Shigellae

Dysentery is an infectious disease characterized by general intoxication of the body, diarrhea and a specific lesion of the mucous membrane of the large intestine. It is one of the most common acute intestinal diseases in the world. Dysentery has been known since ancient times under the name of "bloody diarrhea", but its nature turned out to be different. In 1875, the Russian scientist F. A. Lesh isolated the amoeba Entamoeba histolytica from a patient with bloody diarrhea, in the following 15 years the independence of this disease was established, for which the name amebiasis remained.

The causative agents of dysentery proper are a large group of biologically similar bacteria, united in the genus Shigella. The causative agent was first discovered in 1888 by A. Chantemes and F. Vidal; in 1891 it was described by A. V. Grigoriev, and in 1898 K. Shiga, using serum obtained from a patient, identified the causative agent in 34 patients with dysentery, finally proving the etiological role of this bacterium. However, in subsequent years, other causative agents of dysentery were discovered: in 1900 - by S. Flexner, in 1915 - by K. Sonne, in 1917 - by K. Stutzer and K. Schmitz, in 1932 - by J. Boyd, in 1934 - by D. Large, in 1943 - by A. Sax.

Currently, the genus Shigella includes more than 40 serotypes. All of them are short, non-motile, gram-negative rods that do not form spores or capsules and grow well on ordinary nutrient media, do not grow on a starvation medium with citrate or malonate as the sole carbon source; do not form H2S, do not have urease; the Voges-Proskauer reaction is negative; they ferment glucose and some other carbohydrates to form acid without gas (except for some biotypes of Shigella flexneri: S. manchester and S. newcastle); as a rule, they do not ferment lactose (except for Shigella Sonnei), adonitol, salicin and inositol, do not liquefy gelatin, usually form catalase, do not have lysine decarboxylase and phenylalanine deaminase. The G + C content in DNA is 49-53 mol %. Shigella are facultative anaerobes, the optimum temperature for growth is 37 °C, they do not grow at temperatures above 45 °C, the optimal pH of the medium is 6.7-7.2. Colonies on dense media are round, convex, translucent, in case of dissociation, rough R-form colonies are formed. Growth on MPB is in the form of uniform turbidity, rough forms form a sediment. Freshly isolated cultures of Shigella Sonnei usually form colonies of two types: small round convex (phase I), large flat (phase II). The nature of the colony depends on the presence (phase I) or absence (phase II) of a plasmid with m.m. 120 MD, which also determines the virulence of Shigella Sonnei.

The international classification of Shigella is based on their biochemical characteristics (mannitol-non-fermenting, mannitol-fermenting, slowly lactose-fermenting Shigella) and the features of their antigen structure.

Shigella have O-antigens of varying specificity: common to the Enterobacteriaceae family, generic, species, group and type-specific, as well as K-antigens; they do not have H-antigens.

The classification takes into account only group and type-specific O-antigens. According to these features, the genus Shigella is divided into 4 subgroups, or 4 species, and includes 44 serotypes. Subgroup A (species Shigella dysenteriae) includes shigella that do not ferment mannitol. The species includes 12 serotypes (1-12). Each serotype has its own specific type antigen; antigenic links between serotypes, as well as with other species of shigella, are weakly expressed. Subgroup B (species Shigella flexneri) includes shigella that usually ferment mannitol. Shigella of this species are serologically related to each other: they contain type-specific antigens (I-VI), by which they are divided into serotypes (1-6/' and group antigens, which are found in different compositions in each serotype and by which serotypes are divided into subserotypes. In addition, this species includes two antigenic variants - X and Y, which do not have type antigens, they differ in sets of group antigens. Serotype S.flexneri 6 does not have subserotypes, but it is divided into 3 biochemical types by the features of fermentation of glucose, mannitol and dulcitol.

The lipopolysaccharide antigen O in all Shigella flexneri contains the group antigen 3, 4 as the main primary structure, its synthesis is controlled by a chromosomal gene localized near the his-locus. Type-specific antigens I, II, IV, V and group antigens 6, 7, 8 are the result of modification of antigens 3, 4 (glycosylation or acetylation) and are determined by the genes of the corresponding converting prophages, the integration site of which is located in the lac-pro region of the Shigella chromosome.

The new subserotype S.flexneri 4 (IV:7, 8), which appeared in the country in the 1980s and became widespread, differs from subserotypes 4a (IV;3,4) and 4b (IV:3, 4, 6), and arose from the variant S.flexneri Y (IV:3, 4) as a result of its lysogenization by converting prophages IV and 7, 8.

Subgroup C (Shigella boydix species) includes shigella that usually ferment mannitol. Members of the group are serologically distinct from each other. Antigenic links within the species are weak. The species includes 18 serotypes (1-18), each with its own main type antigen.

Subgroup D (Shigella sonnei species) includes shigella that usually ferment mannitol and are capable of slowly (after 24 hours of incubation and later) fermenting lactose and sucrose. The species S. sonnei includes one serotype, but colonies of phases I and II have their own type-specific antigens. Two methods have been proposed for the intraspecific classification of Shigella sonnei:

• dividing them into 14 biochemical types and subtypes according to their ability to ferment maltose, rhamnose and xylose;
• division into phage types based on sensitivity to a set of corresponding phages.

These typing methods are mainly of epidemiological significance. In addition, Shigella Sonnei and Shigella Flexneri are typed for the same purpose by their ability to synthesize specific colicins (colicin genotyping) and by their sensitivity to known colicins (colicinotyping). To determine the type of colicins produced by Shigella, J. Abbott and R. Shannon proposed sets of typical and indicator strains of Shigella, and to determine the sensitivity of Shigella to known types of colicins, the Set of Reference Colicinogenic Strains of P. Frederick is used.

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Shigella resistance

Shigella have a fairly high resistance to environmental factors. They survive on cotton fabric and paper for 0-36 days, in dried excrement - up to 4-5 months, in soil - up to 3-4 months, in water - from 0.5 to 3 months, on fruits and vegetables - up to 2 weeks, in milk and dairy products - up to several weeks; at a temperature of 60 C they die in 15-20 minutes. They are sensitive to chloramine solutions, active chlorine and other disinfectants.

Pathogenicity factors of Shigella

The most important biological property of shigella, which determines their pathogenicity, is the ability to penetrate into epithelial cells, multiply in them and cause their death. This effect can be detected using a keratoconjunctival test (introduction of one loop of shigella culture (2-3 billion bacteria) under the lower eyelid of a guinea pig causes the development of serous-purulent keratoconjunctivitis), as well as by infecting cell cultures (cytotoxic effect) or chicken embryos (their death), or intranasally white mice (development of pneumonia). The main factors of shigella pathogenicity can be divided into three groups:

• factors determining interaction with the epithelium of the mucous membrane;
• factors that ensure resistance to humoral and cellular defense mechanisms of the macroorganism and the ability of shigella to reproduce in its cells;
• the ability to produce toxins and toxic products that cause the development of the pathological process itself.

The first group includes adhesion and colonization factors: their role is played by pili, outer membrane proteins and LPS. Adhesion and colonization are promoted by enzymes that destroy mucus - neuraminidase, hyaluronidase, mucinase. The second group includes invasion factors that promote the penetration of shigella into enterocytes and their reproduction in them and in macrophages with the simultaneous manifestation of a cytotoxic and (or) enterotoxic effect. These properties are controlled by the genes of the plasmid with m. m. 140 MD (it codes for the synthesis of outer membrane proteins that cause invasion) and chromosomal genes of shigella: kcr A (causes keratoconjunctivitis), cyt (responsible for cell destruction), as well as other genes that have not yet been identified. Protection of shigella from phagocytosis is provided by the surface K-antigen, antigens 3,4 and lipopolysaccharide. In addition, lipid A of shigella endotoxin has an immunosuppressive effect: it suppresses the activity of immune memory cells.

The third group of pathogenicity factors includes endotoxin and two types of exotoxins found in Shigella - Shiga and Shiga-like exotoxins (SLT-I and SLT-II), the cytotoxic properties of which are most pronounced in S. dysenteriae. Shiga and Shiga-like toxins have also been found in other serotypes of S. dysenteriae; they are also produced by S.flexneri, S. sonnei, S. boydii, EHEC and some salmonella. The synthesis of these toxins is controlled by the tox genes of converting phages. Enterotoxins of the LT type have been found in Shigella flexneri, sonnei and boydii. LT synthesis in them is controlled by plasmid genes. Enterotoxin stimulates adenylate cyclase activity and is responsible for the development of diarrhea. Shiga toxin, or neurotoxin, does not react with the adenylate cyclase system, but has a direct cytotoxic effect. Shiga and Shiga-like toxins (SLT-I and SLT-II) have a molecular weight of 70 kDa and consist of subunits A and B (the latter of 5 identical small subunits). The receptor for the toxins is a glycolipid of the cell membrane. The virulence of Shigella sonnei also depends on a plasmid with a molecular weight of 120 MDa. It controls the synthesis of about 40 polypeptides of the outer membrane, seven of which are associated with virulence. Shigella sonnei with this plasmid form phase I colonies and are virulent. Cultures that have lost the plasmid form phase II colonies and are devoid of virulence. Plasmids with a molecular weight of 120-140 MDa were found in Shigella flexneri and Boyd. Shigella lipopolysaccharide is a strong endotoxin.

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Post-infectious immunity

As observations on monkeys have shown, after dysentery, a strong and fairly long-lasting immunity remains. It is caused by antimicrobial antibodies, antitoxins, increased activity of macrophages and T-lymphocytes. Local immunity of the intestinal mucosa, mediated by IgAs, plays a significant role. However, immunity is type-specific, and strong cross-immunity does not occur.

Epidemiology of dysentery

The source of infection is only humans. No animals in nature suffer from dysentery. In experimental conditions, dysentery can only be reproduced in monkeys. The mode of infection is fecal-oral. The routes of transmission are water (predominant for Shigella flexneri), food, with milk and dairy products playing a particularly important role (the predominant route of infection for Shigella sonnei), and contact-household, especially for the species S. dysenteriae.

A feature of dysentery epidemiology is the change in the species composition of pathogens, as well as Sonne biotypes and Flexner serotypes in certain regions. For example, until the end of the 1930s, S. dysenteriae 1 accounted for 30-40% of all cases of dysentery, and then this serotype began to occur less and less often and almost disappeared. However, in the 1960-1980s, S. dysenteriae reappeared on the historical arena and caused a series of epidemics that led to the formation of three hyperendemic foci of it - in Central America, Central Africa and South Asia (India, Pakistan, Bangladesh and other countries). The reasons for the change in the species composition of dysentery pathogens are probably associated with changes in collective immunity and changes in the properties of dysentery bacteria. In particular, the return of S. dysenteriae 1 and its widespread distribution, which caused the formation of hyperendemic foci of dysentery, is associated with its acquisition of plasmids that caused multiple drug resistance and increased virulence.

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Symptoms of dysentery

The incubation period of dysentery is 2-5 days, sometimes less than a day. The formation of an infectious focus in the mucous membrane of the descending colon (sigmoid and rectum), where the causative agent of dysentery penetrates, is cyclical: adhesion, colonization, penetration of shigella into the cytoplasm of enterocytes, their intracellular reproduction, destruction and rejection of epithelial cells, the release of pathogens into the intestinal lumen; after this, another cycle begins - adhesion, colonization, etc. The intensity of the cycles depends on the concentration of pathogens in the parietal layer of the mucous membrane. As a result of repeated cycles, the inflammatory focus grows, the resulting ulcers, joining, increase the exposure of the intestinal wall, as a result of which blood, mucopurulent lumps, polymorphonuclear leukocytes appear in the feces. Cytotoxins (SLT-I and SLT-II) cause cell destruction, enterotoxin - diarrhea, endotoxins - general intoxication. The clinical picture of dysentery is largely determined by the type of exotoxins produced by the pathogen, the degree of its allergenic effect and the immune status of the body. However, many issues of dysentery pathogenesis remain unclear, in particular: the features of the course of dysentery in children of the first two years of life, the reasons for the transition of acute dysentery to chronic, the importance of sensitization, the mechanism of local immunity of the intestinal mucosa, etc. The most typical clinical manifestations of dysentery are diarrhea, frequent urges: in severe cases up to 50 or more times a day, tenesmus (painful spasms of the rectum) and general intoxication. The nature of the stool is determined by the degree of damage to the large intestine. The most severe form of dysentery is caused by S. dysenteriae 1, the mildest is Sonne dysentery.

Laboratory diagnostics of dysentery

The main method is bacteriological. The material for the study is feces. The scheme for isolating the pathogen: sowing on differential diagnostic Endo and Ploskirev media (in parallel on the enrichment medium with subsequent sowing on Endo, Ploskirev media) to isolate isolated colonies, obtaining a pure culture, studying its biochemical properties and, taking into account the latter, identification using polyvalent and monovalent diagnostic agglutinating sera. The following commercial sera are produced.

For Shigella that do not ferment mannitol:

• to S. dysenteriae 1 and 2 (polyvalent and monovalent),
• to S. dysenteriae 3-7 (polyvalent and monovalent),
• to S. dysenteriae 8-12 (polyvalent and monovalent).

To Shigella fermenting mannitol: to typical antigens of S. flexneri I, II, III, IV, V, VI, to group antigens of S.flexneri 3, 4, 6,7,8 - polyvalent, to antigens of S. boydii 1-18 (polyvalent and monovalent), to antigens of S. sonnei phase I, phase II, to antigens of S. flexneri I-VI + S. sonnei - polyvalent.

For rapid identification of Shigella, the following method is recommended: a suspicious colony (lactose-negative on Endo medium) is reseeded onto TSI (triple sugar iron) medium - a three-sugar agar (glucose, lactose, sucrose) with iron to determine H2S production; or onto a medium containing glucose, lactose, sucrose, iron and urea.

Any organism that breaks down urea after 4 to 6 hours of incubation is probably a Proteus organism and can be excluded. An organism that produces H,S or has urease or produces acid on the slant (ferments lactose or sucrose) can be excluded, although strains that produce H2S should be investigated as possible members of the Salmonella genus. In all other cases, the culture grown on these media should be examined and, if it ferments glucose (colour change in the column), isolated in pure form. At the same time, it can be examined in a slide agglutination test with appropriate antisera to the genus Shigella. If necessary, other biochemical tests are carried out to verify belonging to the genus Shigella, and motility is also studied.

The following methods can be used to detect antigens in the blood (including in the CIC), urine and feces: RPGA, RSK, coagglutination reaction (in urine and feces), IFM, RAGA (in blood serum). These methods are highly effective, specific and suitable for early diagnostics.

For serological diagnostics the following can be used: RPGA with the corresponding erythrocyte diagnosticums, immunofluorescence method (in indirect modification), Coombs method (determination of the titer of incomplete antibodies). An allergic test with dysenterin (a solution of protein fractions of shigella flexneri and sonnei) is also of diagnostic value. The reaction is taken into account after 24 hours. It is considered positive in the presence of hyperemia and an infiltrate with a diameter of 10-20 mm.

Treatment of dysentery

The main attention is paid to the restoration of normal water-salt metabolism, rational nutrition, detoxification, rational antibiotic therapy (taking into account the sensitivity of the pathogen to antibiotics). A good effect is given by early use of polyvalent dysentery bacteriophage, especially tablets with a pectin coating, which protects the phage from the action of HCl gastric juice; in the small intestine, pectin dissolves, phages are released and show their effect. For prophylactic purposes, the phage should be given at least once every three days (its survival period in the intestine).

Specific prevention of dysentery

Various vaccines have been used to create artificial immunity against dysentery: from killed bacteria, chemical, alcohol, but all of them turned out to be ineffective and were discontinued. Vaccines against Flexner's dysentery have been created from live (mutant, streptomycin-dependent) Shigella Flexneri; ribosomal vaccines, but they have not found wide application either. Therefore, the problem of specific prevention of dysentery remains unsolved. The main way to combat dysentery is to improve the water supply and sewerage system, ensure strict sanitary and hygienic conditions at food enterprises, especially the dairy industry, in children's institutions, public places and in maintaining personal hygiene.