Microbiology: lecture notes Ksenia Viktorovna Tkachenko

4. Salmonella

The genus Salmonella includes more than 2500 serovars.

Morphology is similar to other members of the family. The bacteria are mobile and do not form spores or capsules.

They grow well on simple nutrient media. They form small transparent colonies.

Biochemical properties:

1) ferment carbohydrates to acid and gas;

2) lactose is not decomposed;

3) deaminate and decarboxylate some amino acids.

Based on biochemical differences, the genus is divided into six groups.

Antigenic structure:

1) O-antigen. According to its structure, Salmonella are divided into 65 serogroups;

2) H-antigen. According to its structure, within the Salmonella serogroup they are divided into serovars.

In humans, salmonella can cause two groups of diseases:

1) anthroponotic – typhoid fever and paratyphoid fever A and B; pathogens: S. typhi, S. paratyphi A, S. paratyphi B;

2) zooanthroponotic – salmonellosis; pathogens: S. typhimurium, S. haifa, S. anatum, S. panama, S. infantis.

Typhoid fever and paratyphoid fever A and B are combined into one group - typhoid paratyphoid diseases - due to a common pathogen, clinical picture, and pathogenesis. The source of infection is the patient (or bacteria carrier).

The disease includes five phases.

1. The phase of introduction of the pathogen into the body, its attachment to the receptors of enterocyte membranes and penetration into cells (corresponds to the incubation period of the disease).

2. Primary localization phase: salmonella penetrate the lymphatic system of the small intestine, sensitize it, and multiply in macrophages; this is accompanied by the death of microorganisms and the release of endotoxin, which enters the blood and causes endotoxemia (corresponds to the prodromal period).

3. Bacteremia phase: the pathogen breaks through the lymphatic barrier and enters the blood, spreading throughout all parenchymal organs (the onset of the disease).

4. Secondary localization phase: typhoid granulomas appear in parenchymal organs (the height of the disease).

5. Excretory-allergic phase: repeated contact of the pathogen with the primary sensitized lymphatic apparatus of the small intestine; ulcers form on the mucous membrane.

The outcome of the disease can be different:

1) recovery;

2) formation of carrier state;

3) lethal.

Diagnosis of typhoparatyphoid diseases:

1) in the bacteremia phase - blood for blood culture (RPHA), if there is a rash - scraping from roseola;

2) in the convalescence phase - bacteriological examination of feces, urine, bile;

3) to identify carriage - serological testing.

Etiotropic therapy: antibiotics taking into account the sensitivity of the pathogen.

Specific prevention: killed typhoid vaccine.

The second group of diseases – salmonellosis – is characterized by a variety of clinical manifestations. Sources of infection are sick animals and contaminated food. The route of infection is nutritional. Most often, salmonellosis occurs as a foodborne illness. In this case, Salmonella infects the enterocytes of the small intestine and becomes fixed in its lymphatic system. When the lymphatic barrier breaks through, bacteremia develops, the pathogen spreads to various organs, and extraintestinal forms of salmonellosis are recorded.

Salmonella identification

After 18-20 hours of incubation of the dishes with differential diagnostic media (bismuth-sulfite agar 48 hours), the growth pattern is taken into account with the selection of 3-5 suspicious colonies on one of the media for primary identification (Kligler, Ressel, Olkenitsky) and on a slant nutrient agar. In the event of an emergency epidemic situation, a culture grown on the specified media is used for subsequent staging of an agglutination reaction. The results of this reaction are indicative and require confirmation at the stage of completion of biochemical identification.

Cultural properties

The commonality of morphology and a number of cultural properties of bacteria of the genus Salmonella does not allow them to be classified according to the indicated characteristics. For this purpose, in addition to morphology and cultural properties, enzymatic properties and antigenic structure are studied, in in some cases perform a biological test on laboratory animals.

The enzymatic properties of bacteria are determined by a set of enzymes that reflect certain nutritional and metabolic conditions characteristic of a given type of microorganism in certain environmental conditions. Bacteria of the genus Salmonella are characterized by the following enzymatic properties: they do not liquefy gelatin, do not decompose adonite and do not ferment sucrose; the vast majority does not break down salicin and does not decompose lactose, does not form indole, does not break down urea, does not give the Voges-Proskauer reaction (reaction to acetylmethylcarbinol); ferments (with a few exceptions maltose, mannitol, sorbitol, breaks down glucose with the formation of gas (S. typhi, S. pullorum usually do not form gas); produces positive reaction with methyl red, utilizes ammonium and reduces nitrates; most of them produce hydrogen sulfide.

To study the enzymatic properties of bacteria of the genus Salmonella, a short colored (variegated) series is usually used, consisting of media with glucose, mannitol, arabinose, dulcitol, rhamnose (Bitter's medium); glycerin fuchsin broth (Stern broth). In addition to the indicated media, media with maltose, inositol, trehalose, and xylose are also used to differentiate serological types of Salmonella; litmus milk (changes in litmus milk during the growth of salmonella allow them to be differentiated by their ability to form acid or alkali). Instead of litmus test, you can use skim milk with bromothymol blue indicator (1 ml of 0.4% solution in 100 ml of milk). The production of hydrogen sulfide by the culture is known to be important for the differentiation of Salmonella. Proteolytic properties are studied by inoculating the studied Salmonella culture on the breast milk and milk.

Due to the similarity of bacteria of the genus Salmonella with other microorganisms of the Enterobacteriaceae family, there is a need to differentiate them. Currently, solid differential diagnostic nutrient media with lactose (Ploskirev, Endo, Levin media) are widely used in bacteriological practice. By the ability of bacteria to ferment lactose, Salmonella is distinguished from the often accompanying E. coli, therefore, when examining material for Salmonella, it is first sown on one of the differential diagnostic media. On these media, E. coli, which ferments lactose with the formation of acid and changes in the color of the indicator, forms colonies that differ in color from Salmonella colonies that do not ferment lactose. On Endo medium, E. coli bacteria produce red colonies, often with a metallic sheen; salmonella are colorless or pale pink (colored in the color of the medium); on Ploskirev's medium E. coli - orange-red colonies, salmonella - transparent or pale pink; on Levin's medium, E. coli form black colonies surrounded by a rim, salmonella are transparent, pale pink or pinkish-violet. To differentiate Salmonella and culturally similar strains, as well as bacteria of the genus Proteus and coliform bacteria, media with urea (Preuss, Ressel, Olkenitsky's medium), SS agar (Salmonella - Shigella - agar), etc. are used. The color of these media is determined unequal intensity of breakdown of nitrogenous substances by microorganisms with the formation of alkaline products. Bacteria of the coli group and Proteus (with the exception of the 0-form), as a rule, do not grow on SS-agape, and salmonella grow in the form of tender, colorless colonies.

Dense differential diagnostic media serve only to determine whether bacteria belong to the genus Salmonella and separate them from the accompanying microflora.

The enzymatic properties of Salmonella are not always stable and can change depending on environmental conditions, therefore, correct typing of Salmonella is possible only as a result of studying a complex of morphological, cultural, enzymatic properties and antigenic structure

In addition to Salmonella, the causative agents of typhoid fever and paratyphoid A and B, more than 2,400 pathogenic Salmonella serovars have been described, causing acute gastroenteritis, typhoid-like and septicopyemic forms of the disease in humans, collectively called salmonellosis. The main causative agents of salmonellosis are Salmonella serovars enteritidis, typhimurium, choleraesuis, haifa etc.

Bacteriological method is the leading method in the laboratory diagnosis of salmonellosis (Scheme 10). Salmonella cultures can most often be isolated from the feces of patients, somewhat less often - from vomit and gastric lavage, and even less often - from blood, urine and bile. Isolation of Salmonella from blood, bone marrow, cerebrospinal fluid, vomit and gastric lavage confirms the diagnosis of salmonellosis. In bacterial carriers, salmonella can be found in feces, urine, and bile.

The test material is inoculated onto plates with bismuth-sulfite agar and into accumulation media (magnesium, selenite), from which, after 6-10 hours, they are transferred to bismuth-sulfite agar. The crops are grown at a temperature of 37 0 C, on the second day black colonies are selected and replanted on Olkenitsky (or Ressel) medium to accumulate a pure culture . On the 3rd day of the study, the isolated pure cultures are subcultured into the “variegated” series media and RA is placed with polyvalent and group (A, B, C, D, E) adsorbed salmonella sera . If a positive result is obtained with one of the groups of sera, RA is performed with adsorbed O-sera characteristic of this group, and then with monoreceptor H-sera (nonspecific and specific phases) to determine the serogroup and serovar of salmonella in accordance with the Kaufman-White scheme.

On the 4th day of the study, changes in the media of the “variegated” series are taken into account (Table 10). The causative agents of salmonellosis are the same as Salmonella paratyphoid B , do not ferment lactose and sucrose, break down glucose, mannitol and maltose to form acid and gas, do not form indole and (with a few exceptions) release hydrogen sulfide.

Scheme 10. Microbiological diagnosis of salmonellosis.

Bioassay. Material from the patient is used to orally infect white mice, which die from septicemia within 1-2 days. When culturing blood from the heart and material from internal organs Salmonella culture is isolated.

Serological study - study using RNGA of paired blood sera of patients taken at intervals of 7-10 days with Salmonella polyvalent and group (groups A, B, C, D, E) diagnostics. An increase in antibody titer by four or more times has diagnostic significance.

Independent work of students

To study the main stages of bacteriological research of the method for diagnosing salmonellosis.

1. Accounting for Salmonella cultures on Levin and Olkenitsky medium (demonstration).

2. Control of the purity of the endowed salmonella culture(a smear was prepared from Olkenitsky’s Wednesday and stained according to Gram). Microscope and draw demonstration micropreparations of the causative agents of salmonellosis S. enterica spp. enterica ser. enteritidis , typhimurium , choleraesuis The causative agents of salmonellosis are gram-negative rods identical in morphology with rounded ends.

3. Identification of the isolated pathogen culture:

- by antigenic properties– accounting indicative reaction agglutination on glass with diagnostic monoreceptor Salmonella agglutinating O- and H sera in accordance with the Kaufman-White scheme;

- by biochemical properties- determination of the biochemical activity of the studied culture according to the “variegated” series of Hiss or Peshkov (demonstration). Pay attention to the breakdown of glucose and the lack of breakdown of lactose and sucrose.

- by phagolysability(counting samples with phage - demonstration)

4. Determination of the sensitivity of an isolated culture to antibiotics using the paper disk method(demonstration).

The genus Salmonella includes more than 2000 representatives, widely distributed in nature. They cause diseases in humans and animals. The Salmonella genus includes the causative agents of typhoid fever, paratyphoid fevers A and B, and foodborne toxic infections.

The causative agent of typhoid fever(S. typhi) was first discovered in 1880 by Ebert in the organs of people who died of typhoid fever. In 1884, Gaffki isolated a pure culture of the microbe. Later, in 1896, Ashar and Bansod found in the pus and urine of patients who had a clinical picture of typhoid fever, bacilli that differed in biochemical and serological properties from the causative agent of typhoid fever. They were called paratyphoid - S. paratyphi A and S. paratyphi B. Of the causative agents of food poisoning, the causative agent of swine cholera was first discovered in 1885 by Salmon - S. cholerae suis. In 1888, Gertner isolated S. enteritidis during an outbreak of foodborne diseases after eating the meat of a sick cow. Subsequently, Salmonella murine typhus - S. typhimurium and other microbes were described, which were similar to each other in a number of characteristics, and were united in the genus Salmonella, named after Salmon.

Morphology and biological properties. Salmonella are short rods with rounded ends, averaging 1-3 microns in size. All of them are motile due to the presence of peritrichial flagella. They do not form spores or capsules. They stain well with aniline dyes and are gram negative. Facultative aerobes. They grow well on simple nutrient media at temperatures of 20–40°C and pH from 5.0 to 8.0, with an optimum of 37°C and pH 7.2–7.4. Pa liquid media give uniform turbidity. On meat-peptone agar, the colonies are smaller than those of E. coli, tender, and translucent. On the differential diagnostic media Endo, Levin, Ploskirev, the colonies are small and colorless. Colonies on bismuth sulfite agar are black.

The enzymatic properties of Salmonella (see Table 3) are quite constant: they do not decompose lactose and sucrose, they ferment glucose and mannitol with the formation of acid and gas, although there are types that ferment them only to acid (for example, Salmonella typhus). Most salmonella break down proteins with the formation of hydrogen sulfide, do not form indole, and do not liquefy gelatin. Salmonella contains endotoxin of lipopolysaccharide-protein nature. It is heat stable and has antigenic properties.

Sustainability. Salmonella is resistant in the external environment. They can be stored in dust, ice, or clean water for up to 3 months. At a temperature of 70°C they die within 5-10 minutes, at 10°C - instantly. In salted and smoked meat, Salmonella remains viable for 27.2 months. They can reproduce in milk. Under the influence of a 1% solution of sublimate, 3-5% solution of carbolic acid and chloramine they die within a few minutes.

Antigenic structure and classification. Salmonella contains two main antigenic complexes: O-somatic and N-flagellate. O-antigen is a lipopolysaccharide-protein complex, thermostable, inactivated by formaldehyde, corresponds to endotoxin bacterial cell. H-antigen is protein in nature, thermolabile, and is easily destroyed by alcohol and phenol. Resistant to formaldehyde. The production of N-diagioscum is based on this property. O- and H-antigens in various representatives of Salmonella are heterogeneous, which was the basis for the classification of these bacteria developed by Kaufman and White (Table 4).

They divided all salmonella by O-antigens into groups: A, B, C, D, E, etc. Each group is characterized by the presence of a specific O-antigen (for example, in group B it is “4”). Some groups have common O-antigens (for example, group A, B and D - “1, 12”). Salmonella typhus contains a Vi antigen, which is located more superficially than the O antigen and can interfere with agglutination with O serum. Its loss leads to the restoration of O-agglutination. Vi-antigen is easily destroyed by boiling the culture for 10 minutes, adding phenol to the medium, or growing the microbe on artificial media.

In Salmonella H-antigens, phases I and II are distinguished. The first phase of H-antigens is different for serotypes belonging to the same group (for example, in the group S. paratyphi B - “c”, and Salm. typhimurium - “i”). This separation helps to differentiate individual types of Salmonella in the glass agglutination reaction with monoreceptor Salmonella sera. In the agglutination reaction, when H-antigens interact with the corresponding antibodies, coarse H-agglutination appears; O- and Vi-agglutination is fine-grained.

In addition to serological typing of Salmonella, phage types are sometimes determined using specific Salmonella bacteriophages, of which more than 100 are currently known. It has been established that some phages lyse Salmonella containing O-antigen, others (Vi-phages) - only strains containing Vi-antigen . Salmonella phagotypes are stable. The Salmonella phage typing method is used for epidemiological analysis to identify the source of infection.

Pathogenicity. Among Salmonella there are types that are pathogenic only for humans: Salmonella typhoid, paratyphoid A and B. There are types that cause diseases only in animals. Most are pathogenic for both humans and animals. The variety of clinical forms of diseases caused by Salmonella depends on the properties of the pathogen, the severity of the infection, the state of the host’s defenses and other reasons.

The genus Salmonella includes the causative agent of typhoid fever S. typhi, paratyphoid fever - S. paratyphi A, S. schottmuelleri and the causative agents of food toxic infections, called salmonellosis.

Morphology and tinctorial properties. S. typhi, S. paratyphi A, S. schottmuelleri - short rods with rounded ends, have peritrichous flagella, gram-negative.

Cultivation and enzymatic properties. On differential diagnostic media (Endo, Levin) they form transparent, colorless colonies. Salmonella have saccharolytic properties: S. typhi ferments glucose, maltose, mannitol to acid, S. paratyphi and S. schottmuelleri - those. the same as sugar, but before acid and gas, which serves as a differential diagnostic feature. The decomposition of S. typhi and S. schottmuelleri proteins produces hydrogen sulfide. Gelatin is not liquefied.

Antigenic structure and toxin formation of Salmonella. Salmonella contains an O-antigen-lipopolysaccharide-protein complex identical to endotoxin, an H-antigen and a K-antigen - surface, shell, capsular. Identification of salmonella is carried out by antigenic properties, according to the classification proposed by Kaufman and White and built on the following principle: all salmonella, according to the commonality of O-antigens, are divided into groups designated by capital letters of the Latin alphabet (A, B, C, D, E, etc. .); within each O-group, Salmonella are divided into serological variants based on the different structure of H-antigens, designated by a lowercase letter of the Latin alphabet and numbers. There are two phases in H-antigens: the first - specific and the second - non-specific. When identifying Salmonella, diagnostic agglutinating adsorbed sera are used.

Complex antigenic structure S. typhi includes O-, H- and Vi-antigens, but such antigenically complete bacteria are isolated only at the height of the disease, and during the period of convalescence and during subcultures in laboratory conditions, the Vi-antigen is lost.

Resistance. Salmonella is quite stable in the external environment. Depending on conditions (temperature, humidity, insolation, etc.), they can last up to a year. Disinfectants cause the death of salmonella within a few minutes, heating to 60 °C - after 10-15 minutes.

Pathogenicity for animals. Typhoid fever and paratyphoid fever only affect people.

Clinical symptoms of the disease are characterized by a gradual increase in temperature, general malaise, which turns into a state of deep intoxication of the body, for which the disease is called typhus. This condition is caused by the action of endotoxin, which at the height of the disease is released in large quantities as a result of the massive death of microbes, which is facilitated by the resulting antibodies.

Bacteria travel with the blood to the liver, spleen, bone marrow, and lymph nodes of the small intestine, where they multiply, enriching the blood with pathogens. The phase of parenchymal diffusion begins. Salmonella, accumulating in large quantities in the gallbladder, enters the small intestine for the second time and multiplies in lymphatic formations, which become inflamed, necrotic, ulcerated, which can lead to destruction of the intestinal wall, i.e. perforation is one of the most severe complications of typhoid fever. Typhoid bacteria find particularly favorable conditions for reproduction in the gallbladder, where they linger for the longest time and, when released into the intestine, intensify its pathological lesions. The excretory-allergic phase of the release of microbes constitutes the next stage of pathogenesis. Bacteria are released from the body not only in feces, but also in the urine and milk of a nursing mother. The excretory stage passes into the recovery stage, accompanied by an increase in the titer of specific antibodies.

Immunity. There is no innate immunity to infections caused by typhoid and paratyphoid pathogens. Once the disease has been transmitted, it leaves a strong immunity and cases of recurrent disease are rare. However, relapses are possible, and those who have had typhoid fever become chronic bacteria carriers.

Laboratory diagnostics of salmonella. When diagnosing, epidemiological data and clinical symptoms are taken into account. From the first day of illness it is necessary to examine the blood. The blood culture method is a decisive and early method. The best selective nutrient medium is a medium containing bile, which neutralizes antibodies and the bactericidal properties of blood serum. It is possible to isolate a culture of the pathogen from bone marrow, urine, roseola, but these methods are rarely used. During the period of convalescence, the pathogen is isolated from feces. Isolated cultures are identified by biochemical and antigenic properties.

When determining the type of Salmonella, first an agglutination reaction is performed with a polyvalent serum, which makes it possible to determine whether the isolated culture belongs to the Salmonella genus, then the group is determined using separate group adsorbed sera, and then the type of Salmonella is determined in an agglutination reaction with monoreceptor specific H-sera.

The study of patients' serum can be carried out in the first days of the disease by detecting incomplete antibodies using the Coombs test and from the 4-5th day - complete antibodies using the Widal test, which is repeated after a few days in order to determine the increase in antibody titer. However, along with the advantages of performing the Widal reaction (simplicity of the reaction, obtaining a quick response, the possibility of a retrospective diagnosis), the method also has disadvantages: it is not early; antibodies can be present in both the person who has been ill (anamnestic reaction) and the vaccinated person (“vaccine Vidal”). It is also necessary to take into account the fact that H-antibodies persist for a long time, and O-antibodies disappear from the blood of a person who has recovered from the disease quite quickly.

In order to detect antibodies in the blood sera of patients with typhoid fever, the indirect hemagglutination reaction (IRHA) is used.

It is very difficult to identify bacteria carriers. Bacteriological studies, which are carried out on a massive scale during periodic examinations of workers at food enterprises and children's institutions, take a lot of time and are not always effective, since it is difficult to isolate a microbe even from a known carrier. Currently, the serological method of Vi-hemagglutination reaction with carrier serum is widely used to detect the carrier. Vi-antigen is a polysaccharide by its chemical nature; it is extracted in its pure form and adsorbed on the surface of human blood group 0 erythrocytes. The carrier's serum causes a hemagglutination reaction when interacting with Vi-erythrocyte diagnosticum. The carrier has a positive reaction in small dilutions. Carriers identified by the Vi-hemagglutination reaction are examined by bacteriological methods (culture isolation from feces, duodenal contents).

If necessary, for the epidemiological deciphering of diseases, differentiation of isolated cultures is carried out using Vi-bacteriophages, which have strict type specificity. Phagotyping uses 78 types of Vi-bacteriophages, with the help of which the same number of phagotypes of these bacteria is identified. The phage typing method makes it possible to establish or exclude suspected sources of infection, trace epidemiological connections, distinguish local cases from “imported” ones and sporadic diseases from epidemic ones.

Epidemiology of Salmonella. The source and reservoir of infection is only a person: the patient or the carrier. The mechanism of transmission is fecal-oral, the route of spread is through direct contact with contaminated food (milk), water, contaminated wastewater. With a relatively low incidence, the main role now is through contact and household transmission - through contaminated hands, dishes, linen, etc. The incidence is characterized by summer-autumn seasonality.

Specific treatment and prevention. Broad-spectrum antibiotics, the tetracycline group, and chloramphenicol are used to treat patients. Antibiotic therapy, however, does not prevent relapses and prolonged bacterial carriage. Soviet and foreign researchers, studying the mechanism of action of antibiotics in clinical settings, drew attention to the fact that their effectiveness is closely related to the degree of development of specific immunity. Therefore, in addition to antibiotics, it is recommended to treat with agents that specifically stimulate immunogenesis (corpuscular vaccine, partial antigens). With complex immunoantibiotic therapy (antibiotics and separately monovaccine and Vi-antigen), the frequency of relapses in patients with typhoid fever is significantly reduced, and the formation of bacterial carriage is absent.

For the purpose of prevention, typhoid alcohol vaccine enriched with Vi-antigen is used (according to epidemic indications). To immunize military personnel, a chemical sorbed typhoid-paratyphoid-tetanus vaccine (TABle) is used, containing complex antigens isolated from typhoid, paratyphoid A and B bacteria and purified tetanus toxoid.