Scientific classification of staphylococcus:
Domain:
Type: Firmicutes
Class: bacilli
Order: Lactobacillales (Lactobacilli)
Family: Streptococcaceae (Streptococcus)
View: Pneumococcus (Streptococcus pneumoniae)
International scientific name: Streptococcus pneumoniae
Pneumococcus (lat. Streptococcus pneumoniae) is a spherical or egg-shaped bacterium belonging to the Streptococcus family (Streptococcaceae).
Other names for pneumococcus: Weixelbaum's diplococcus, Frenkel's diplococcus.
Pneumococcus is the most common causative agent of such diseases as -. The mortality rate of pneumonia is up to 5% of cases. Other diseases of pneumococcal etiology include otitis media, sinusitis, laryngitis, tracheitis, bronchitis, meningitis, sepsis and others. Especially pneumococcal infection often causes exacerbation of bronchopulmonary diseases in children.
Characteristics of streptococci
Like other types of streptococci, pneumococci most often exist in pairs, sometimes lining up in chains. The size of the bacterium is 0.5-1.25 microns. Behaviorally, pneumococcal infection is nonmotile, anaerobic, and gram-positive. Rapid reproduction occurs with an increase in carbon dioxide. The basis of pneumococcus is peptidoglycan, together with surface proteins, carbohydrates, lipoproteins and teichoic acids, and all this is contained in a protective, powerful polysaccharide capsule that prevents opsonization.
The classification of pneumococci includes up to 100 strains of these bacteria.
Diseases that can cause pneumococci
The most popular pneumococcal diseases are:
- Septic arthritis;
- Otitis media;
- (rhinitis (runny nose), sinusitis, ethmoiditis, sphenoiditis and frontal sinusitis);
- Pneumonia (community acquired);
The most popular pneumococcal diseases are pneumonia (about 70%), otitis media (about 25%), meningitis (5 to 15%) and endocarditis (about 3%).
In addition, pneumococcal infection can join existing diseases of other types of infection -, etc.
How to inactivate pneumococcus?
The pneumococcus bacterium dies when:
- their treatment with solutions of antiseptics and disinfectants;
- exposure to antibacterial agents.
How is pneumococcus transmitted? The conditions under which a person begins to develop pneumococcal diseases usually have two parts - exposure to infection and weakened immunity. However, a person can become seriously ill through normal contact with this type of bacteria, when its amount in the air is in high concentration.
Let's consider the most popular ways of contracting pneumococcal infection:
How can pneumococcus enter the body?
Airborne path. The main route of infection with pneumococcal infection is airborne droplets. Cough and a person standing next to you are the root causes of most. The insidiousness of pneumococcal infection lies in the fact that its carrier is often unaware of his role, because it may not cause any symptoms in its carrier. It is also worth noting that during the period, in the air, especially in enclosed spaces, the concentration of other species increases. That is why the first victims are people who often stay or work in crowded places.
Airborne dust path. Dust, including house dust, consists of many particles - plant pollen, animal hair, particles of exfoliated skin and paper, as well as viruses, bacteria, fungi and other infections. A person's presence in rooms where there is little or infrequent cleaning is another factor contributing to infection.
Contact and household path. Most types of infection do not die on their own, therefore, sharing the same kitchen utensils and personal hygiene items with a sick person increases the risk of getting sick.
Hematogenous route. Infection occurs when human blood comes into contact with an infected object. Frequent patients are people who use injection drugs.
Medical path. Infection occurs when using, for example during routine examination, contaminated medical equipment/instruments.
How can pneumococcus seriously harm a person's health, or what weakens the immune system?
As we said, the second factor that contributes to the development of pneumococcal disease is a weakened immune system, which performs the body’s protective function. So, when an infection enters the body, the immune system produces special antibodies, which, when they reach the source of infection or the infection has settled, stop it and destroy it. If the immune system is weakened, there is no one to fight the infection except medicines.
Let's consider the main causes of weakened immunity:
- The presence of chronic diseases - any disease in the body of a chronic form indicates that the immune system cannot cope with it on its own, while the disease gradually continues to harm health;
- The presence of other infectious diseases - sinusitis, bronchopulmonary diseases,;
- Insufficient amount of vitamins and minerals in the body ();
- Bad habits – smoking, drugs;
- Sedentary lifestyle;
- Lack of healthy sleep, chronic fatigue;
- Abuse of certain medications, especially antibiotics;
- Very often, pneumococcal infection is brought into the home by children from school and kindergarten. This is facilitated by close contact between children, as well as not fully developed immunity. Further, if certain preventive measures are not followed at home, the disease develops in adults.
At-risk groups
Let's consider a group of people who have an increased risk of contracting pneumococcal diseases:
- Elderly people, over 60 years old and children;
- Persons working in crowded places - office workers, drivers and conductors of public transport, employees of large enterprises, employees of medical institutions, employees of nursing homes and educational institutions, military personnel.
- People who have chronic diseases and systems, as well as diseases such as diabetes, emphysema, kidney disease, HIV.
- Persons who drink alcoholic beverages and smokers.
- Persons who like to walk in frosty and/or cool damp weather without a hat, wearing short jackets, thin trousers and other clothing that exposes the body to hypothermia.
- Persons who have had other infectious diseases - acute respiratory viral infections, acute respiratory infections, influenza, and others.
Symptoms of pneumococcus
The symptoms (clinical picture) of pneumococcal diseases are very extensive, and largely depend on the place (organ) in which the infection settled, the strain of pneumococcus, the person’s health and the state of his immunity.
Common symptoms of pneumococcus may include:
- , malaise, and ;
- Difficulty breathing, sneezing, shortness of breath;
- Increased and high body temperature;
- , sometimes strong;
- , impaired consciousness;
- Photophobia;
- Impaired sense of smell;
- , sometimes with ;
- All types – , and ;
- Diseases of the respiratory system: pharyngitis, laryngitis, tracheitis, bronchitis and pneumonia;
Complications of pneumococcus:
- Inflammation of the heart muscle - endocarditis;
- Purulent otitis;
- Decreased or loss of voice or hearing;
- Lung abscess;
- Sepsis;
- Mental retardation;
- Stiffness of movements;
- Epilepsy;
- Death.
Important! Some clinical complications can sometimes accompany a person for the rest of his life.
Diagnosis of pneumococcus
The test for pneumococcus is usually taken from swabs taken from the oropharynx (for upper respiratory tract diseases), nasal sputum and blood.
Thus, the following tests and methods for examining the body for pneumococcal infection are distinguished:
- Bacteriological culture of sputum and smears taken from the nasal cavity and oropharynx;
- internal organs;
- lungs;
How to treat pneumococcus? Treatment of pneumococcus usually consists of several points:
1. Antibacterial therapy;
2. Strengthening the immune system;
3. Restoration of normal intestinal microflora, which is usually disrupted when using antibacterial drugs;
4. Detoxification of the body;
5. Antihistamines - prescribed to children with allergies to antibiotics;
6. Symptomatic therapy;
7. If there are other diseases at the same time, they are also treated.
Treatment of pneumococcal diseases in any case begins with a visit to the doctor and the patient undergoing diagnostics. This must be done to exclude other types of infection, as well as to check the resistance (susceptibility) of the infection to a particular antibacterial drug.
Before considering antibiotics for pneumococcal diseases, let's look at their interactions (resistance).
Antibiotic resistance
Doctors note a not very favorable trend in the treatment of pneumococcal infection. Thus, from year to year, throughout the world, resistance (resistance) of pneumococci to antibacterial drugs of the penicillin and tetracycline series, as well as macrolides, has been observed, and immunity to antibiotics is gradually increasing. The most resistant pneumococci are found in America, Western Europe, Asia, and the least resistant in Germany and the Netherlands. If we talk about superficial reasons, this was largely due to the availability of antibiotics to anyone, even without prescriptions. The fact is that incorrectly selected antibiotics, or a course of therapy with this group of drugs, contribute to the infection developing a certain immunity to these drugs in the future, the bacteria mutate, and new strains develop. In some countries, for example Germany, it is impossible to purchase antibiotics without a doctor’s prescription, and therefore many infectious diseases of a bacterial nature are more easily treatable, and the number of complications and, accordingly, deaths is much less.
The highest resistance of pneumococci in the territories of Russia and Ukraine is observed in relation to tetracycline (40%) and co-trimoxazole (50%).
1. Antibacterial therapy
Important! Before using antibiotics, be sure to consult with your doctor.
In parentheses, after the name of the antibiotic, the percentage of bacterial resistance to the drug is indicated (in Russia, as of 2002-2012).
Antibiotics against pneumococci for internal use:"Amoxicillin" and "Amoxicillin-Clavulanate" (0.5%), "Vancomycin" (1%), "Levofloxacin" (1%), "Rifampicin" (1%), "Clindamycin" (2%), "Cefotaxime" "(2%), "Cefepime" (2%), "Ciprofloxacin" (2%), macrolides (from 7 to 26% - " ", "Claritomycin", "Midecamycin", "Spiramycin", " "), Chloramphenicol (5%), “Penicillin” (29%), “ ” (40%), “Co-trimoxazole” (50%).
The course of antibacterial therapy is prescribed individually by the attending physician. Usually it is 5-10 days.
Antibiotics against pneumococci for local use:"Bioparox", "Gexoral".
Important! Often, to treat a disease, a doctor selects a combination of 2 antibacterial drugs that must be taken simultaneously.
2. Strengthening the immune system
To strengthen the immune system and stimulate its functioning, immunostimulants are prescribed in combination with antibiotics: “Immunal”, “IRS-19”, “Imudon”.
A natural immunostimulant is, which is present in large quantities in cranberries and sea buckthorn.
3. Restoration of normal intestinal microflora
When taking antibacterial drugs, they also enter the intestines and destroy beneficial microflora that contribute to the normal absorption of food and participate in other important processes of the body. Therefore, when taking antibacterial drugs, recently taking probiotics, which restore normal intestinal microflora, has become increasingly popular.
Among the probiotics we can highlight: “Acipol”, “Bifiform”, “Linex”.
4. Detoxification of the body
Pneumococcal infection, while remaining inside the body, poisons it with the products of its vital activity. Intoxication with infectious enzymes contributes to the worsening of the disease, causing symptoms such as nausea, vomiting, loss of strength, hallucinations and delirium.
To remove waste products of infection from the body, detoxification therapy is prescribed, which includes:
- drink plenty of fluids (up to 3 liters of liquid per day, preferably with the addition of vitamin C);
- rinsing the nose and oropharynx with a weak saline solution or furacillin solution;
- taking detoxification drugs: Atoxil, Albumin, Enterosgel.
5. Antihistamines
Antihistamines are prescribed if, when taking antibiotics, a person experiences an allergic reaction - skin itching, rash, redness and other manifestations.
Among the antihistamines we can highlight: “”, “”, “Cetrin”.
6. Symptomatic therapy
To relieve the symptoms of pneumococcal diseases and alleviate their course, symptomatic therapy is prescribed.
At high body temperature: cool compresses on the forehead, neck, wrists, armpits. Among the drugs we can highlight - “”, “”.
For nasal congestion- vasoconstrictor drugs: Noxprey, Farmazolin.
Important!
Before using folk remedies, be sure to consult your doctor.
Pneumococci were first described by R. Koch (1871).
Morphology. Pneumococci are diplococci in which the sides of the cells facing each other are flattened, and the opposite sides are elongated, so they have a lanceolate shape, reminiscent of a candle flame (see Fig. 4). The size of pneumococci is 0.75-0.5 × 0.5-1 microns, they are located in pairs. In liquid nutrient media they often form short chains, becoming similar to streptococci. Preumococci are immobile, do not have spores, and in the body form a capsule surrounding both cocci. The capsule contains a heat-resistant substance antifagin (protecting pneumococcus from phagocytosis and the action of antibodies). When growing on artificial nutrient media, pneumococci lose their capsule. Pneumococci are gram-positive. Gram-negative bacteria are found in older cultures.
Cultivation. Pneumococci are facultative anaerobes. They grow at a temperature of 36-37° C and a pH of 7.2-7.4. They are demanding on media, since they cannot synthesize many amino acids, so they grow only on media with the addition of native protein (blood or serum). On serum agar they form small, delicate, rather transparent colonies. On blood agar, moist greenish-gray colonies grow, surrounded by a green zone, which is the result of the conversion of hemoglobin to methemoglobin. Pneumococci grow well in broth with the addition of 0.2% glucose and in broth with whey. Growth in liquid media is characterized by diffuse turbidity and dusty sediment at the bottom.
Enzymatic properties. Pneumococci have quite pronounced saccharolytic activity. They break down: lactose, glucose, sucrose, maltose, inulin to form acid. Mannitol is not fermented. Their proteolytic properties are weakly expressed: they curdle milk, do not liquefy gelatin, and do not form indole. Pneumococci dissolve in bile. The breakdown of inulin and dissolution in bile is an important diagnostic feature that distinguishes Streptococcus pneumoniae from Streptococcus pyogenes.
Pathogenicity factors. Pneumococci produce hyaluronidase, fibrinolysin, etc.
Toxin formation. Pneumococci produce endotoxin, hemolysin, and leukocidin. The virulence of pneumococci is also associated with the presence of antiphagin in the capsule.
Antigenic structure and classification. In the cytoplasm of pneumococci there is a protein antigen common to the entire group, and in the capsule there is a polysaccharide antigen. Based on the polysaccharide antigen, all pneumococci are divided into 84 serovars. Among those pathogenic for humans, serovars I, II, and III are most common.
Pneumococci belong to the group of unstable microorganisms. A temperature of 60° C kills them in 3-5 minutes. They are quite resistant to low temperatures and drying. In dried sputum they remain viable for up to 2 months. They can be stored on a nutrient medium for no more than 5-6 days. Therefore, when cultivating, it is necessary to reseed every 2-3 days. Conventional solutions of disinfectants: 3% phenol, sublimate in a dilution of 1:1000 destroy them in a few minutes.
Pneumococci are especially sensitive to optochin, which kills them at a dilution of 1:100,000.
Animal susceptibility. The natural host of pneumococci is humans. However, pneumococci can cause disease in calves, lambs, piglets, dogs and monkeys. Of the experimental animals, white mice are highly sensitive to pneumococcus.
Sources of infection
Transmission routes. Airborne droplets, maybe airborne dust.
Entrance gate. Mucous membrane of the upper respiratory tract, eyes and ears.
Diseases in humans. Pneumococci can cause purulent-inflammatory diseases of various localizations. Specific for pneumococci are:
1) lobar pneumonia; 2) creeping corneal ulcer;
The most common disease is lobar pneumonia, which affects one, less often two or three lobes of the lung. The disease is acute, accompanied by high fever and cough. It usually ends critically.
Immunity. After the illness, unstable immunity remains, since pneumonia is characterized by relapses.
Prevention. It comes down to sanitary and preventive measures. Specific prevention has not been developed.
Treatment. Antibiotics are used - penicillin, tetracycline, etc.
Control questions 1. Morphology of pneumococci. Cultivation and enzymatic properties.
2. What factors determine the pathogenicity of pneumococci and what protects pneumococci from phagocytosis?
3. What are the main gates of pneumococcal infection. What diseases do pneumococci cause?
Microbiological examination
Purpose of the study: identification of pneumococcus.
Material for research
1. Sputum (pneumonia).
2. Mucus from the throat (sore throat).
3. Discharge from an ulcer (creeping corneal ulcer).
4. Discharge from the ear (otitis media).
5. Pus (abscess).
6. Pleural punctate (pleurisy).
7. Blood (suspicion of sepsis).
Methods of collecting material
Methods of collecting material
1 (It is better to take morning sputum (with specific pneumonia, the sputum has a rusty color).)
Basic research methods
1. Microscopic.
2. Microbiological.
3. Biological.
Progress of the study
First day of the study
First day of the study
Biological sample. A little (3-5 ml of sputum) is emulsified in a sterile broth, 0.5 ml of this mixture is injected intraperitoneally into a white mouse. After 6-8 hours, the mouse shows signs of disease. At this time, pneumococcus can already be detected in the exudate of the abdominal cavity. The exudate is taken with a sterile syringe. Smears are made from it, stained with Gram and examined under a microscope. To isolate a pure culture, the exudate is inoculated onto serum agar. If the mouse dies or becomes ill, blood from the heart is cultured onto serum agar to isolate a pure culture. The crops are placed in a thermostat.
Accelerated method for determining the type of pneumococcus(microagglutination reaction). 4 drops of exudate from the abdominal cavity of an infected mouse are applied to a glass slide. Type I agglutinating serum is added to the first drop, type II serum is added to the second, type III is added to the third, and isotonic sodium chloride solution (control) is added to the fourth.
Type I and II serums are pre-diluted in a ratio of 1:10, and type III serum - 1:5. All drops are stirred, dried, fixed and stained with diluted fuchsin. If the result is positive, microbial crowding (agglutination) is noted in one of the drops.
Second day of the study
The cultures are removed from the thermostat, examined, and smears are made from suspicious colonies. If gram-positive lanceolate diplococci are present in the smears, 2-3 colonies are isolated on a serum agar slant to obtain a pure culture. The crops are placed in a thermostat. Smears are made from the broth, stained with Gram and examined under a microscope.
Third day of the study The crops are removed from the thermostat. They check the purity of the culture - make smears, Gram stain and microscope. If there are gram-positive lanceolate diplococci in the isolated culture, the isolated culture is identified by culturing:
1) on Hiss media (lactose, glucose, sucrose, maltose) sowing is carried out in the usual way - by injection into the medium;
2) on a medium with inulin; 3) on a medium with optochin;
4) perform a bile test.
Inulin test. The culture under study is sown on a nutrient medium containing inulin and litmus tincture and placed in a thermostat. After 1824 hours, the crops are removed from the thermostat. In the presence of pneumococci, the medium turns red (streptococci do not change the consistency and color of the medium).
Determination of sensitivity to optochin. The isolated culture is inoculated on 10% blood agar containing optochin 1:50000. Pneumococci, unlike streptococci, do not grow on media containing optochin.
Bile test. 1 ml of the test broth culture is poured into agglutination tubes. A drop of rabbit bile is added to one of them, the second tube serves as a control. Both test tubes are placed in a thermostat. After 18-24 hours, lysis of pneumococci occurs, which is expressed in the clearing of a cloudy broth. In the control, the suspension remains cloudy.
A bile sample can be performed on a solid nutrient medium. To do this, a grain of dry bile is applied to a colony of pneumococci grown in dishes with agar and serum - the colony dissolves and disappears.
Fourth day of the study The results are recorded (Table 26).
Table 26. Differentiation of pneumococcus from viridans streptococcus
Note. j - breakdown of carbohydrates with the formation of acid.
Currently, serological research methods (RSK and RIGA) are widely used to determine streptococcal antibodies. Determination of the group and serovar of the isolated culture is carried out using fluorescent antibodies.
Determination of pneumococcal virulence. A daily broth culture of pneumococcus is diluted with 1% peptone water from 10 -2 to 10 -8, 0.5 ml of each dilution is administered to two white mice. The culture that caused the death of mice at a dilution of 10 -7 is assessed as virulent, at a dilution of 10 -4 -10 -
6 is considered moderately virulent. A culture that does not cause the death of mice is avirulent.
Control questions
1. What methods of isolating a pure culture of pneumococci do you know?
2. Which animal is most sensitive to pneumococcus?
3. What reactions are performed with the exudate of an infected mouse and for what purpose?
4. From which representatives of pyogenic cocci should pneumococcus be differentiated and using what test?
5. How to determine the virulence of pneumococci? Exercise
Make a diagram of the sputum examination, indicating its stages by day.
Culture media
Serum agar(see chapter 7).
Broth with whey(see chapter 7).
Blood agar(see chapter 7).
Hiss media(dry).
Sample medium with inulin. To 200 ml of distilled water add 10 ml of inactivated bovine serum, 18 ml of litmus tincture and 3 g of inulin. Sterilize with flowing steam at 100° C for 3 days in a row. Bile broth (see Chapter 7).
Chapter 16. Meningococci
The genus Neisseria includes two species of microbes that are pathogenic to humans: N. meningitidis and N. gonorrhoeae. Neisseria meningitidis were isolated from the cerebrospinal fluid of a patient by Wekselbaum (1887).
Morphology. Meningococci are paired cocci, consisting of two bean-shaped cocci, lying with concave sides to each other, their outer walls are convex (see Fig. 4). The size of each coccus is 0.6-0.8 × 1.2-1.5 microns. They are polymorphic. Meningococci are immobile, do not have spores, and form a capsule. Gram negative. In pure cultures they are arranged in tetrads and in the form of individual cocci without a specific order, and in smears prepared from cerebrospinal fluid they are often arranged in pairs. In purulent material they are located inside a leukocyte.
Cultivation. Meningococci are aerobes. They are demanding on nutrient media and reproduce only on media containing native protein (serum, blood). They grow at a temperature of 36-37° C (at 25° C, growth stops), the pH of the medium is 7.4-7.6. For their reproduction, a moist environment and an increased amount of carbon dioxide (a factor that stimulates their growth) are required. Sowing should be done on freshly prepared medium.
On solid nutrient media, meningococci form small, 2-3 mm in diameter, tender, translucent, bluish, viscous colonies. In broth with whey, meningococci produce a slight turbidity and a small sediment. Freshly isolated strains in S-form. Old cultures can dissociate and form rough R-form colonies.
Enzymatic properties. Biochemically, meningococci are little active. They break down glucose and maltose to form acid. They do not have pronounced proteolytic properties (they do not curdle milk, they do not liquefy gelatin).
The pathogenicity of meningococci is determined by the presence of a capsule that prevents phagocytosis, pili that promote attachment of the microbe to the surface of epithelial cells, and the formation of enzymes: hyaluronidase and neuraminidase.
Toxin formation. When bacterial cells are destroyed, a strong heat-stable endotoxin, which is a lipopolysaccharide of the cell wall, is released. When the disease occurs, it is found in the blood and cerebrospinal fluid of patients. The severity of the disease often depends on the amount of toxin accumulated.
Antigenic structure. Based on the polysaccharide (capsular) antigen, meningococci are divided into serogroups: A, B, C, D, X, Y U-135 29E (a total of nine serogroups).
According to the international classification, the main groups are A, B and C. Group A meningococci often cause generalized processes and are of greatest epidemiological significance. Meningococci of groups B and C cause sporadic diseases. The remaining serogroups have been poorly studied.
Resistance to environmental factors. Meningococci are not resistant. A temperature of 70°C kills them in 2-3 minutes, 55°C in 5 minutes. Unlike other cocci in this group, they do not tolerate low temperatures and are especially sensitive to temperature fluctuations.
Regular concentrations of disinfectant solutions destroy them quickly.
Animal susceptibility. Under natural conditions, animals are not sensitive to meningococci. But subdural injection of meningococci into monkeys can cause disease in them.
Intraperitoneal infection of guinea pigs and white mice causes their death due to the action of endotoxin.
Sources of infection. A sick person and a bacteria carrier.
Transmission routes. The main route is airborne.
Diseases in humans:
1) nasopharyngitis;
2) meningococcemia;
3) cerebrospinal epidemic meningitis.
Pathogenesis. Once on the mucous membrane of the nasopharynx, meningococci can be localized there, causing carriage or causing acute nasopharyngitis. If they penetrate the lymphatic vessels, blood and are generalized, they cause profound changes in parenchymal organs due to the action of endotoxin. Meningococcemia develops. When meningococci penetrate into the meninges, purulent inflammation occurs - meningitis. With meningococcal meningitis, the cerebrospinal fluid is cloudy (unlike tuberculous meningitis). During a spinal puncture, fluid flows out in a stream due to increased intracranial pressure. Meningeal phenomena are characterized by headache, neck stiffness, vomiting, etc. Meningitis most often affects children. In adults, infection is more often limited to carriage or nasopharyngitis.
Immunity. Post-infectious immunity is intense, it is caused by opsonins, complement-fixing and bactericidal antibodies. The course of the disease depends on the intensity of the formation of antibodies to polysaccharide and protein antigens.
Prevention. It comes down to early identification of carriers, isolation of those sick with nasopharyngitis. Patients are subject to hospitalization.
Specific prevention. A chemical vaccine has been developed, consisting of polysaccharides of serogroups A and C. Immunoglobulin is used for emergency prevention.
Treatment. Antibacterial drugs - penicillin, chloramphenicol, ampicillin.
Control questions
1. What are the morphological properties of meningococci?
2. On what media are meningococci grown and what conditions are necessary for their reproduction?
3. What is the biochemical activity of meningococci and their stability in the external environment?
4. What diseases are caused by meningococci?
5. By what antigen are meningococci divided into serogroups?
Pneumococci (synonym: Pneumococcus Talamon - Frankel, Streptococcus lanceolatus Pasteur, Micrococcus pneumoniae, Diplococcus pneumoniae Frankel, Streptococcus pneumoniae) are lanceolate diplococci isolated in human pneumonia. Discovered in 1881 by L. Pasteur and independently by G. M. Sternberg in the USA. The etiological relationship of pneumococci to human pneumonia was established by Frenkel and Weichselbaum (A. Frankel, A. Weichselbaum) in 1884.
Pneumococci isolated from the human or animal body are oval or lanceolate cocci, arranged in pairs; stained positively by Gram, with a value of about 1 micron. Each pair is surrounded by a thick capsule, revealed by eosin staining [T. J. Mackie, J. E. McCartney]. Pneumococci usually grow on artificial nutrient media in the form of chains. The chain of pneumococcus is usually shorter than that of streptococcus pyogenes. In culture, pneumococci are less lanceolate and more round, nonmotile and do not form spores. The capsule of pneumococcus is clearly visible on preparations from animal and human exudates, when growing on nutrient media to which blood, blood serum or ascitic fluid is added, but is poorly visible when growing on ordinary nutrient media. Pneumococci are aerobes or facultative anaerobes, easily stained with ordinary aniline dyes and Gram positive, although they become Gram negative in old cultures.
The growth of pneumococci on ordinary nutrient media is poor, but is significantly improved by adding glucose (0.1%), blood, serum or ascitic fluid to the nutrient medium. Pneumococci grow well in an atmosphere containing 5-10% carbon dioxide. The optimal growth temperature is 37°, maximum 42°, minimum 25°. Pneumococci are sensitive to changes in the pH of the nutrient medium; the optimal pH is 7.8, the acidity limit is 6.5, and the alkalinity limit is 8.3. On nutrient agar, pneumococci grow, forming small colonies 1 mm in diameter, tender, translucent, resembling dewdrops, not merging with each other. Colonies of pneumococci on special nutrient media, for example on blood agar (5%), are small, moist, transparent, with well-demarcated edges, exhibit α-hemolysis, appear surrounded by a greenish discolored zone, similar to that observed when viridans streptococcus (Streptococcus) grows on blood agar viridans).
When growing on colored nutrient media, pneumococci ferment carbohydrates with the formation of acid, but without the formation of gas. Fermentation of inulin is an important distinguishing feature of pneumococci (viridans streptococcus does not have the ability to decompose inulin). Pneumococci exhibit the ability to undergo rapid autolysis under the influence of bile salts. Bile or bile salts dissolve pneumococcus, which also distinguishes it from streptococcus.
Pneumococci are more sensitive than many other microorganisms to the bactericidal action of quinine and some of its derivatives. For example, optochin (ethylhydrocuprein) kills pneumococci at a concentration of 1:500,000, and streptococci at a concentration of 1:5000.
Pneumococci quickly lose virulence when stored in ordinary nutrient media, but can survive for months in the cold in the vacuum-dried spleen of a white mouse that died from pneumococcal septicemia. The most sensitive to pneumococci are white mice and rabbits, guinea pigs are less sensitive, and cats, dogs, chickens and pigeons are highly resistant. Pneumococcus does not produce a true toxin, but produces hemolysins that are active against red blood cells of sheep, guinea pigs and humans, as well as hyaluronidase, lencocidin and a necrotizing substance. The virulence of pneumococcus does not depend on these toxic formations, but on the presence of a specific soluble substance inherent in the pneumococcus of the corresponding type.
Pneumococci contain several antigens. Deep in the microbial cell there is a nucleoprotein component associated with species specificity. Closer to the surface is a species-specific polysaccharide (C-antigen) - a somatic antigen that is immunologically identical in all pneumococci. A type-specific protein (M-antigen), similar to the M-antigen of hemolytic streptococci, is also located close to the surface of the microbial cell. The capsule located more superficially consists entirely or partially of a polysaccharide specific to each type of pneumococcus and is closely related to the virulence of the living microbe. This antigen - a polysaccharide hapten or specific soluble substance (SSS) - is type-specific and serves to differentiate the immunological types of pneumococci.
Each type has an individual antigenic structure and virulence. Pneumococci isolated during pneumonia are divided on the basis of immunological reactions into types I, I, III and IV. Type IV pneumococci are immunologically heterogeneous. This type includes all pneumococci that do not belong to the first three types. Typing of pneumococci was important in its time due to the fact that the effect of serotherapy for pneumonia with specific serum was directly dependent on the type of pathogen.
Microbiological diagnosis of pneumococci consists of microscopic examination and isolation of pneumococci on artificial nutrient media. The type of pneumococcus is determined by: the capsule swelling reaction, the microagglutination reaction on glass (Sabin method) and the macroscopic agglutination reaction. If for some reason the white mouse cannot be used, then sputum or other pathological material is inoculated into blood broth with glucose, which is then used as an antigen in the same immunological reactions.
Pneumococci are found on the mucous membranes of the mouth and upper respiratory tract more often than in the human environment. Pneumococci are transmitted by airborne droplets. Type IV pneumococci are much more common than types I, II and III. Recently, the incidence of pneumococci in pneumonia has sharply decreased, while at the same time the incidence of staphylococci has increased significantly. With a significant decrease in the inoculation rate of pneumococci and, consequently, with a decrease in their importance as etiological agents, Escherichia coli, enterococci, proteus and other microorganisms began to be isolated in larger quantities.
Acquired immunity to pneumococcus is obviously associated with a capsular antigen, upon immunization with which a clear correlation of resistance is established with an antibody response to this antigen. See also Bacteria.
MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION KAZAN STATE MEDICAL UNIVERSITY DEPARTMENT OF MICROBIOLOGY
PNEUMOCOCCUS
KAZAN 1999
UDC 576.851.21(07)
Published by decision of the Central Coordination and Methodological Council of the Kazan State Medical University.
Compiled by:
(Head of the Department of Microbiology, Doctor of Medical Sciences, Professor O.K. Pozdeev, Assistant of the Department of Microbiology, Candidate of Medical Sciences, E.R. Fedorova.
Reviewers:
Head of the Department of Epidemiology, Kazan State Medical University, Doctor of Medical Sciences, Associate Professor M.Sh. Shafeev, Head of the Department of Epidemiology, Kazan State Medical Academy, Doctor of Medical Sciences, Professor V.E. Grigoriev.
Pneumococci /O.K. Pozdeev, E.R. Fedorov - Kazan: KSMU, 1999. - 14 s.
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Kazan State Medical University, 1999
Pneumococcus (Streptococcus pneumoniae) was first isolated by Pasteur (1881) while working on an anti-rabies vaccine and was initially considered the causative agent of rabies. The etiological role of the microorganism in the development of pneumonia in humans was proven by Frenkel and Weichselbaum (1884). The bacteria colonize human and animal organisms and belong to the group of so-called “oral” streptococci. They are the main causative agents of pneumonia, and can also cause pleurisy, meningitis, creeping corneal ulcers, purulent inflammation of the middle ear, septic conditions and other diseases. In the IX edition of Bergey's guide to bacteria (1994), pneumococci are included in group 17, “Gram-positive cocci.”
Epidemiology of lesions. Pneumococcus is one of the main causative agents of bacterial pneumonia recorded outside hospitals (2-4 cases per 1000 people); Every year, at least 500,000 cases of pneumococcal pneumonia are observed in the world (the real value is much higher). Children and the elderly are most susceptible to infection. The reservoir of infection is patients and carriers (20-50% of preschool children and 20-25% of adults); the main transmission route is contact; during outbreaks it is also airborne. The peak incidence occurs in the cold season. In the vast majority of cases, clinical forms of infection develop when the body’s resistance is impaired (including due to cold stress), as well as against the background of other pathologies (sickle cell anemia, Hodgkin’s disease, HIV infection, myeloma, diabetes mellitus, conditions after splenectomy, etc.) or with alcoholism. Options 1, 2 and 3 play the greatest epidemiological significance in pathology in adults; for children - 1, 2, 3 and 14 options. The virulence of serovars in descending order is variants 3, 1, 2, 5, 7 and 8. White mice (if infected, they die from septicemia within 24 hours), calves, lambs, piglets, dogs and monkeys are sensitive to pneumococci.
Morphology. Pneumococci are immobile, they do not form spores, and have a slightly elongated shape, reminiscent of the contours of a candle flame. In smears of clinical material, they are arranged in pairs, each of which is surrounded by a thick capsule. In smears from culture media, they may be located in short chains and be more rounded. On simple media they form a thin capsule; its development is stimulated by the introduction of blood, serum or ascitic fluid. Capsule formation is most pronounced in type III bacteria. When arranged in chains, the capsule may be common.
Cultural properties. Pneumococci are aerobes or facultative anaerobes; When cultivating, capnophilic conditions (5-10% CO2) are preferred. They are chemoorganogrophs and grow well on blood or serum media supplemented with 0.1% glucose. They can grow in the temperature range of 28-42 °C, optimum - 37 °C. Optimum pH -7.6-7.8. On dense media they form delicate, translucent, clearly defined colonies with a diameter of about 1 mm. Sometimes they can be flat with a central depression; like other streptococci, colonies never fuse
between themselves. On blood agar they form small translucent greenish-gray colonies. The center of the colonies is darker, the periphery is lighter. Under the colony and along its periphery, a zone of a-hemolysis is visible in the form of a greenish discolored zone (which is due to the transition of hemoglobin to methemoglobin). Colonies of type III pneumococcus often have a mucous consistency and are up to 2 mm in size. They are a bit cloudy and can merge with each other. They form S- and R-forms of colonies. When transitioning from the S- to the R-form, they lose the ability to synthesize the capsule. On liquid media with serum and 0.2% glucose they give uniform turbidity and a small flocculent sediment. With prolonged cultivation, the sediment increases.
Sustainability. Pneumococci belong to the group of unstable microorganisms. They persist in dry sputum for up to two months. Able to be stored for a long time at low temperatures; at a temperature of 60 °C they die within 3-5 minutes. A 3% solution of carbolic acid kills them in 1-2 minutes. Optochin (at a concentration of 1:100,000) and bile have a detrimental effect on pneumococci, which is used to identify bacteria.
Pneumococci differ from other microorganisms in a number of properties (Table 1).
Table 1 Biochemical properties of pneumococci
|
Test substrate |
Result |
Test substrate |
Result |
|
Growth at 100°C |
|||
|
Raffinose |
|||
|
Wednesday with 6.5% Nad |
|||
|
a-hemolysis |
|||
|
B-hemolysis |
Trehalose |
||
|
Phosphatase |
|||
|
Hippurate |
β-galactosidase |
||
|
Glycerol |
|||
|
Designations: “+” - 90% or more strains are positive; (+) - 80-89% of strains are positive; d - 21-79% of strains are positive; (-) - 11-20% of strains are positive; “- - 90% or more of the strains are negative. |
|||
Antigenic structure. Several types of antigens have been found in pneumococci: polysaccharide, 0-somatic antigen, located in the cell wall; polysaccharide capsular K-antigens and M-protein. The polysaccharide somatic antigen is similar to the C-substance of other streptococci. The relationship determines the similarity in the chemical structure of ribitteichoic acids associated with choline phosphate. Capsule antigens also have a polysaccharide nature, consisting of monosaccharides repeated in various combinations: D-glucose, D-galactose and L-rhamnose. Based on the structure of capsular antigens, pneumococci are divided into 84 serovars. It should be remembered that capsular antigens cross-react with antisera to antigens of group A and B streptococci, as well as with sera to Klebsiella and Escherichia antigens. During the transition from S to R form, capsular antigens are lost. For serotyping of pneumococci, group sera are produced, designated by Latin letters (A, B, C, D, etc.) and serovariant sera, designated by Roman numerals. Agglutinating serum III is not included in serum mixtures. It is released separately and cannot be bred. In humans, pneumococci of serovariants I, II and III are most often isolated. They are the most virulent for humans, so the agglutination test is initially performed using antisera to these variants. If the result is negative, the agglutination reaction is performed with a mixture of sera A, B, C, etc. (until a positive result is obtained), and then with separate antisera. For faster identification of serovars, the Neufeld reaction (immune capsule swelling) is used. The method is based on the ability of pneumococcal capsules to increase in volume in the presence of homologous antiserum, which is recorded by light optical microscopy. Capsular polysaccharides have sensitizing properties, manifested in the development of a delayed-type hypersensitivity reaction, detected using skin tests.
Pathogenicity factors. The main factor is the capsule, which protects bacteria from the microbicidal potential of phagocytes and diverts them from the action of opsonins. Non-encapsulated strains are practically avirulent and are rare.
The majority of the pool of antipneumococcal ATs consists of ATs to Ag capsules. An important function of the capsule and M-protein is also to ensure adhesion to the mucosa. Substance C is essential, as it specifically interacts with C-reactive protein. The consequence of such a reaction is the activation of the complementary cascade and the release of mediators of the acute phase of inflammation; their accumulation in the lung tissue stimulates the migration of polymorphonuclear phagocytes. The formation of powerful inflammatory infiltrates is accompanied by a disruption of the homeostasis of the lung tissue and its hepatization. Pneumococci produce endotoxin, a- and beta-hemolysins (pneumolysins), and leukocidin. α-pneumolysin is a thermolabile protein capable of neutralizing O-streptolysin,
erythrogenic substance, neuraminidase. Pneumococci also synthesize a number of enzymes that contribute to the pathogenesis of lesions - muramidase, hyaluronidase (promotes the spread of microorganisms in tissues), peptidase (breaks down IgA).
Clinical manifestations. Classic pneumococcal pneumonia begins suddenly; They note a rise in body temperature, a productive cough and chest pain. In weakened persons and the elderly, the disease develops slowly, with slight fever, impaired consciousness and signs of pulmonary heart failure.
Streptococcal meningitis is registered in all age groups; they are characterized by a violent onset with a rise in body temperature, stiffness of the neck muscles, headache, nausea and vomiting. Lesions of the vessels of the meninges are often accompanied by loss of consciousness; among children and the elderly, mortality can reach 80%. Quite often, hematogenous pneumococcal lesions, as well as sinusitis, mastoiditis, otitis media, endocarditis and peritonitis, are noted in people with immunodeficiencies (for example, HIV-infected) or patients with splenectomy. After an illness, unstable immunity develops, which is type-specific and is caused by the appearance of antibodies against the typical capsular polysaccharide.
Treatment. The basis of treatment for pneumococcal infection is antibiotics - penicillin, tetracycline, chloramphenicol, vancomycin, rifampicin, ceftriaxone.
Prevention.
Nonspecific prevention of pneumococcal infections is aimed at identifying patients and carriers with their subsequent treatment. For specific prevention of infection, children over two years of age, adults at risk, as well as healthy individuals during a disease outbreak are vaccinated with the polyvalent polysaccharide vaccine "Pneumovex 23". The drug contains 23 capsular polysaccharide antigens of pneumococci (1, 2, 3, 4, 5, 6B 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 1-9F, 19A, 20, 22F , 23F, 33F). Antigens
Laboratory diagnostics. The “gold standard” is pathogen isolation. It should be remembered that the material must be examined quickly, because bacteria are prone to rapid autolysis due to the activity of intracellular enzymes. The material for the study is sputum, pleural effusion and other exudates, cerebrospinal fluid, blood, mucus from the nose and pharynx, discharge from eye ulcerations, discharge from the ear, urine, pieces of organs (in case of death of the patient). A signal response to pneumococcal infection can be issued when neutrophils and gram-positive lanceolate diplococci (at least 10 per field of view) are detected in sputum smears. Otherwise, they resort to isolating the pathogen.
The first stage of the study. Pathological material is subjected to preliminary bacterioscopy (except blood). The sputum is placed in a sterile Petri dish, washed, a purulent mucous lump is captured with a loop, ground on a glass slide, dried and stained with a Gram stain. The smear reveals gram-positive lancet-shaped or oval-shaped cocci surrounded by a capsule (capsule formation is observed only in pneumococci isolated from sick and infected animals). Detection of pneumococcal capsules can be carried out using the Burri-Gins method. Inoculation of pathological material is carried out on 5-10% blood or serum agar and on enrichment medium (8-10% whey broth). If sepsis of a pneumococcal nature is suspected, 5-10 ml of the patient's blood is inoculated into 45-90 ml of whey broth. The cerebrospinal fluid, if it is clear, is centrifuged and a few drops from the sediment are inoculated onto nutrient media. Semi-solid whey agar is used as an enrichment medium. The crops are incubated at 37 °C for 24 hours. The best method for isolating a pure culture of pneumococci is to infect white mice with pathological material. Sputum, washed in a Petri dish with sterile saline, is ground in a sterile mortar with a sterile pestle or broken glass while adding saline in a ratio of 1:2-1:5. The suspension is settled, the supernatant liquid in an amount of 0.5-1 ml is administered intraperitoneally to white mice. If pneumococci are present in the material, mice die within 72 hours. Typical pneumococci are found in smears from organs and blood. Organs and blood are also cultured on whey broth and on Petri dishes with blood or serum agar.
Second stage of the study. The growth pattern on nutrient media is studied. On blood agar, colonies of pneumococci are small, round, with smooth
edges, tender, surrounded by a zone of greening of the medium (which is very reminiscent of the growth of viridans streptococci). On serum agar, the colonies are delicate, translucent and transparent. During bacterioscopy of Gram-stained smears. Gram-positive diplococci without capsules are detected. After bacterioscopy, colonies suspected of pneumococci are subcultured onto slanted serum or blood agar or into whey broth. When microscopying smears from the enrichment medium, along with various microflora, gram-positive cocci can be detected, arranged in pairs or short chains. The material from the enrichment medium is transferred to solid nutrient media. The crops are incubated at 37°C for 24 hours.
The third stage of the study. On slants of blood agar, pneumococci form a delicate, thin, translucent coating. In whey broth, pneumococci cause turbidity and a slight sediment. In smears from solid culture media, pneumococci can have different appearances. Along with diplococci of elongated shape with pointed outer ends, reminiscent of a candle flame, there are cells of regular oval and round shape. In broth culture, pneumococci are often arranged in chains. Based on the morphological and cultural properties of pneumococci, it is difficult to distinguish from viridans streptococci, therefore a set of special tests has been proposed for their differentiation:
Solubility in bile (deoxycholate test);
Ability to decompose inulin;
Sensitivity to optochin;
Agglutination reaction with specific antipneumococcal sera;
The ability to decompose glucose, maltose, sucrose, lactose, mannitol, sorbitol and salicin.
The most accessible methods for differentiating pneumococci from other streptococci are a test with optochin (inhibits their growth); They are distinguished from viridans streptococci by their ability to ferment inulin, as well as sensitivity to bile.
Deoxycholate test. After preliminary bacterioscopy, 10 drops of isolated pure culture (preferably broth) are added to a test tube with 5 drops of sterile bovine bile. The control is a culture added to a test tube with 5 drops of saline solution. After 30-60 minutes of incubation at 37 °C, complete lysis of the culture is observed in the form of clearing in the test tube with bile; in the control tube the mixture remains cloudy. It should be remembered that avirulent pneumococcal cultures are resistant to bile.
Bile resistance can also be tested by culture in 10% bile broth. The test material is added to the medium, and the broth becomes cloudy. After 24 hours of incubation at 37 °C, the presence of pneumococci will be indicated by clearing of the broth as a result of lysis of bacteria.
You can also use discs soaked in a 20% bile solution. The discs are placed on the grown culture in a dish and incubated for 1-2 hours at 37 °C. In the presence of pneumococci, colonies are lysed around the disc at a distance of 1-2 mm.
Test for inulin. The pneumococcal culture is inoculated on a medium with inulin. To do this, add 200 ml of sterile distilled water, 18 ml of litmus tincture and 3 g of inulin to 100 ml of bovine serum heated at 56 °C for 30 minutes, and sterilize with running steam for 30 minutes. The crops are incubated at 37 °C for 24 hours. Pneumococcus decomposes inulin, causing the medium to turn red. Viridans streptococcus does not cause redness of the environment.
Test with optochin. The test pneumococcal culture is inoculated on whey broth with optochin at a dilution of 1:100,000 or 1:200,000. Pneumococcus does not grow on such a medium. You can also determine sensitivity to optochin by plating on 10% blood agar containing optochin at a dilution of 1:50,000. The control is to inoculate the culture on blood agar. Pneumococci do not grow on the medium with optochin; growth of pneumococci is observed on the control medium. You can use disks soaked in 6 μg of optochin, which are applied to the surface of the medium after inoculation. In pneumococci, a zone of growth inhibition with a diameter of at least 18 mm is formed around the disc.
Virulence test. A daily culture of pneumococcus grown in whey broth is diluted with 1% sterile peptone water (pH - 7.6) or slightly alkaline broth to 1:10. The diluted culture is administered intraperitoneally to white mice weighing 16-20 g in a volume of 0.5 ml and observed for 72 hours. The organs of a dead mouse are inoculated onto nutrient media and the fingerprint smears are examined microscopically. Highly virulent cultures include pneumococci, which cause the death of mice after the introduction of a culture at a dilution of 1:10. Avirulent cultures do not cause death in mice.
Serotyping of pneumococci. The 18-hour culture is tested in the Sabin microagglutination reaction. 4 drops of pneumococcal culture are applied to a glass slide. To 1 drop add a drop of antipneumococcal serum type 1, to the 2nd - type II serum, to the 3rd - serum - 111, to the 4th - a drop of normal serum. The mixtures on glass are mixed with a loop and examined under a magnifying glass or microscope at low magnification. In a positive case, agglutination is observed in one of the first three drops. The type of pneumococcus is determined by an agglutination reaction with specific agglutinating sera of the first three fixed types. Cultures that are not agglutinated by these types of sera are classified as X-group. The reaction is set up as follows. Pour 0.5 ml of 18-hour broth culture into test tubes. Then an equal volume of serum is added, diluted with saline in a ratio of 1:5. The controls are 2 test tubes, one of which contains the test culture mixed with
normal rabbit serum, and the other - only the test culture. The contents of the tubes are thoroughly shaken and placed in a thermostat at 37 °C for 2 hours, after which a preliminary calculation of the reaction is carried out. Final results are noted after additional storage at room temperature for 20 hours. Agglutination is assessed as four pluses if the contents of the tubes are completely cleared and the agglutination culture is a dense film that does not break when shaken; three pluses if, when the contents of the tube are completely cleared, the agglutinating culture easily breaks into parts; two pluses - if clearing does not occur, particles of the agglutinated culture are clearly visible to the naked eye in the turbid contents of the test tube; with agglutination for one plus, a fine-grained mixture of glued pneumococci is found in the test tube. In case of a negative reaction visible to the eye, agglutination is not observed;
The contents of the test tubes after shaking are uniformly cloudy.
Typing of X-group pneumococci is carried out using group
sera containing a mixture of typical agglutinating sera taken
in equal volumes. Prepare the following group sera by
mixing equal volumes of undiluted standard diagnostic
serums (Lund, I960):
A -1, II, IV, V, XVIII serovars;
B - VI, VIII, XIX serovars;
C - VII, XX. XXIV, XXXI, XL serovars;
D - IX, XI, XVI, XXXVI. XXXVII serovars;
E - X, XXI. XXXIII, XXXIX serovars;
F - XII. XVII. XXII, XXXVII, XXXII, XLI serovars;
G - XIII, XXV. XXIX, XXXIV, XXXV, XXXVIII, XLII, XLVII serovars;
J - XLIII. XLIV, XLV, XLVI serovars.
Type III agglutinating serum is used per se (without mixing with other standard sera) due to the difficulty of obtaining it in a sufficiently high titer. Typing is carried out in two stages: first with the help of group sera, and then with individual sera of the group with which a positive reaction was obtained. Serotyping of pneumococci is used primarily for epidemiological studies of the results of specific serotherapy and seroprophylaxis.
Microagglutination of pneumococci using the Sabin method can be obtained by mixing anti-pneumococcal sera with exudate from the abdominal cavity of a mouse contaminated with the sputum of a patient. Already four hours after infection, a pure culture of pneumococci is detected in the exudate, giving a positive Sabin agglutination.
Accelerated methods for detection and typing of pneumococci. 1. Neufeld's method or the phenomenon of swelling of the pneumococcal capsule. One lump of freshly secreted sputum of the patient is applied to three
coverslips, to each of them add a drop of undiluted specific antipneumococcal serum (types 1, II, III) and a drop of Loeffler's blue. The drops are thoroughly mixed and covered with a glass slide with a well smeared around the edges with Vaseline. After two minutes, the hanging drops are examined under a microscope with an immersion system. In a positive case, a sharp increase in pneumococcal capsules is visible. If the result is negative, the capsules are hardly treasured. The swelling reaction is specific and does not give a positive result with other capsular bacteria. I do not use it for examining sputum from patients treated with sulfonamides and antibiotics, because in this case, non-capsular pneumococci can be isolated.
2. Precipitation method. 5-10 ml of sputum is boiled in a water bath until a dense clot is obtained. The clot is ground and a small amount of saline is added and boiled again for several minutes to extract the specific polysaccharide from pneumococci. The suspension is centrifuged, and a ring precipitation reaction is performed with the resulting clear liquid and specific standard sera in precipitation tubes. The appearance of a ring at the interface between the liquids indicates a positive result.
3. Determination of pneumococcal capsules according to Burri. A drop of the test material and a drop of ink are applied to the end of the slide. The mixture is mixed and a smear is made, dried in air and, without fixing, examined under a microscope. The background of the preparation is dark smoky; microbial bodies and their capsules are not stained. The preparation prepared according to Burri can be fixed with Nikiforov’s mixture, rinsed with water, and stained with Ziel fuchsin diluted 1:3 for 3-5 minutes. Against the dark background of the smear, unpainted capsules stand out, inside of which there are bacteria of a bright crimson color (Hins method).
Page 40 of 91
The causative agent of lobar pneumonia (pneumonia) is pneumococcus - Diplococcus pneumoniae, first discovered by Pasteur in the saliva of a person who died of rabies (1881).
Morphology and tinctorial properties. Pneumococci (Fig. 67 and 68 in the inset) are paired cocci with an elongated lancet-like shape. Therefore, they are otherwise called lanceolate diplococci. Forming short chains, pneumococci become similar to streptococci, and therefore II. F. Gamaleya named them Streptococcus lanceolatus. The cell size ranges from 0.5X0.75 to 1X1.5 μm. They do not have spores or flagella. A distinctive feature of pneumococcus is the formation of a capsule, which can be clearly expressed in pathological materials (sputum, blood, etc.). When cultivated on nutrient media, the capsule is lost. Pneumococci easily accept aniline dyes and stain positively on the Gram.
Cultural and biochemical properties.
Rice. 68. Pneumococci in sputum smear.
Pneumococci are aerobes and facultative anaerobes. Temperature optimum is about 37°. They grow on media containing animal protein (blood or serum agar, ascitagar).
After 24 hours, small colonies are formed on the surface of the agar, reminiscent of streptococcal colonies, but smaller and more transparent.
On slanted agar, with abundant inoculation, a very delicate transparent coating is obtained, consisting of tiny, non-merging colonies; on broth, there is slight turbidity and a small flaky sediment.
Freshly isolated strains do not grow on gelatin. Old laboratory strains of pneumococci can produce small whitish colonies already at 18-22°. Gelatin is not liquefied.
They grow well in milk, curdling it to form acid.
On blood agar, a zone of incomplete hemolysis with a greenish-brown coloration of the medium forms around the colonies. 
Rice. 67. Pneumococci in pure culture from broth.
Pneumococci degrade sucrose, raffinose and lactose. The most important feature is the decomposition of inulin. Most streptococci do not have this property. Virulent pneumococci are bile soluble.
Antigenic structure and serological types of pneumococci. The cytoplasm of pneumococci contains a protein antigen common to all pneumococci. This antigen determines their species specificity. The capsule contains specific polysaccharide antigens (hapten), which differ in their chemical composition among different pneumococci (type antigens). Based on these typical antigens, using the agglutination and precipitation reaction, all pneumococci are divided into three main groups (I, II, III) and a fourth group (X-group). The X-group includes more than 70 types.
Resistance. On artificial nutrient media, pneumococci die quickly (4-7 days). Under a layer of petroleum jelly in liquid and semi-liquid media containing protein, they remain viable for 3-12 months.
Pneumococci tolerate drying well: they persist in dry sputum in diffuse light for up to 2 months. When heated to 52-55° they die in 10 minutes, at 60° they die even faster. In a solution of carbolic acid (3%), pneumococci die within 1-2 minutes.
Pneumococci are especially sensitive to optochin. Under the influence of the latter, they die at a concentration of 1: 1,000,000.
Toxin formation and pathogenicity for animals. Pneumococcal venom is an endotoxin. Among laboratory animals, white mice and rabbits are more sensitive to pneumococcus. Parenteral administration of virulent pneumococci after 24-48 hours causes the death of animals with symptoms of sepsis. Upon autopsy, fibrinous exudate is found at the injection site; the spleen is enlarged and hyperemic.
Pathogenesis and diseases in humans. The entry point for infection is usually the mucous membrane of the pharynx. The introduction of pneumococci into the body and their penetration into the lung tissue can apparently occur both through the lymphatic and circulatory system, and directly through the branches of the bronchi. The most common disease is lobar pneumonia, which is characterized by a sudden onset, high fever, sometimes with chills, pain in the side when breathing, headache, sometimes loss of consciousness, delirium, and severe agitation. Subsequently, a cough appears with characteristic rusty-red sputum. In the lungs, a process is observed that often involves one, less often two or three lobes.
The sources of infection are the sick person and the bacteria carrier. Infection from the outside occurs both aerogenously - by droplets from the carrier, and through dust infection. Pneumococci can persist in dried sputum for a long time (about 2 months) and enter the air with dust.
When examining healthy people, pathogenic pneumococci are often found in the nasopharynx, so the possibility of autoinfection cannot be excluded, and factors that weaken the body's resistance, such as hypothermia, play a significant role.
In addition to lobar pneumonia, pneumococci cause inflammation of the middle ear, meninges (meningitis), as well as the mucous membrane of the nose and air sinuses, tonsillitis, creeping corneal ulcers and inflammation of the lacrimal sac.
Immunity. Having pneumonia does not provide immunity. The disease can recur several times. This is explained by the presence of many types of pneumococci and the fact that past pneumonia increases the body's sensitivity to pneumococci.
The serum of those who have recovered contains antibodies (agglutinins, etc.).
By the time of the crisis with pneumonia, the concentration of antibodies in the blood reaches a significant titer, and phagocytosis increases sharply (I. Ya. Chistovich). Based on these data, immunity in pneumonia should be considered primarily as phagocytic, in which antibodies (bacteriotropins) play a major role.
Microbiological diagnostics. The materials for research in pneumococcal diseases are sputum, blood and pus taken from various lesions, and less often cerebrospinal fluid.
Pathological material (excluding blood) is examined bacterioscopically, bacteriologically and by infecting white mice. The latter method has to be resorted to because the source material, especially sputum, usually contains abundant foreign microflora, which, when directly inoculating the material on nutrient media, makes it difficult to isolate pneumococcus.
Smears from sputum, pus, etc. are Gram stained. Under a microscope, lanceolate diplococci surrounded by a capsule are found, Gram stained positively.
To isolate cultures, inoculate them on blood agar or ascig agar. After 24-48 hours of growth at 37°C, in the presence of pneumococcus, characteristic colonies appear. Colonies are sown on slants of whey or ascites agar, and the isolated culture is tested for solubility in bile and the ability to decompose inulin.
Infecting a white mouse is the most reliable way to isolate a pneumococcal culture. Material from a patient or corpse (sputum, pus, a piece of organ, etc.) is placed in a sterile cup, then ground in a sterile mortar, with 1-2 ml of sterile broth and 0.5 ml of this suspension is injected intraperitoneally into a white mouse. After the death of the mouse, which occurs within 12-48 hours, blood cultures are taken from the heart, and in almost all cases a pure culture of pneumococcus is obtained.
If sepsis is suspected, 10-20 ml of blood is inoculated into ascitic or serum broth. After enrichment, the broth is inoculated onto blood agar and the isolated pure culture is identified by morphological and biochemical characteristics.
Specific therapy and chemotherapy. Currently, sulfonamide drugs and antibiotics (penicillin, biomycin, tetracycline, etc.) are used with great success to treat lobar pneumonia.