition of intestinal microflora.
- Efficient vaccines for prevention of shigellosis are not available yet.
- Non-specific prophylaxis of the disease comprises thorough control of water and food microbial contamination, isolation and adequate treatment of patients with laboratory confirmation of the recovery, the detection and treatment of carriers, adequate disinfection measures, the maintenance of sanitary and hygienic regimens according to the actual regulation acts, etc.