Laboratory diagnosis of staphylococcal infection.

Gram-positive cocci

There are several types of symbiosis between cocci and the human body. Saprophytic and conditionally pathogenic types of staphylococci and streptococci live on the skin, mucous membranes, and in the respiratory tract. Meningococci may be harboured for long periods in the nasopharynx, and faecal streptococci (enterococci) in the intestine.

When body resistance is lowered or the skin and mucous membranes are injured, these bacteria penetrate the body tissues and cause infection. The various cocci possess different organotropic ability. This is distinctly manifest in meningococci, and gonococci but less so in staphylococci and streptococci. Cocci belong to the families Micrococcaceae, Streptococcaceae Peptococcaceae, Neisseriaceae.

Staphylococci. The staphylococcus, Staphylococcus aureus, was discovered by R. Koch (18,78), and later isolated from furuncle pus by L. Pasteur (1880). It has been described as the causative agent of numerous suppurative processes by A. Ogston (1881), and has been studied in detail by F. Rosenbach (1884).

Morphology. Staphylococci are spherical in shape, 0.8-1 mem in diameter, and form irregular clusters resembling bunches of grapes (Fig. 1). In smears from cultures and pus the organisms occur in short chains, in pairs, or as single cocci. Large spherical (L-forms) or very small (G-forms) and even filterable forms may be seen in cultures which have been subjected to various physical, chemical, and biological (antibiotics) factors.

Figure 1. Staphylococci.

Staphylococci are Gram-positive organisms which possess no flagella and do not form spores. A macrocapsule can be seen on ultrathin sections of Staphylococci isolated from infected mice. The nucleoid occupies most of the cytoplasm and is filled with DNA fibrils. The G+C content in DNA ranges between 30.7 and 39.0 per cent.

Cultivation. Staphylococci are facultative-anaerobes. They grow well on ordinary nutrient media with a pH of 7.2-7.4 at a temperature of 37 °C but do not grow at temperatures below 10 °C and above 45 °C. At room temperature with adequate aeration and subdued light – the organisms produce golden, white, lemon-yellow, and other pigments known as lipochromes. These pigments do not dissolve in water but are soluble in ether, benzene, acetone, chloroform, and alcohol. They are most readily formed on milk agar and potatoes at a temperature of 20-25° C.

On meat peptone agar Staphylococci produce well defined colonies with smooth edges, measuring from 1-2 to 2.5 mm in diameter. Under the microscope the course granular nature of the colonies can be seen, the latter are opaque and have a dense centre. Their colour epends on the pigment produced by the organisms. Besides the typical colony types, Staphylococci produce R-, G-, and L-forms. Growth of Staphylococci in meat-peptone broth produces diffuse opacity throughout the medium and, subsequently, a precipitate. In some cases when there is sufficient aeration, the organisms form a pellicle on the surface of the broth. Staphylococci grow well on potatoes and coagulated serum. After 24-48 hours of incubation there is usually abundant growth along the inoculation stab and liquefaction of gelatin media. On the fourth or fifth day the gelatin medium resembles a funnel filled with fluid.

On blood agar pathogenic Staphylococci cause haemolysis of the erythrocytes. Rabbit and sheep erythrocytes are the most sensitive to the staphylococcal haemotoxin.

Fennentative properties. Staphylococci produce enzymes which cause the lysis of proteins and sugars (see Table 1). There is no indole production in young cultures. The organisms liquefy gelatin, coagulate milk and occasionally serum, reduce nitrates to nitrites, produce urease, catalase, phosphatase, ammonia, and hydrogen sulphide. They ferment glucose, levulose, maltose, lactose, saccharose, mannitol, and glycerin, with acid formation. A connection has been revealed between arginase activity and the level of g-toxin formation.

Toxin production. Staphylococci produce a-, b-, d- and g-haemolysins which are characterized by lethal, haemolytic, and necrotic activity. Filtrates of staphylococcal broth cultures contain an enterotoxin which causes food poisoning on entry into the gastro-intestinal tract. Staphylococci excrete exofoliatines which cause staphylococcal impetigo and pemphigus neonatomm in children.

Leucocidin, a substance which destroys leucocytes, haematoblasts of the bone marrow and nerve cells, is also produced by the Staphylococci. The organisms also coagulate blood plasma. Their ability to coagulate, plasma is a stable property and is used for differentiating various strains. Coagulase is thermoresistant. It can be isolated from staphylococcal broth cultures.

Staphylococci produce fibrinolysin which when added to a blood clot dissolves the latter within 24-48 hours.

The Staphylococci produce hyaluronidase which breaks down hyaluronic acid, a component of connective tissue.

Coagulase, fibrinolysin, lecithinase, hyaluronidasa and phosphatasa all belong to the group of enzymes possessing destructive properties. Lecithinase destroys the lecithin protective membranes of the colloidal particles of a substance found in human, sheep, and rabbit erythrocytes. An anticoagulant which inhibits blood coagulation has also been derived from the staphylococcal cultures. This staphylococcal anticoagulant is produced in exudates of inflamed tissues, occurring during staphylococcal infections. Haemagglutmins which cause the agglutination of rabbit erythrocytes have also been found in staphylococcal culture filtrates. Virulent Staphylococci inhibit the phagocytic activity of leucocytes.

Many microbiologists believe that the Staphylococci isolated from patients produce alpha-haemolysins, while the organisms pathogenic for animals (e. g. itesponsible for mastitis in cows) more often produce beta-haemolysin.

Staphylococcal exotoxin, inactivated by treatment with 0.3-0.5 per cent formalin at 37 °C for 7-28 days, and injected parenterally to humans and animals, stimulates the production of a specific antitoxin capable of reacting with the toxin.

Antigenic structure. Polysaccharides A and B have been obtained from a staphylococcal suspension by treating the latter alternately with acid and alkali and removing the proteins with trichloracetic acid.

Polysaccharide A was extracted from pathogenic strains isolated from patients with septicaemia, furunculosis, osteomyelitis, and acute conjunctivitis, etc. Polysaccharide B is found in avirulent, non-pathogenic strains. Polysaccharides A and B differ not only in their serological reactions but also in their chemical structures.

Antigen C, containing a specific polysaccharide, has been recently isolated. Staphylococcal polysaccharides demonstrate a marked type specificity. Even in a 1 :1000 000 dilution they give a distinct precipitin reaction. The protein antigen is common to all species and types of staphylococci.

Three types (I, II, III) of staphylococci have been revealed by the agglutination test and precipitin reaction. However, quite a number of cultures are unsuitable for serological typing. Recent studies have revealed fifteen type-specific staphylococcal antigens.

Classification. Staphylococci are included in the class Bacteria, family Micrococcaceae, genus Staphylococcus.

According to the contemporary classification, staphylococci are subdivided into three species: S. aureus, S. epidermidis, and S. saprophyticus.

Differentiation of Staphylococci

Main characteristics	Species
	S. aureus	S. epidermidis;	S. saprophyticus
Plasmacoagulase	+	—	—
Phosphatase	+	+	—
Arginine dihydrolase nitrate	+	+	—
Reductase	+	+	—
Protein A in superficial antigen	+	—	—
Oxidation
Mannitol	+	—	+
Trehalose	+	—	+
Galactose	+	+	—
Ribose	+	—	—
Production of alpha-toxin	+	–	–
Resistance to novobiocin	S	S	R
Growth in the presence of biotin	–	+	NT

Note: S, sensitive; R, resistant, NT, not tested.

Certain strains of the family Micrococcaceae are strict anaerobes. Peptococcus niger, Peptococcus anaerobius, Peptococcus asaccharolyticus and others are usually conditionally pathogenic for human beings. They live in the mouth mucosa, in the intestine, urogenital tract, and in other parts of the human body. In weakened individuals and in people suffering from chronic diseases the anaerobic micrococci may give rise to various diseases and complications.

Resistance. Staphylococci are characterized by a relatively strong resistance to desiccation, freezing, sunlight, and chemical substances. After desiccation they can survive for more than 6 months. Repeated freezing .and thawing do not kill the organisms. They survive for many hours under direct sunlight. Staphylococci maintain their viability for more than 1 hour at 70 °C. At a temperature of 80 °C they are destroyed within 10-60 minutes and at boiling point, they instantly perish. A 5 per cent phenol solution kills the organisms in 15-30 minutes. Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant green which is used for treating pyogenic skin diseases caused by these organisms. Staphylococci possess high resistance to antibacterial agents, in 70-80 per cent of cases they are resistant simultaneously to 4-5 agents. Cross resistance to antibiotics of the macrolide group (erythromycin, oleandomycin, etc.) is encountered.

Pathogenicity for animals. Horses, cattle, sheep, goats, pigs, and, among laboratory animals, rabbits, white mice, and kittens are susceptible to pathogenic staphylococci.

An intracutaneous injection of a culture of pathogenic staphylococci produces inflammation and subsequent necrosis in the skin of the rabbit. An intravenous injection of a staphylococcal culture filtrate causes a condition similar to acute poisoning in rabbits, which is characterized by motonc excitation, respiratory disorders, convulsions, paralysis of the hind extremities, and sometimes, by diarrhoea and urine discharge. After complete fatigue the animal shortly dies.

Staphylococci or their toxin will cause vomiting, diarrhoea, and weakness in kittens if introduced per os or intraperitoneally. Functional disorders of the digestive tract arise owing to the effect of tne enterotoxin which is distinguished from the other fractions of the staphylococcal toxin by its thermoresistance. It withstands a temperature of 100 °C for 30 minutes. The most reliable test for the presence of enterotoxin is an intravenous injection to adult cats.

Pathogenesis and diseases in man. Staphylococci enter the body through the skin and mucous membranes. When they overcome the lymphatic barrier and penetrate the blood, staphylococcal septicaemia sets in. Both the exotoxins and the bacterial cells play an important role in pathogenesis of diseases caused by these organisms. Consequently, staphylococcal diseases should be regarded as toxinfections.

The development of staphylococcal diseases is also influenced by the resulting allergy which in many cases is the cause of severe clinical forms of staphylococcal infections which do not succumb to treatment.

Staphylococci are responsible for a number of local lesions in humans: hidradenitis, abscess, paronychia, blepharitis, furuncle, carbuncle, periostitis, osteomyelitis, folliculitis, sycosis, dermatitis, eczema, chronic pyodermia, peritonitis, meningitis, appendicitis, and cholecystitis.

Diabetes mellitus, avitaminosis, alimentary dystrophy, excess perspiration, minor occupational skin abrasions, as well as skin irritation caused by chemical substances, are some examples of the conditions conducive to the formation of pyogenic lesions of the skin and furunculosis.

In some cases staphylococci may give rise to secondary infection in individuals suffering from smallpox, influenza, and wounds, as well as postoperative suppurations. Staphylococcal sepsis and staphylococcal pneumonia in children are particularly severe diseases. Ingestion of foodstuffs (cheese, curds, milk, rich cakes and pastry, ice cream, etc.) contaminated with pathogenic staphylococci may result in food poisoning.

Staphylococci play an essential part in mixed infections, and are found together with streptococci in cases of wound infections, diphtheria, tuberculosis, actinomycosis, and angina.

The wide use of antibacterial agents, antibiotics in particular, led to considerable changes in the severity and degree of the spread of staphylococcal lesions. Growth in the incidence of diseases and intrahospital infections m obstetrical, surgical and children's in-patient institutions, intensive spread of the causative agent, and increase in the number of carriers among the medical staff and population have been noted in all countries of the world. Intrauterme and extrauterine contamination of children with staphylococci has been registered, with the development of vesiculopustular staphyloderma, pemphigus, infiltrates, abscesses, conjunctivitis, nasopharyngitis, otitis, pneumonia, and other diseases.

It has been established that staphylococci become adapted rapidly to chemical agents and antibiotics due to the spread of R-plasmids among these bacteria. The high concentration of drugs in the body of humans and in the biosphere has resulted in essential disturbance in the microflora and the extensive spread of resistant strains possessing more manifest virulence. The L-forms of staphylococci are especially marked by increased degree of resistance to antibiotics.

Immunity. The tendency to run a chronic flaccid course or relapse is regarded as a characteristic symptom of staphylococcal infections. This peculiarity gives a basis for concluding that postmfectional immunity following staphylococcal diseases is of low grade and short duration.

Immunity acquired after staphylococcal diseases is due to phagocytosis and the presence of antibodies (antitoxins, precipitins, opsonins, and agglutinins).

The inflammation restricts the staphylococci to the site of penetration and obstructs their spreading throughout the body. At the centre of inflammation the organisms undergo phagocytosis. Neutralization of the staphylococcal toxin by the antitoxin is an important stage of the immunity complex. As a result of capillary permeability, the antitoxin penetrates from the blood into the inflammation zone and renders harmless the toxin produced by staphylococci. Thus, the phagocytic and humoral factors act together and supplement each other.

The presence in the human organs and tissues of antigens which are also common in the staphylococci (mimicry antigens) is among the causes of low immunity. This causes a state of immunological tolerance to staphylococci and their toxins, which provides favourable conditions for uninhibited reproduction of the causative agent in the patient's body. The wide use of antibacterial agents promotes intensive selection of staphylococcal strains resistant to the natural inhibitors of the microorganism.

Laboratory diagnosis. Test material may be obtained from pus, mucous membrane discharge, sputum, urine, blood, foodstuffs (cheese, curds, milk, pastry, cakes, cream, etc.), vomit, lavage fluids, and faeces.

The material is examined for the presence of pathogenic staphylococci. Special rules are observed when collecting the material since non-pathogenic strains are widespread in nature.

Laboratory studies comprise the determination of the main properties of the isolated staphylococci (i. e their morphologic, cultural and biochemical characteristics), as well as their virulence. For this purpose the following procedures are carried out. Smears are made from pus and stained by the Gram method. Pus is inoculated onto blood agar and meat-peptone agar containing crystal violet. In cases of septicaemia blood is inoculated into glucose broth.The isolated pure culture is tested for its haemolytic (by inoculation onto blood agar plates), plasmacoagulative (by inoculation into citrated rabbit plasma), and hyaluronidase activities. Virulence is determined in rabbits by intracutaneous injection of 400 million microbial cells. Necrosis develops at the site of injection within 24-48 hours.

Pigment production of the isolated culture is also taken into account. For revealing sources of infection, particularly food poisoning and outbreaks of sepsis in maternity hospitals, serological typing and phage typing are carried out. To ensure effective therapy the isolated cultures are examined for sensitivity to antibiotics.

In cases of food poisoning presence of the enterotoxin in th isolated staphylococcal culture is tested for by intravenous injection of the culture filtrate to adult cats. In cases when intoxication is due to ingestion of the milk of a cow suffering from mastitis, the culture grown on starch medium is tested directly for toxin production as a means of detecting staphylococci of animal origin. When the causative agent cannot be detected (osteomyelitis and other diseases), the patients' serum is tested for agglutinins.

Treatment. Staphylococcal diseases are treated with antibiotics (penicillin, phenoxymethyl penicillin, tetracycline, gramicidin, etc.), sulphonamides (norsulphazol, sulphazol, etc.), and antistaphylococcal gamma-globulin.

When tr eating patients suffering from staphylococcal infections one should bear in mind that it is necessary to relieve intoxication and improve the immunological defence forces of the body (infusion of glucose, plasma, blood transfusion, injection of cardiac stimulants).

In cases of chronic staphylococcal lesions specific therapy is recommended: autovaccines, staphylococcal anatoxin, antitoxic serum, and diphage containing staphylococcal and streptococcal phages.

Staphylococd produce strains resistant to sulphonamides, antibiotics, and bacteriophage, which advances the wide distribution of staphylococcal infection. This variability is of particular importance in the therapy of staphylococcal pyogenic diseases. The medical services produce semisynthetic preparations of penicillin and tetracycline which are effectively used for the treatment of these diseases.

Prophylaxis. The general precautionary measures include: hygiene in working and everyday-life conditions, treatment of vitamin deficiency, prevention of traumatism and excess perspiration, observance of rules of hygiene in maternity hospitals, surgical departments, children's institutions, industrial plants and enterprises, particularly canneries, observance of personal hygiene and frequent washing of hands in warm water with soap.

Routine disinfection of hospital premises (surgical departments, maternity wards) and bacteriological examination of the personnel for carriers of pathogenic staphylococci resistant to antibiotics are also necessary.

To prevent pyoderma protective ointments and pastes are used at industrial enterprises. For the treatment of microtraumas, besides iodine tincture and alcohol solutions of aniline dyes, solutions are widely applied which dry in one or two minutes and form an elastic film protecting the wound surface from dirt and infection. Patients with bums are kept in soft (plastic) isolating compartments which protect them against the entry of microflora from the external environment. In some cases specific prophylaxis by means of immunization with the staphylococcal anatoxin may be recommended for individuals subject to injury or infection with antibiotic-resistant staphylococci.

Streptococci. The streptococcus {Streptococcus pyogenes) was discovered by T. Billroth (1874) in tissues of patients with erysipelas and wound infections and by L. Pasteur and others (1880) in patients with sepsis. A. Ogston described the organisms in studies of suppurative lesions (1881). A pure culture of the organism was isolated by F. Fehleisen (1883) from a patient with erysipelas and by F. Rosenbach (1884) from pus. Streptococci belong to the family Streptococcaceae.

Morphology. Streptococci are spherical in shape, 0.6 to 1 mem in diameter, and form chains. They are non-motile (although motile forms are encountered), do not form spores and are Gram-positive. Some strains are capsulated. In smears from cultures grown on solid media the streptococci are usually present in pairs or in short chains, while in smears from broth cultures they form long chains or clusters.

Figure. Streptococci

The capsule is clearly demonstrated at the end of the phase of logarithmic growth. The microcapsule is seen on ultrathin sections, which forms in the phases of logarithmic and stationary growth. Under the microcapsule there is a three-layer cell wall 100-300 nm thick and then a three-layer cytoplasmic membrane which forms invaginations directed into the cytoplasm.

The cytoplasm is microgranular and contains ribosomes, some inclusions, and vacuoles with an electron-dense material resembling volutin granules. The nucleoid occupies most of the cytoplasm. Division of the cell begins with protrusion of the cytoplasmic membrane after which a septum forms in the middle. New division of the cell begins before the previous one is completed, as the result of which dumbbell-like cells form. The G+C content in DNA ranges from 34.5 to 38.5 per cent.

Cultivation. Streptococci are facultatively aerobic, and there are also anaerobic species. The optimal temperature for growth is 37° C, and no growth occurs beyond the limits of 20-40° C for enterococci the limits are 10-45 °C).

The organisms show poor growth on ordinary meat-peptone agar, and grow well on sugar, blood, serum and ascitic agar and broth, when the pH of the media is 7.2-7.6. On solid media they produce small (0.5-1.0 mm in diameter), translucent, grey or greyish-white, and granular colonies with poorly defined margins. Some streptococcal strains cause haemolysis on blood agar (Fig, 2, 2), others produce a green coloration surrounding the colony 1-2 mm in diameter as result a conversion of haemoglobin into methaemoglobin, while others do not cause any change in the erythrocytes. On sugar broth medium growth is in the form of fine-granular precipitates on the walls and at the bottom of the tube and only rarely does the broth become turbid.

Fermentative properties. Streptococci are non-proteolytic, do not liquefy gelatin, and do not reduce nitrates to nitrites. They coagulate milk, dissolve fibrin, ferment glucose, maltose, lactose, saccharose, mannitol (not always constantly), and break down salicin and trehalose, with acid formation.

Toxin production. Streptococci produce exotoxins with various activities:

(1) haemolysin (haemotoxin, 0- and S-streptolysm) which loses its activity after 30 minutes at a temperature of 55 °C; disintegrates erythrocytes; produces haemoglobinaemia and haematuria in rabbits following intravenous injection;

(2) leucocidin which is destructive to leucocytes; occurs in highly virulent strains and is rendered harmless by a temperature of 70 °C

(3) lethal (dialysable) toxin which produces necrosis in rabbits when injected intracutaneously; it also causes necrosis in other tissues, particularly in the hepatic cells;

(4) erythrogenic toxin produces inflammation in humans who have no antitoxins in their blood;

(5) Streptococcus pneumoniae produces alpha-hae molysin secretedinto the culture fluid and beta-haemolysin which is released after lysis of the streptococci.

Other substances produced by streptococci are harmful enzymes. They include hyaluronidase (which facilitates the spread of the organisms throughout the tissues and organs of the affected animal), fibrinolysin, desoxyribonuclease, ribqnuclease, proteinase, amylase, lipase, and diphosphopyridine nucleotidase. Streptococcal phages possess transduction properties and may be responsible for increased toxigenicity of C. diphtheriae and increased virulence of other bacteria occurring in association with streptococci.

Endotoxins, characterized by their thermoresistance and specific activity, are responsible for the pathogenic properties of streptococci together with the exotoxins and aggressive enzymes.

Antigenic structure. The study of the antigenic structure of streptococci is based on serologic examinations. F. Griffith used th e agglutination test, while R. Lancefield employed the precipitin reaction with an extract of a broth culture precipitate.

Four antigenic fractions were recovered from streptococci: the type-specific protein (M- and T-substances); group-specific polysaccharide (C-substance), and nucleoprotein (P-substance). The M-substance is a protein which confers type specificity, virulence, and immunogenicity. The T-substance contains 0-, K-, and L-antigens. The C-substance is a polysaccharide common to the whole group of haemolytic streptococci. The P-substance belongs to the nucleoprotein fraction, being non-specific for haemolytic streptococci; it contains nucleoproteins common to other groups of streptococci, as well as staphylococci.

Group A and, partly, group C and G streptococci possess extracellular antigens: streptolysin 0, a protein which causes erythrocyte haemolysis, and streptolysin S, a lipoprotein complex possessing erythrocytolytic activity.

Classification. By means of the precipitation reaction founded on the detection of group specific carbohydrates, streptococci are subdivided into groups which are designated by capital letters from A to H and from K to T.

Five out of the known Streptococcal species cannot be related to any antigenic group. The haemolytic streptococci, rec overed from sick human beings, weresubdivided by F. Griffith into 51 serovars. He attributed 47 serovars to group A, serovars 7, 20, and 21 to group C, and serovar 16 to group G.

Streptococcus faecalis (enterococci) are pleomorphic oval cells which occur in pairs or in short chains. Some are oval or spear-shaped in form. The organisms are from 0.5 to 1 mem in diameter. Haemolytic types (Streptococcus faecalis) and organisms liquefying gelatin (S. faecalis, var. liquefaciens) are found. According to their antigenic structure, enterococci are divided into six 0-groups among which there are strains with K-antigens (capsular antigens). Some enterococcal and lactic streptococci possess identical antigens. On solid media enterococcal growths form a thin pellicle with smooth edges. On sugar broth they produce turbidity and precipitate. Certain enterococci are highly motile. Some of the strains produce a yellow pigment, and the pathogenic enterococci produce fibrinolysin. The organisms grow at tempera hires ranging from 10 to 45 °C. They are resistant to high temperature (e. g. withstand exposure to 60 °C for half an hour). Enterococci can be grown in broth containing 6.5 per cent common salt at pH 9.6 and on blood agar containing 40 per cent bile or an equivalent amount of bile salts. They ferment glucose, maltose, lactose, mannitol, trehalose, salicin, and inulin, with acid formation. They reduce and coagulate litmus milk in the presence of 0.1 per cent methylene blue. Enterococci differ from other streptococci in their ability to grow over a wide range of temperatures (10-45 °C) and in a medium of pH 9.6, in their resistance to high concentrations of salt and to penicillin (a number of strainsshow growth in media containing 0.5-1 U of antibiotic per 1 ml of media). All enterococci decarboxylate tyrosine.

Enterococci inhabit the small and large intestine of man and warm-blooded animals. The organisms possess properties antagonistic to dysentery, enteric fever, and paratyphoid bacteria, and to the coli bacillus. In the child's intestine the enterococci are more numerous than the E. coli. In lesions of the duodenum, gall bladder, and urinary tract enterococci are found as a result of dysbacteriosis. Isolation of enterococci serves as a criterion of contamination of water, sewage, and foodstuffs with faeces.

Streptococcus pneumonias (Diplococcus pneumoniae) belongs to the family Streptomycetaceae. For many years it was called pneumococcus. These are lanceolate or slightly elongated cocci measuring up to 0.5-1.25 mem in diameter and occurring in pairs, sometimes as single organisms or arranged in chains. In the body of humans and animals they have a capsule: they are Gram-positive, but young and old cultures are Gram-negative. The organisms are non-motile and do not form spores. S. pneumoniae is a facultative anaerobe, the optimum temperature for growth is 37° C and the organism grows at 28-42 °C. The organisms are poorly cultivated on ordinary media but develop readily on serum or^ascitic agar at pH 7.2-7.6 as small colonies 1.0 mm in diameter. On blood agar they form small, rounded succulent colonies on a green medium. On sugar broth they produce turbidity and a precipitate. The organisms grow readily on broth to which 0.2 per cent of glucose is added. They usually do not form capsules on artificial media, but the addition of animal protein to fluid medium promotes the formation of a capsule. There are 80 variants which are agglutinated ony by the corresponding type sera.

Among farm animals the young ones (calves, piglets, and lambs) and in vivariums guinea pigs may contract pneumonia. Among experimental animals albino mice and rabbits are most susceptible to the disease. Variants I, II, and III S. pneumoniae cause lobar pneumonia in man, which is characterized by an acute course occurring in cycles.

The organisms may also cause septicaemia, meningitis, affection of the joints, ulcerative endocarditis, otitis, peritonitis, rhinitis, highmoritis, creeping comeal ulcer, tonsillitis, and acute catarrhs of the upper respiratory passages.

Of special interest are the alpha-streptococci (the viridans group). They are responsible for haemometamorphosis on blood agar (greenish discoloration of the media) and produce no soluble haemolysin. The organisms of this group usually ferment raffinose and do not ferment mannitol. They are always isolated from the mouth and throat of healthy people and have low virulence for humans and animals. The viridans streptococci are found in pyogenic and inflammatory lesions of the teeth and gums and are responsible for subacute endocarditis.

Anaerobic streptococci (Peptostreptococcus putridus, Peptostreptococcus anaerobius, and others) are the cause of severe puerperal septic infections (puerperal sepsis). They are isolated from pyogenic and gangrenous lesions which have a putrefactive odour.

Resistance. Streptococci live for a long time at low temperatures, are resistant to desiccation, and survive for many months in pus and sputum. When exposed to a temperature of 70 °C, they are destroyed within one hour. A 3-5 per cent phenol solution kills the organisms within 15 minutes.

Pathogenicity for animals. Cattle, horses, and, among laboratory animals, rabbits and white mice are susceptible to the pathogenic streptococci.

The virulence of streptococci is tested on rabbits. The animals are infected by rubbing a culture suspension into a scratch made on the skin of the ear or on the back. This results in a local inflammation with the appearance of hyperaemia and swelling. An intravenous injection of pathogenic streptococci causes septicaemia or selectively affects the lungs, liver, kidneys, or joints.

Pathogenesis and diseases in man. The pathogenesis of streptococcal infections is brought about by the effect of the exotoxin and the-bacterial cells.The reactivity of the infected body and its previous resistance play an important part in the origin and development of streptococcal diseases. Such diseases as endocarditis, polyarthritis, highmoritis, chronic tonsillitis, and erysipelas are associated with abnormal body reactivity, hyperergia. This condition may persist for a long period of time and serve as the main factor for the development of chronic streptococcal diseases.

With an exogenous mode of infection streptococci invade the human body from without (from sick people, and animals, various contaminated objects and foodstuffs). They gain access through injured skin and mucous membranes or enter the intestine with the food. Streptococci are mainly spread by the air droplet route. When the natural body resistance is weakened, conditionally pathogenic streptococci normally present in the human body become pathogenic. Penetrating deep into the tissues they produce local pyogenic inflammations, such as streptoderma, abscesses, phlegmons, lymphadenitis, lymphangitis, cystitis, pyelitis, cholecystitis, and peritonitis. Erysipelas (inflammation of the superficial lymphatic vessels) and tonsillitis (inflammation of the pharyngeal and tonsillar mucosa) are among the diseases caused by streptococci. Invading the blood, streptococci produce a serious septic condition. They are more commonly the cause of puerperal sepsis than other bacteria.

Streptococci may cause secondary infections in patients with diphtheria, smallpox, whooping cough, measles, and other diseases. Chronic tonsillitis is attributed to the viridans streptococci and adenoviruses. Contamination of wounds with streptococci during war results in wound suppurations, abscess formation, phlegmons, and traumatic sepsis.

Immunity. Immunity acquired after streptococcal infections is ofa low grade and short duration. Relapses of erysipelas, fre quent tonsilitis, dermatitis, periostitis, and osteomyelitis occur as a result of sensitization of the body. This is attributed to low immunogenic activity and high allergen content of the streptococci, as well as to the presence of numerous types of the organisms against which no cross immunity is produced.

Immunity following streptococcal infections is of an anti-infectious nature. It is associated with antitoxic and antibacterial factors. The antitoxins neutralize the streptococcal toxin and together with the opsonins facilitate phagocytosis.

Laboratory diagnosis. Test material is obtained from the pus of wounds, inflammatory exudate. tonsillar swabs, blood, urine, and foodstuffs. Procedures are the same as for staphylococcal infections. Tests include microscopy of pus smears, inoculation of test material onto blood agar plates, isolation of the pure culture and its identification. Blood is sown on sugar broth if sepsis is suspected. Virulence is tested on rabbits by an intracutaneous injection of 200-400 million microbial cells. Toxicity is determined by injecting them intracutaneously with broth culture filtrate.

The group and type of the isolated streptococcus and its resistance to the medicaments used are also determined. In endocarditis there are very few organisms present in the blood in which they appear periodically. For this reason blood in large volumes (20-50 ml) is inoculated into vials containing sugar broth. If possible, the blood should be collected while the patient has a high temperature. In patients with chronic sepsis an examination of the centrifuged urine precipitate and isolation of the organism in pure culture are recommended.

Besides, the group and type of the isolated streptococcus are identified by means of fluorescent antibodies. Serological methods are also applied to determine the increase in the titre of antibodies, namely streptolysins O and antihyaluronidase.

Treatment. Usually penicillin is used. For penicillin-resistant strains,and when penicillin is contraindicated, streptomycin, and erythromycin are required. Vaccine therapy (autovaccines and polyvalent vaccines) and phage therapy are recommended in chronic conditions.

In some countries diseases caused by beta-haemolytic streptococci of groups A, C, G, and H and by alpha-streptococci (endocarditis) are treated with anti-infectious (antitoxic and antibacterial) streptococcal sera together with antibiotics and sulphonamides.

Prophylaxis. Streptococcal infections are prevented by the practice of general hygienic measures at factories, children's institutions, maternity hospitals, and surgical departments, in food production, agricultural work, and everyday life.

Maintaining appropriate sanitary levels of living and working condi- tions, raising the cultural level of the population, and checking personal hygiene are of great importance.

Since streptococci and the macro-organism share antigenic structures in common and because streptococci are marked by weak immunogenic ability and there are a great number of types among them which do not possess the property of producing cross immunity, specific prophylaxis of streptococcal diseases has not been elaborated. Vaccines prepared from M-protein fractions of streptococci are being studied.

Role of Streptococcus in the Aetiology of Scarlet Fever

Scarlet fever has long been known as a widespread disease but at the present time its aetiology has not yet been ascertained. Four different theories were proposed: streptococcal, allergic, viral, and combined (viral-streptococcal). Most scientists and medical practitioners favoured the streptococcal theory. G. Gabrichevsky in 1902 was the first to point out the aetiological role of the haemolytic streptococcus in scarlet fever. Usually he recovered the organisms from the pharynx of scarlet fever patients and from blood contained in the heart of those that had died of the disease. In 1907 he prepared vaccine from killed scarlet fever haemolytic streptococcal cultures. This vaccine was widely used for human vaccination.

In 1905 I. Savchenko, cultivating scarlet fever streptoco cci, obtained the toxin and used it for hyperimmunization of horses. The antitoxic antiscarlatinal serum was effectively used for treating people suffering from scarlet fever.

Data presented by Gabrichevsky and Savchenko concerning the streptococcal theory were confirmed by studies carried out in 1923-24 by G. Dick and G. Dick and by many other scientists.

The streptococcal aetiology o f scarlet fever is supported by the following arguments: (1) all people suffering from scarlet fever are found to harbour in their throats haemolytic streptococci which are agglutinated by the sera of convalescents; (2) a subcutaneous injection of the scarlet fever toxin into susceptible people (volunteers) in some cases is followed by the appearance of a characteristic skin rash, vomiting, fever, tonsillitis, and other scarlatinal symptoms; (3) an intracutaneous injection of the toxin into susceptible children produces a local erythematous and oedematous reaction; the toxin produces no reaction in children who had previously suffered from scarlet fever and were im-mune to the disease; (4) if 0.1 ml of antitoxic antistreptococcal serum or convalescent serum is introduced into the skin of a scarlet fever patient in the area of the rash, the latter turns pale (is 'extinguished'); (5) hyper-immunization of animals with the scarlet fever toxin leads to the production of antitoxins, and a neutralization reaction takes place between the toxin and antitoxins; (6) therapy with antitoxic sera and prophylaxis with combined vaccines consisting of the toxin and haemolytic streptococcal cells result in the appearance of less severe cases and decrease in morbidity and mortality.

At present many investigators accept the streptococcal theory in scarlet fever aetiology. In postwar years this theory has been confirmed by a number of investigations. Arguments against the streptococcal theory are as follows: (1) people inoculated with scarlet fever streptococci or their toxins do not always display the characteristic symptoms of the disease, e. g. there is no peeling, only rarely are there instances of tonsillitis, and phlegmon, sepsis, and erysipelas occasionally develop; (2) in severe hypertoxic forms the antitoxic ssswm has little effect, while the serum of convalescents gives better results; (3) the skin toxin test (Dick test) sometimes gives a negative reaction with susceptible children and produces a positive reaction with those who are immune; (4) immunity acquired after scarlet fever is very stable and of long duration, while that acquired after other streptococcal diseases is unstable, of short duration, and is frequently accompanied by an increased susceptibility to streptococci.

It is assumed that scarlet fever is caused by group A beta-haemolytic streptococci which possess M-antigen and produce erythrogenic exotoxin. People become infected by the air droplet route. Sic k people, convalescents, and carriers of the causative agent of scarlet fever are all sources of infection. The disease is most commonly encountered in children from 1 to 8 years of age.

The causative agent sometimes enters the body through wounds on the skin and mucous membranes of the genitalia. This form of scarlet fever is known as extrabuccal or extrapharyngeal (traumatic, combustion, surgical, and puerperal). Certain objects (e. g. utensils, toys, books, etc.) as well as foodstuffs (e. g. milk), contaminated by adult carriers, may also be sources of infection. Of great importance in the epidemiology of scarlet fever are the patients with atypical, unrecognizable forms of the disease. In its initial stage scarlet fever is chiefly characterized by intoxication, while in the second stage it is accompan ied by septic and allergic conditions.

Scarlet fever produces a relatively stable immunity. Reinfections are very rare. They have increased in number in the last years as a result of wide use of antibiotics which reduce the immunogenic activity of the pathogen and its toxin.

Data concerning the correlation between a positive Dick test and susceptibility to scarlet fever provide evidence of the antitoxic nature of immunity acquired after scarlet fever. Children from 1 to 5 years are most susceptible.

Scarlet fever is recognized mainly by its clinical course and on epidemiological grounds. Laboratory diagnosis for the detection of haemolytic streptococci and their typing is employed only in certain cases. This method is of no practical value since haemolytic streptococci are often isolated from people with various diseases and frequently from healthy individuals.

The phenomenon of rash 'extinguishmenf is employed as an auxiliary diagnostic method. In the case of scarlet fever, the rash at the site of injection will disappear within 12-20 hours and the skin will turn pale.

Certain physicians apply the Dick test with the thermolabile fraction of the toxin. The diagnosis of scarlet fever is verified to a certain extent if on a second injection of the toxin a positive Dick test reverts to a negative reaction.

Scarlet fever may also be diagnosed by detecting precipitins in the urine (urine precipitation test). A layer of type-specific streptococcal sera or convalescent serum is transferred onto freshly filtered urine of patients in the first days of the disease. The appearance of a greyish-white ring at the interface of the two fluids designates a positivereaction.

Scarlet fever patients are treated with penicillin, tetracycline, sulphonamides (norsulphazol, etc.), and gamma-globulin from human blood. The wide use of antibiotics has led to a significant decrease in the morbidity and mortality rate of scarlet fever and to a milder course of the disease. This fact also confirms the definite role played by haemolytic streptococci in the aetiology and pathogenesis of scarlet fever, since it is known that these organisms are extremely sensitive to penicillin and other antibiotics. In the recent years, however, an increase in the incidence of scarlet fever and a more severe course of the disease are noted.

Prophylaxis consists of early diagnosis, isolation of patient s and hospitalization in the presence of epidemiological and clinical indications. Extremely hygienic cleaning and ventilation and observance of correct hospital regime are also necessary. If cases of scarlet fever occur in children's institutions, the children concerned must be isolated. Debilitated children who have been in contact with scarlet fever patients must be injected with 1.5-3.0 ml of human serum gammaglobulin.

Role of Streptococcus in the Aetiology of Rheumatic Fever

The majority of authors maintain that rheumatic fever develops as a result of the body becoming infected by group A beta-haemolytic streptococci. Acute or chronic tonsillitis and pharyngitis produce a change in the immunological reactivity of the body and this gives rise to characteristic clinical symptoms and a pathological reaction. It should be noted, however, that recent research shows the leading role in rheumatism of virus agents with persisting properties. The active phase with an acute and subacute course is attended with virusaemia. Rheumatism is characterized by the virus remaining in the body for a long period of time: the viral antigen penetrates the leucocytes and sensitizes them. Diminution of the specific and non-specific reactions to the virus leads to tolerance of the body. Autoimmune reactions are encountered in rheumatism. Streptococci and other exogenous and endogenous factors contribute to exacerbation of rheumatism. Virusaemia is almost always revealed in the patients during exacerbation. Identical viruses are detected in the mother's blood and in the blood of premature infants, stillborns, and in infants who die soon after birth.

No streptococci, streptococcal antigen or antibodies to them are found in some patients and penicillin therapy proves ineffective. On the grounds of this, it is considered that the diagnosis of rheumatism should be made and penicillin prescribed only if the presence of streptococcal infection is sufficiently verified.

The prevalence of rheumatic fever depends on the time of the year. The highest number of cases occur in October-November and March- April. Acute and chronic tonsillitis, pharyngitis, and catarrh are also most prevalent in these months.

The allergic reaction produced in the body as a result of re-invasion by antigens (streptococcal exo- and endotoxms, autoantigens, and complexes consisting of streptococcal toxins and components of tissue and blood proteins of sick people) is an important factor in the pathogenesis of the disease. It is known that blood of individuals who have suffered from a streptococcal infection contains antibodies against beta-haemolytic streptococci. In 1 -3 per cent of these cases the formation of antibodies does not produce immunity, and a secondary invasion of the body by specific and non-specific antigens leads to the development of hyperergia. Experiments have shown that streptococci bring about the formation of autoantigens which cause the production of autoantibodies in the body. These autoantibodies are responsible for lesions in certain tissues and organs.

Studies of high-molecular gamma-globulins and their complexes in rheumatic fever have shown that the normal human gamma-globulin contains two fractions (7S and 19S) which differ in their pre cipitate constant. The majority of the common antibodies are associated with the 7S gamma-globulin fraction, while isoagglutinins, Rh-agglutinins, and complement-fixing antibodies are contained in the 19S fraction. The gamma-globulin fraction, rich in 19S, is found to contain the rheumatoid factor. An interaction has been demonstrated between the rheumatoid factor and the antigen-antibody precipitate, the latter possessing antigenic properties. Thus, in its reaction with the antigen-antibody complex the rheumatoid factor behaves as the complement. Alternatively, the rheumatoid factor may act as an antibody to gamma-globulin or to the antigen-antibody complex, the latter acting as an antigen.

Antigen-antibody reactions result in the injury of the interstitial connective tissue, release of histamine, and inflammation. Disturbances of coordination in the hypophysis-adrenal system are encountered in rheumatic fever. For this reason supporters of the Selye theory consider rheumatic fever to be an adaptational disease. However, the above mentioned aspects on the mechanism of rheumatic fever can by no means cover all the complex processes involved in the pathogenesis of this disease.

According to its clinical course, rheumatic fever is differentiated into active and inactive phases. The active phase is characterized by acute rheumocarditis without valvular defects and relapsing rheumocarditis accompanied by valvular defects, polyarthritis, chorea, pleuritis, peritonitis, nephritis, he patitis, pneumonia, lesions in the skin and subcutaneous tissue, eyes, and other systems. The inactive phase develops in the form of rheumatic myocardiosclerosis, heart defects, and conditions following extracardial affections.

Three periods can be distinguished during the development of rheumatic fever: (1) period of acute streptococcal infection and initial sensitization; (2) penod ofhyperergic reactions, resulting frominteraction between antigens and antibodies, which are accompanied by pnmary rheumatic polyarthritis or carditis; (3) period of stable allergic reacts ity accompanied by pronounced manifestations of parallergy and autosensitization, profound and stable immunogenic disturbances, and relapses.

Laboratory diagnosis is made on the basis of determination of an increase in antistreptolysin, antifibrinolysin, and antihyaluronidase titres and detection of C-reactive protein.

Treatment of rheumatic patients is accomplished by several measures aimed at desensitization of the body, abatement of inflammatory conditions, recovery of normal body reactivity, condition of the nervous system, and disturbed processes, and control of local infections.

Prophylaxis includes prevention of streptococcal infections, strengthening of general resistance, and creation of favourable conditions for everyday life and work. In addition, all people suffering from rheumatic fever and those susceptible to the disease should be given prophylactic treatment with penicillin and tetracycline preparations in spring and autumn.

Additional materials about laboratory diagnosis

STAPHYLOCOCCAL INFECTION. In diseases caused by pathogenic staphylococci (Staphylococcus aureus) the materials to be examined include pus, mucosal secretions, blood, sputum, urine, and cerebrospinal fluid. In cases of food intoxications, vomit, faeces and food remnants are also. studied.

In open suppurative lesions the material is taken with a cotton wool swab after removing the superficial layer of the pus, which may contain non-pathogenic staphylococci and other microorganisms usually present on the skin and in the air. When purulent foci are unruptured, they are punctured and the pus from the syringe is poured out into a sterile test tube. Mucosal secretion is obtained with a tampon. Urine and sputum are collected into sterile test tubes and jars. Blood withdrawn from a patient's ulnar vein with a syringe as well as aseptically obtained cerebrospinal fluid are inoculated at the patient's bedside into a vessel containing 100-200 ml of sugar broth (pH 7,2–7,4). Staphylococci propagate quite readily in simple media too but the use of sugar broth is preferable since septicaemia may be secondary not only to staphylococci but also to other microorganisms which are more demanding with regard to nutrient media.

Laboratory diagnosis of staphylococcal infection.

Gram-positive cocci

There are several types of symbiosis between cocci and the human body. Saprophytic and conditionally pathogenic types of staphylococci and streptococci live on the skin, mucous membranes, and in the respiratory tract. Meningococci may be harboured for long periods in the nasopharynx, and faecal streptococci (enterococci) in the intestine.

When body resistance is lowered or the skin and mucous membranes are injured, these bacteria penetrate the body tissues and cause infection. The various cocci possess different organotropic ability. This is distinctly manifest in meningococci, and gonococci but less so in staphylococci and streptococci. Cocci belong to the families Micrococcaceae, Streptococcaceae Peptococcaceae, Neisseriaceae.

Staphylococci. The staphylococcus, Staphylococcus aureus, was discovered by R. Koch (18,78), and later isolated from furuncle pus by L. Pasteur (1880). It has been described as the causative agent of numerous suppurative processes by A. Ogston (1881), and has been studied in detail by F. Rosenbach (1884).

Morphology. Staphylococci are spherical in shape, 0.8-1 mem in diameter, and form irregular clusters resembling bunches of grapes (Fig. 1). In smears from cultures and pus the organisms occur in short chains, in pairs, or as single cocci. Large spherical (L-forms) or very small (G-forms) and even filterable forms may be seen in cultures which have been subjected to various physical, chemical, and biological (antibiotics) factors.

Figure 1. Staphylococci.

Staphylococci are Gram-positive organisms which possess no flagella and do not form spores. A macrocapsule can be seen on ultrathin sections of Staphylococci isolated from infected mice. The nucleoid occupies most of the cytoplasm and is filled with DNA fibrils. The G+C content in DNA ranges between 30.7 and 39.0 per cent.

Cultivation. Staphylococci are facultative-anaerobes. They grow well on ordinary nutrient media with a pH of 7.2-7.4 at a temperature of 37 °C but do not grow at temperatures below 10 °C and above 45 °C. At room temperature with adequate aeration and subdued light – the organisms produce golden, white, lemon-yellow, and other pigments known as lipochromes. These pigments do not dissolve in water but are soluble in ether, benzene, acetone, chloroform, and alcohol. They are most readily formed on milk agar and potatoes at a temperature of 20-25° C.

On meat peptone agar Staphylococci produce well defined colonies with smooth edges, measuring from 1-2 to 2.5 mm in diameter. Under the microscope the course granular nature of the colonies can be seen, the latter are opaque and have a dense centre. Their colour epends on the pigment produced by the organisms. Besides the typical colony types, Staphylococci produce R-, G-, and L-forms. Growth of Staphylococci in meat-peptone broth produces diffuse opacity throughout the medium and, subsequently, a precipitate. In some cases when there is sufficient aeration, the organisms form a pellicle on the surface of the broth. Staphylococci grow well on potatoes and coagulated serum. After 24-48 hours of incubation there is usually abundant growth along the inoculation stab and liquefaction of gelatin media. On the fourth or fifth day the gelatin medium resembles a funnel filled with fluid.

On blood agar pathogenic Staphylococci cause haemolysis of the erythrocytes. Rabbit and sheep erythrocytes are the most sensitive to the staphylococcal haemotoxin.

Fennentative properties. Staphylococci produce enzymes which cause the lysis of proteins and sugars (see Table 1). There is no indole production in young cultures. The organisms liquefy gelatin, coagulate milk and occasionally serum, reduce nitrates to nitrites, produce urease, catalase, phosphatase, ammonia, and hydrogen sulphide. They ferment glucose, levulose, maltose, lactose, saccharose, mannitol, and glycerin, with acid formation. A connection has been revealed between arginase activity and the level of g-toxin formation.

Toxin production. Staphylococci produce a-, b-, d- and g-haemolysins which are characterized by lethal, haemolytic, and necrotic activity. Filtrates of staphylococcal broth cultures contain an enterotoxin which causes food poisoning on entry into the gastro-intestinal tract. Staphylococci excrete exofoliatines which cause staphylococcal impetigo and pemphigus neonatomm in children.

Leucocidin, a substance which destroys leucocytes, haematoblasts of the bone marrow and nerve cells, is also produced by the Staphylococci. The organisms also coagulate blood plasma. Their ability to coagulate, plasma is a stable property and is used for differentiating various strains. Coagulase is thermoresistant. It can be isolated from staphylococcal broth cultures.

Staphylococci produce fibrinolysin which when added to a blood clot dissolves the latter within 24-48 hours.

The Staphylococci produce hyaluronidase which breaks down hyaluronic acid, a component of connective tissue.

Coagulase, fibrinolysin, lecithinase, hyaluronidasa and phosphatasa all belong to the group of enzymes possessing destructive properties. Lecithinase destroys the lecithin protective membranes of the colloidal particles of a substance found in human, sheep, and rabbit erythrocytes. An anticoagulant which inhibits blood coagulation has also been derived from the staphylococcal cultures. This staphylococcal anticoagulant is produced in exudates of inflamed tissues, occurring during staphylococcal infections. Haemagglutmins which cause the agglutination of rabbit erythrocytes have also been found in staphylococcal culture filtrates. Virulent Staphylococci inhibit the phagocytic activity of leucocytes.

Many microbiologists believe that the Staphylococci isolated from patients produce alpha-haemolysins, while the organisms pathogenic for animals (e. g. itesponsible for mastitis in cows) more often produce beta-haemolysin.

Staphylococcal exotoxin, inactivated by treatment with 0.3-0.5 per cent formalin at 37 °C for 7-28 days, and injected parenterally to humans and animals, stimulates the production of a specific antitoxin capable of reacting with the toxin.

Antigenic structure. Polysaccharides A and B have been obtained from a staphylococcal suspension by treating the latter alternately with acid and alkali and removing the proteins with trichloracetic acid.

Polysaccharide A was extracted from pathogenic strains isolated from patients with septicaemia, furunculosis, osteomyelitis, and acute conjunctivitis, etc. Polysaccharide B is found in avirulent, non-pathogenic strains. Polysaccharides A and B differ not only in their serological reactions but also in their chemical structures.

Antigen C, containing a specific polysaccharide, has been recently isolated. Staphylococcal polysaccharides demonstrate a marked type specificity. Even in a 1 :1000 000 dilution they give a distinct precipitin reaction. The protein antigen is common to all species and types of staphylococci.

Three types (I, II, III) of staphylococci have been revealed by the agglutination test and precipitin reaction. However, quite a number of cultures are unsuitable for serological typing. Recent studies have revealed fifteen type-specific staphylococcal antigens.

Classification. Staphylococci are included in the class Bacteria, family Micrococcaceae, genus Staphylococcus.

According to the contemporary classification, staphylococci are subdivided into three species: S. aureus, S. epidermidis, and S. saprophyticus.

Differentiation of Staphylococci

Main characteristics	Species
	S. aureus	S. epidermidis;	S. saprophyticus
Plasmacoagulase	+	—	—
Phosphatase	+	+	—
Arginine dihydrolase nitrate	+	+	—
Reductase	+	+	—
Protein A in superficial antigen	+	—	—
Oxidation
Mannitol	+	—	+
Trehalose	+	—	+
Galactose	+	+	—
Ribose	+	—	—
Production of alpha-toxin	+	–	–
Resistance to novobiocin	S	S	R
Growth in the presence of biotin	–	+	NT

Note: S, sensitive; R, resistant, NT, not tested.

Certain strains of the family Micrococcaceae are strict anaerobes. Peptococcus niger, Peptococcus anaerobius, Peptococcus asaccharolyticus and others are usually conditionally pathogenic for human beings. They live in the mouth mucosa, in the intestine, urogenital tract, and in other parts of the human body. In weakened individuals and in people suffering from chronic diseases the anaerobic micrococci may give rise to various diseases and complications.

Resistance. Staphylococci are characterized by a relatively strong resistance to desiccation, freezing, sunlight, and chemical substances. After desiccation they can survive for more than 6 months. Repeated freezing .and thawing do not kill the organisms. They survive for many hours under direct sunlight. Staphylococci maintain their viability for more than 1 hour at 70 °C. At a temperature of 80 °C they are destroyed within 10-60 minutes and at boiling point, they instantly perish. A 5 per cent phenol solution kills the organisms in 15-30 minutes. Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant green which is used for treating pyogenic skin diseases caused by these organisms. Staphylococci possess high resistance to antibacterial agents, in 70-80 per cent of cases they are resistant simultaneously to 4-5 agents. Cross resistance to antibiotics of the macrolide group (erythromycin, oleandomycin, etc.) is encountered.

Pathogenicity for animals. Horses, cattle, sheep, goats, pigs, and, among laboratory animals, rabbits, white mice, and kittens are susceptible to pathogenic staphylococci.

An intracutaneous injection of a culture of pathogenic staphylococci produces inflammation and subsequent necrosis in the skin of the rabbit. An intravenous injection of a staphylococcal culture filtrate causes a condition similar to acute poisoning in rabbits, which is characterized by motonc excitation, respiratory disorders, convulsions, paralysis of the hind extremities, and sometimes, by diarrhoea and urine discharge. After complete fatigue the animal shortly dies.

Staphylococci or their toxin will cause vomiting, diarrhoea, and weakness in kittens if introduced per os or intraperitoneally. Functional disorders of the digestive tract arise owing to the effect of tne enterotoxin which is distinguished from the other fractions of the staphylococcal toxin by its thermoresistance. It withstands a temperature of 100 °C for 30 minutes. The most reliable test for the presence of enterotoxin is an intravenous injection to adult cats.

Pathogenesis and diseases in man. Staphylococci enter the body through the skin and mucous membranes. When they overcome the lymphatic barrier and penetrate the blood, staphylococcal septicaemia sets in. Both the exotoxins and the bacterial cells play an important role in pathogenesis of diseases caused by these organisms. Consequently, staphylococcal diseases should be regarded as toxinfections.

The development of staphylococcal diseases is also influenced by the resulting allergy which in many cases is the cause of severe clinical forms of staphylococcal infections which do not succumb to treatment.

Staphylococci are responsible for a number of local lesions in humans: hidradenitis, abscess, paronychia, blepharitis, furuncle, carbuncle, periostitis, osteomyelitis, folliculitis, sycosis, dermatitis, eczema, chronic pyodermia, peritonitis, meningitis, appendicitis, and cholecystitis.

Diabetes mellitus, avitaminosis, alimentary dystrophy, excess perspiration, minor occupational skin abrasions, as well as skin irritation caused by chemical substances, are some examples of the conditions conducive to the formation of pyogenic lesions of the skin and furunculosis.

In some cases staphylococci may give rise to secondary infection in individuals suffering from smallpox, influenza, and wounds, as well as postoperative suppurations. Staphylococcal sepsis and staphylococcal pneumonia in children are particularly severe diseases. Ingestion of foodstuffs (cheese, curds, milk, rich cakes and pastry, ice cream, etc.) contaminated with pathogenic staphylococci may result in food poisoning.

Staphylococci play an essential part in mixed infections, and are found together with streptococci in cases of wound infections, diphtheria, tuberculosis, actinomycosis, and angina.

The wide use of antibacterial agents, antibiotics in particular, led to considerable changes in the severity and degree of the spread of staphylococcal lesions. Growth in the incidence of diseases and intrahospital infections m obstetrical, surgical and children's in-patient institutions, intensive spread of the causative agent, and increase in the number of carriers among the medical staff and population have been noted in all countries of the world. Intrauterme and extrauterine contamination of children with staphylococci has been registered, with the development of vesiculopustular staphyloderma, pemphigus, infiltrates, abscesses, conjunctivitis, nasopharyngitis, otitis, pneumonia, and other diseases.

It has been established that staphylococci become adapted rapidly to chemical agents and antibiotics due to the spread of R-plasmids among these bacteria. The high concentration of drugs in the body of humans and in the biosphere has resulted in essential disturbance in the microflora and the extensive spread of resistant strains possessing more manifest virulence. The L-forms of staphylococci are especially marked by increased degree of resistance to antibiotics.

Immunity. The tendency to run a chronic flaccid course or relapse is regarded as a characteristic symptom of staphylococcal infections. This peculiarity gives a basis for concluding that postmfectional immunity following staphylococcal diseases is of low grade and short duration.

Immunity acquired after staphylococcal diseases is due to phagocytosis and the presence of antibodies (antitoxins, precipitins, opsonins, and agglutinins).

The inflammation restricts the staphylococci to the site of penetration and obstructs their spreading throughout the body. At the centre of inflammation the organisms undergo phagocytosis. Neutralization of the staphylococcal toxin by the antitoxin is an important stage of the immunity complex. As a result of capillary permeability, the antitoxin penetrates from the blood into the inflammation zone and renders harmless the toxin produced by staphylococci. Thus, the phagocytic and humoral factors act together and supplement each other.

The presence in the human organs and tissues of antigens which are also common in the staphylococci (mimicry antigens) is among the causes of low immunity. This causes a state of immunological tolerance to staphylococci and their toxins, which provides favourable conditions for uninhibited reproduction of the causative agent in the patient's body. The wide use of antibacterial agents promotes intensive selection of staphylococcal strains resistant to the natural inhibitors of the microorganism.

Laboratory diagnosis. Test material may be obtained from pus, mucous membrane discharge, sputum, urine, blood, foodstuffs (cheese, curds, milk, pastry, cakes, cream, etc.), vomit, lavage fluids, and faeces.

The material is examined for the presence of pathogenic staphylococci. Special rules are observed when collecting the material since non-pathogenic strains are widespread in nature.

Laboratory studies comprise the determination of the main properties of the isolated staphylococci (i. e their morphologic, cultural and biochemical characteristics), as well as their virulence. For this purpose the following procedures are carried out. Smears are made from pus and stained by the Gram method. Pus is inoculated onto blood agar and meat-peptone agar containing crystal violet. In cases of septicaemia blood is inoculated into glucose broth.The isolated pure culture is tested for its haemolytic (by inoculation onto blood agar plates), plasmacoagulative (by inoculation into citrated rabbit plasma), and hyaluronidase activities. Virulence is determined in rabbits by intracutaneous injection of 400 million microbial cells. Necrosis develops at the site of injection within 24-48 hours.

Pigment production of the isolated culture is also taken into account. For revealing sources of infection, particularly food poisoning and outbreaks of sepsis in maternity hospitals, serological typing and phage typing are carried out. To ensure effective therapy the isolated cultures are examined for sensitivity to antibiotics.

In cases of food poisoning presence of the enterotoxin in th isolated staphylococcal culture is tested for by intravenous injection of the culture filtrate to adult cats. In cases when intoxication is due to ingestion of the milk of a cow suffering from mastitis, the culture grown on starch medium is tested directly for toxin production as a means of detecting staphylococci of animal origin. When the causative agent cannot be detected (osteomyelitis and other diseases), the patients' serum is tested for agglutinins.

Treatment. Staphylococcal diseases are treated with antibiotics (penicillin, phenoxymethyl penicillin, tetracycline, gramicidin, etc.), sulphonamides (norsulphazol, sulphazol, etc.), and antistaphylococcal gamma-globulin.

When tr eating patients suffering from staphylococcal infections one should bear in mind that it is necessary to relieve intoxication and improve the immunological defence forces of the body (infusion of glucose, plasma, blood transfusion, injection of cardiac stimulants).

In cases of chronic staphylococcal lesions specific therapy is recommended: autovaccines, staphylococcal anatoxin, antitoxic serum, and diphage containing staphylococcal and streptococcal phages.

Staphylococd produce strains resistant to sulphonamides, antibiotics, and bacteriophage, which advances the wide distribution of staphylococcal infection. This variability is of particular importance in the therapy of staphylococcal pyogenic diseases. The medical services produce semisynthetic preparations of penicillin and tetracycline which are effectively used for the treatment of these diseases.

Prophylaxis. The general precautionary measures include: hygiene in working and everyday-life conditions, treatment of vitamin deficiency, prevention of traumatism and excess perspiration, observance of rules of hygiene in maternity hospitals, surgical departments, children's institutions, industrial plants and enterprises, particularly canneries, observance of personal hygiene and frequent washing of hands in warm water with soap.

Routine disinfection of hospital premises (surgical departments, maternity wards) and bacteriological examination of the personnel for carriers of pathogenic staphylococci resistant to antibiotics are also necessary.

To prevent pyoderma protective ointments and pastes are used at industrial enterprises. For the treatment of microtraumas, besides iodine tincture and alcohol solutions of aniline dyes, solutions are widely applied which dry in one or two minutes and form an elastic film protecting the wound surface from dirt and infection. Patients with bums are kept in soft (plastic) isolating compartments which protect them against the entry of microflora from the external environment. In some cases specific prophylaxis by means of immunization with the staphylococcal anatoxin may be recommended for individuals subject to injury or infection with antibiotic-resistant staphylococci.

Streptococci. The streptococcus {Streptococcus pyogenes) was discovered by T. Billroth (1874) in tissues of patients with erysipelas and wound infections and by L. Pasteur and others (1880) in patients with sepsis. A. Ogston described the organisms in studies of suppurative lesions (1881). A pure culture of the organism was isolated by F. Fehleisen (1883) from a patient with erysipelas and by F. Rosenbach (1884) from pus. Streptococci belong to the family Streptococcaceae.

Morphology. Streptococci are spherical in shape, 0.6 to 1 mem in diameter, and form chains. They are non-motile (although motile forms are encountered), do not form spores and are Gram-positive. Some strains are capsulated. In smears from cultures grown on solid media the streptococci are usually present in pairs or in short chains, while in smears from broth cultures they form long chains or clusters.

Figure. Streptococci

The capsule is clearly demonstrated at the end of the phase of logarithmic growth. The microcapsule is seen on ultrathin sections, which forms in the phases of logarithmic and stationary growth. Under the microcapsule there is a three-layer cell wall 100-300 nm thick and then a three-layer cytoplasmic membrane which forms invaginations directed into the cytoplasm.

The cytoplasm is microgranular and contains ribosomes, some inclusions, and vacuoles with an electron-dense material resembling volutin granules. The nucleoid occupies most of the cytoplasm. Division of the cell begins with protrusion of the cytoplasmic membrane after which a septum forms in the middle. New division of the cell begins before the previous one is completed, as the result of which dumbbell-like cells form. The G+C content in DNA ranges from 34.5 to 38.5 per cent.

Cultivation. Streptococci are facultatively aerobic, and there are also anaerobic species. The optimal temperature for growth is 37° C, and no growth occurs beyond the limits of 20-40° C for enterococci the limits are 10-45 °C).

The organisms show poor growth on ordinary meat-peptone agar, and grow well on sugar, blood, serum and ascitic agar and broth, when the pH of the media is 7.2-7.6. On solid media they produce small (0.5-1.0 mm in diameter), translucent, grey or greyish-white, and granular colonies with poorly defined margins. Some streptococcal strains cause haemolysis on blood agar (Fig, 2, 2), others produce a green coloration surrounding the colony 1-2 mm in diameter as result a conversion of haemoglobin into methaemoglobin, while others do not cause any change in the erythrocytes. On sugar broth medium growth is in the form of fine-granular precipitates on the walls and at the bottom of the tube and only rarely does the broth become turbid.

Fermentative properties. Streptococci are non-proteolytic, do not liquefy gelatin, and do not reduce nitrates to nitrites. They coagulate milk, dissolve fibrin, ferment glucose, maltose, lactose, saccharose, mannitol (not always constantly), and break down salicin and trehalose, with acid formation.

Toxin production. Streptococci produce exotoxins with various activities:

(1) haemolysin (haemotoxin, 0- and S-streptolysm) which loses its activity after 30 minutes at a temperature of 55 °C; disintegrates erythrocytes; produces haemoglobinaemia and haematuria in rabbits following intravenous injection;

(2) leucocidin which is destructive to leucocytes; occurs in highly virulent strains and is rendered harmless by a temperature of 70 °C

(3) lethal (dialysable) toxin which produces necrosis in rabbits when injected intracutaneously; it also causes necrosis in other tissues, particularly in the hepatic cells;

(4) erythrogenic toxin produces inflammation in humans who have no antitoxins in their blood;

(5) Streptococcus pneumoniae produces alpha-hae molysin secretedinto the culture fluid and beta-haemolysin which is released after lysis of the streptococci.

Other substances produced by streptococci are harmful enzymes. They include hyaluronidase (which facilitates the spread of the organisms throughout the tissues and organs of the affected animal), fibrinolysin, desoxyribonuclease, ribqnuclease, proteinase, amylase, lipase, and diphosphopyridine nucleotidase. Streptococcal phages possess transduction properties and may be responsible for increased toxigenicity of C. diphtheriae and increased virulence of other bacteria occurring in association with streptococci.

Endotoxins, characterized by their thermoresistance and specific activity, are responsible for the pathogenic properties of streptococci together with the exotoxins and aggressive enzymes.

Antigenic structure. The study of the antigenic structure of streptococci is based on serologic examinations. F. Griffith used th e agglutination test, while R. Lancefield employed the precipitin reaction with an extract of a broth culture precipitate.

Four antigenic fractions were recovered from streptococci: the type-specific protein (M- and T-substances); group-specific polysaccharide (C-substance), and nucleoprotein (P-substance). The M-substance is a protein which confers type specificity, virulence, and immunogenicity. The T-substance contains 0-, K-, and L-antigens. The C-substance is a polysaccharide common to the whole group of haemolytic streptococci. The P-substance belongs to the nucleoprotein fraction, being non-specific for haemolytic streptococci; it contains nucleoproteins common to other groups of streptococci, as well as staphylococci.

Group A and, partly, group C and G streptococci possess extracellular antigens: streptolysin 0, a protein which causes erythrocyte haemolysis, and streptolysin S, a lipoprotein complex possessing erythrocytolytic activity.

Classification. By means of the precipitation reaction founded on the detection of group specific carbohydrates, streptococci are subdivided into groups which are designated by capital letters from A to H and from K to T.

Five out of the known Streptococcal species cannot be related to any antigenic group. The haemolytic streptococci, rec overed from sick human beings, weresubdivided by F. Griffith into 51 serovars. He attributed 47 serovars to group A, serovars 7, 20, and 21 to group C, and serovar 16 to group G.

Streptococcus faecalis (enterococci) are pleomorphic oval cells which occur in pairs or in short chains. Some are oval or spear-shaped in form. The organisms are from 0.5 to 1 mem in diameter. Haemolytic types (Streptococcus faecalis) and organisms liquefying gelatin (S. faecalis, var. liquefaciens) are found. According to their antigenic structure, enterococci are divided into six 0-groups among which there are strains with K-antigens (capsular antigens). Some enterococcal and lactic streptococci possess identical antigens. On solid media enterococcal growths form a thin pellicle with smooth edges. On sugar broth they produce turbidity and precipitate. Certain enterococci are highly motile. Some of the strains produce a yellow pigment, and the pathogenic enterococci produce fibrinolysin. The organisms grow at tempera hires ranging from 10 to 45 °C. They are resistant to high temperature (e. g. withstand exposure to 60 °C for half an hour). Enterococci can be grown in broth containing 6.5 per cent common salt at pH 9.6 and on blood agar containing 40 per cent bile or an equivalent amount of bile salts. They ferment glucose, maltose, lactose, mannitol, trehalose, salicin, and inulin, with acid formation. They reduce and coagulate litmus milk in the presence of 0.1 per cent methylene blue. Enterococci differ from other streptococci in their ability to grow over a wide range of temperatures (10-45 °C) and in a medium of pH 9.6, in their resistance to high concentrations of salt and to penicillin (a number of strainsshow growth in media containing 0.5-1 U of antibiotic per 1 ml of media). All enterococci decarboxylate tyrosine.

Enterococci inhabit the small and large intestine of man and warm-blooded animals. The organisms possess properties antagonistic to dysentery, enteric fever, and paratyphoid bacteria, and to the coli bacillus. In the child's intestine the enterococci are more numerous than the E. coli. In lesions of the duodenum, gall bladder, and urinary tract enterococci are found as a result of dysbacteriosis. Isolation of enterococci serves as a criterion of contamination of water, sewage, and foodstuffs with faeces.

Streptococcus pneumonias (Diplococcus pneumoniae) belongs to the family Streptomycetaceae. For many years it was called pneumococcus. These are lanceolate or slightly elongated cocci measuring up to 0.5-1.25 mem in diameter and occurring in pairs, sometimes as single organisms or arranged in chains. In the body of humans and animals they have a capsule: they are Gram-positive, but young and old cultures are Gram-negative. The organisms are non-motile and do not form spores. S. pneumoniae is a facultative anaerobe, the optimum temperature for growth is 37° C and the organism grows at 28-42 °C. The organisms are poorly cultivated on ordinary media but develop readily on serum or^ascitic agar at pH 7.2-7.6 as small colonies 1.0 mm in diameter. On blood agar they form small, rounded succulent colonies on a green medium. On sugar broth they produce turbidity and a precipitate. The organisms grow readily on broth to which 0.2 per cent of glucose is added. They usually do not form capsules on artificial media, but the addition of animal protein to fluid medium promotes the formation of a capsule. There are 80 variants which are agglutinated ony by the corresponding type sera.

Among farm animals the young ones (calves, piglets, and lambs) and in vivariums guinea pigs may contract pneumonia. Among experimental animals albino mice and rabbits are most susceptible to the disease. Variants I, II, and III S. pneumoniae cause lobar pneumonia in man, which is characterized by an acute course occurring in cycles.

The organisms may also cause septicaemia, meningitis, affection of the joints, ulcerative endocarditis, otitis, peritonitis, rhinitis, highmoritis, creeping comeal ulcer, tonsillitis, and acute catarrhs of the upper respiratory passages.

Of special interest are the alpha-streptococci (the viridans group). They are responsible for haemometamorphosis on blood agar (greenish discoloration of the media) and produce no soluble haemolysin. The organisms of this group usually ferment raffinose and do not ferment mannitol. They are always isolated from the mouth and throat of healthy people and have low virulence for humans and animals. The viridans streptococci are found in pyogenic and inflammatory lesions of the teeth and gums and are responsible for subacute endocarditis.

Anaerobic streptococci (Peptostreptococcus putridus, Peptostreptococcus anaerobius, and others) are the cause of severe puerperal septic infections (puerperal sepsis). They are isolated from pyogenic and gangrenous lesions which have a putrefactive odour.

Resistance. Streptococci live for a long time at low temperatures, are resistant to desiccation, and survive for many months in pus and sputum. When exposed to a temperature of 70 °C, they are destroyed within one hour. A 3-5 per cent phenol solution kills the organisms within 15 minutes.

Pathogenicity for animals. Cattle, horses, and, among laboratory animals, rabbits and white mice are susceptible to the pathogenic streptococci.

The virulence of streptococci is tested on rabbits. The animals are infected by rubbing a culture suspension into a scratch made on the skin of the ear or on the back. This results in a local inflammation with the appearance of hyperaemia and swelling. An intravenous injection of pathogenic streptococci causes septicaemia or selectively affects the lungs, liver, kidneys, or joints.

Pathogenesis and diseases in man. The pathogenesis of streptococcal infections is brought about by the effect of the exotoxin and the-bacterial cells.The reactivity of the infected body and its previous resistance play an important part in the origin and development of streptococcal diseases. Such diseases as endocarditis, polyarthritis, highmoritis, chronic tonsillitis, and erysipelas are associated with abnormal body reactivity, hyperergia. This condition may persist for a long period of time and serve as the main factor for the development of chronic streptococcal diseases.

With an exogenous mode of infection streptococci invade the human body from without (from sick people, and animals, various contaminated objects and foodstuffs). They gain access through injured skin and mucous membranes or enter the intestine with the food. Streptococci are mainly spread by the air droplet route. When the natural body resistance is weakened, conditionally pathogenic streptococci normally present in the human body become pathogenic. Penetrating deep into the tissues they produce local pyogenic inflammations, such as streptoderma, abscesses, phlegmons, lymphadenitis, lymphangitis, cystitis, pyelitis, cholecystitis, and peritonitis. Erysipelas (inflammation of the superficial lymphatic vessels) and tonsillitis (inflammation of the pharyngeal and tonsillar mucosa) are among the diseases caused by streptococci. Invading the blood, streptococci produce a serious septic condition. They are more commonly the cause of puerperal sepsis than other bacteria.

Streptococci may cause secondary infections in patients with diphtheria, smallpox, whooping cough, measles, and other diseases. Chronic tonsillitis is attributed to the viridans streptococci and adenoviruses. Contamination of wounds with streptococci during war results in wound suppurations, abscess formation, phlegmons, and traumatic sepsis.

Immunity. Immunity acquired after streptococcal infections is ofa low grade and short duration. Relapses of erysipelas, fre quent tonsilitis, dermatitis, periostitis, and osteomyelitis occur as a result of sensitization of the body. This is attributed to low immunogenic activity and high allergen content of the streptococci, as well as to the presence of numerous types of the organisms against which no cross immunity is produced.

Immunity following streptococcal infections is of an anti-infectious nature. It is associated with antitoxic and antibacterial factors. The antitoxins neutralize the streptococcal toxin and together with the opsonins facilitate phagocytosis.

Laboratory diagnosis. Test material is obtained from the pus of wounds, inflammatory exudate. tonsillar swabs, blood, urine, and foodstuffs. Procedures are the same as for staphylococcal infections. Tests include microscopy of pus smears, inoculation of test material onto blood agar plates, isolation of the pure culture and its identification. Blood is sown on sugar broth if sepsis is suspected. Virulence is tested on rabbits by an intracutaneous injection of 200-400 million microbial cells. Toxicity is determined by injecting them intracutaneously with broth culture filtrate.

The group and type of the isolated streptococcus and its resistance to the medicaments used are also determined. In endocarditis there are very few organisms present in the blood in which they appear periodically. For this reason blood in large volumes (20-50 ml) is inoculated into vials containing sugar broth. If possible, the blood should be collected while the patient has a high temperature. In patients with chronic sepsis an examination of the centrifuged urine precipitate and isolation of the organism in pure culture are recommended.

Besides, the group and type of the isolated streptococcus are identified by means of fluorescent antibodies. Serological methods are also applied to determine the increase in the titre of antibodies, namely streptolysins O and antihyaluronidase.

Treatment. Usually penicillin is used. For penicillin-resistant strains,and when penicillin is contraindicated, streptomycin, and erythromycin are required. Vaccine therapy (autovaccines and polyvalent vaccines) and phage therapy are recommended in chronic conditions.

In some countries diseases caused by beta-haemolytic streptococci of groups A, C, G, and H and by alpha-streptococci (endocarditis) are treated with anti-infectious (antitoxic and antibacterial) streptococcal sera together with antibiotics and sulphonamides.

Prophylaxis. Streptococcal infections are prevented by the practice of general hygienic measures at factories, children's institutions, maternity hospitals, and surgical departments, in food production, agricultural work, and everyday life.

Maintaining appropriate sanitary levels of living and working condi- tions, raising the cultural level of the population, and checking personal hygiene are of great importance.

Since streptococci and the macro-organism share antigenic structures in common and because streptococci are marked by weak immunogenic ability and there are a great number of types among them which do not possess the property of producing cross immunity, specific prophylaxis of streptococcal diseases has not been elaborated. Vaccines prepared from M-protein fractions of streptococci are being studied.

Role of Streptococcus in the Aetiology of Scarlet Fever

Scarlet fever has long been known as a widespread disease but at the present time its aetiology has not yet been ascertained. Four different theories were proposed: streptococcal, allergic, viral, and combined (viral-streptococcal). Most scientists and medical practitioners favoured the streptococcal theory. G. Gabrichevsky in 1902 was the first to point out the aetiological role of the haemolytic streptococcus in scarlet fever. Usually he recovered the organisms from the pharynx of scarlet fever patients and from blood contained in the heart of those that had died of the disease. In 1907 he prepared vaccine from killed scarlet fever haemolytic streptococcal cultures. This vaccine was widely used for human vaccination.

In 1905 I. Savchenko, cultivating scarlet fever streptoco cci, obtained the toxin and used it for hyperimmunization of horses. The antitoxic antiscarlatinal serum was effectively used for treating people suffering from scarlet fever.

Data presented by Gabrichevsky and Savchenko concerning the streptococcal theory were confirmed by studies carried out in 1923-24 by G. Dick and G. Dick and by many other scientists.

The streptococcal aetiology o f scarlet fever is supported by the following arguments: (1) all people suffering from scarlet fever are found to harbour in their throats haemolytic streptococci which are agglutinated by the sera of convalescents; (2) a subcutaneous injection of the scarlet fever toxin into susceptible people (volunteers) in some cases is followed by the appearance of a characteristic skin rash, vomiting, fever, tonsillitis, and other scarlatinal symptoms; (3) an intracutaneous injection of the toxin into susceptible children produces a local erythematous and oedematous reaction; the toxin produces no reaction in children who had previously suffered from scarlet fever and were im-mune to the disease; (4) if 0.1 ml of antitoxic antistreptococcal serum or convalescent serum is introduced into the skin of a scarlet fever patient in the area of the rash, the latter turns pale (is 'extinguished'); (5) hyper-immunization of animals with the scarlet fever toxin leads to the production of antitoxins, and a neutralization reaction takes place between the toxin and antitoxins; (6) therapy with antitoxic sera and prophylaxis with combined vaccines consisting of the toxin and haemolytic streptococcal cells result in the appearance of less severe cases and decrease in morbidity and mortality.

At present many investigators accept the streptococcal theory in scarlet fever aetiology. In postwar years this theory has been confirmed by a number of investigations. Arguments against the streptococcal theory are as follows: (1) people inoculated with scarlet fever streptococci or their toxins do not always display the characteristic symptoms of the disease, e. g. there is no peeling, only rarely are there instances of tonsillitis, and phlegmon, sepsis, and erysipelas occasionally develop; (2) in severe hypertoxic forms the antitoxic ssswm has little effect, while the serum of convalescents gives better results; (3) the skin toxin test (Dick test) sometimes gives a negative reaction with susceptible children and produces a positive reaction with those who are immune; (4) immunity acquired after scarlet fever is very stable and of long duration, while that acquired after other streptococcal diseases is unstable, of short duration, and is frequently accompanied by an increased susceptibility to streptococci.

It is assumed that scarlet fever is caused by group A beta-haemolytic streptococci which possess M-antigen and produce erythrogenic exotoxin. People become infected by the air droplet route. Sic k people, convalescents, and carriers of the causative agent of scarlet fever are all sources of infection. The disease is most commonly encountered in children from 1 to 8 years of age.

The causative agent sometimes enters the body through wounds on the skin and mucous membranes of the genitalia. This form of scarlet fever is known as extrabuccal or extrapharyngeal (traumatic, combustion, surgical, and puerperal). Certain objects (e. g. utensils, toys, books, etc.) as well as foodstuffs (e. g. milk), contaminated by adult carriers, may also be sources of infection. Of great importance in the epidemiology of scarlet fever are the patients with atypical, unrecognizable forms of the disease. In its initial stage scarlet fever is chiefly characterized by intoxication, while in the second stage it is accompan ied by septic and allergic conditions.

Scarlet fever produces a relatively stable immunity. Reinfections are very rare. They have increased in number in the last years as a result of wide use of antibiotics which reduce the immunogenic activity of the pathogen and its toxin.

Data concerning the correlation between a positive Dick test and susceptibility to scarlet fever provide evidence of the antitoxic nature of immunity acquired after scarlet fever. Children from 1 to 5 years are most susceptible.

Scarlet fever is recognized mainly by its clinical course and on epidemiological grounds. Laboratory diagnosis for the detection of haemolytic streptococci and their typing is employed only in certain cases. This method is of no practical value since haemolytic streptococci are often isolated from people with various diseases and frequently from healthy individuals.

The phenomenon of rash 'extinguishmenf is employed as an auxiliary diagnostic method. In the case of scarlet fever, the rash at the site of injection will disappear within 12-20 hours and the skin will turn pale.

Certain physicians apply the Dick test with the thermolabile fraction of the toxin. The diagnosis of scarlet fever is verified to a certain extent if on a second injection of the toxin a positive Dick test reverts to a negative reaction.

Scarlet fever may also be diagnosed by detecting precipitins in the urine (urine precipitation test). A layer of type-specific streptococcal sera or convalescent serum is transferred onto freshly filtered urine of patients in the first days of the disease. The appearance of a greyish-white ring at the interface of the two fluids designates a positivereaction.

Scarlet fever patients are treated with penicillin, tetracycline, sulphonamides (norsulphazol, etc.), and gamma-globulin from human blood. The wide use of antibiotics has led to a significant decrease in the morbidity and mortality rate of scarlet fever and to a milder course of the disease. This fact also confirms the definite role played by haemolytic streptococci in the aetiology and pathogenesis of scarlet fever, since it is known that these organisms are extremely sensitive to penicillin and other antibiotics. In the recent years, however, an increase in the incidence of scarlet fever and a more severe course of the disease are noted.

Prophylaxis consists of early diagnosis, isolation of patient s and hospitalization in the presence of epidemiological and clinical indications. Extremely hygienic cleaning and ventilation and observance of correct hospital regime are also necessary. If cases of scarlet fever occur in children's institutions, the children concerned must be isolated. Debilitated children who have been in contact with scarlet fever patients must be injected with 1.5-3.0 ml of human serum gammaglobulin.

Role of Streptococcus in the Aetiology of Rheumatic Fever

The majority of authors maintain that rheumatic fever develops as a result of the body becoming infected by group A beta-haemolytic streptococci. Acute or chronic tonsillitis and pharyngitis produce a change in the immunological reactivity of the body and this gives rise to characteristic clinical symptoms and a pathological reaction. It should be noted, however, that recent research shows the leading role in rheumatism of virus agents with persisting properties. The active phase with an acute and subacute course is attended with virusaemia. Rheumatism is characterized by the virus remaining in the body for a long period of time: the viral antigen penetrates the leucocytes and sensitizes them. Diminution of the specific and non-specific reactions to the virus leads to tolerance of the body. Autoimmune reactions are encountered in rheumatism. Streptococci and other exogenous and endogenous factors contribute to exacerbation of rheumatism. Virusaemia is almost always revealed in the patients during exacerbation. Identical viruses are detected in the mother's blood and in the blood of premature infants, stillborns, and in infants who die soon after birth.

No streptococci, streptococcal antigen or antibodies to them are found in some patients and penicillin therapy proves ineffective. On the grounds of this, it is considered that the diagnosis of rheumatism should be made and penicillin prescribed only if the presence of streptococcal infection is sufficiently verified.

The prevalence of rheumatic fever depends on the time of the year. The highest number of cases occur in October-November and March- April. Acute and chronic tonsillitis, pharyngitis, and catarrh are also most prevalent in these months.

The allergic reaction produced in the body as a result of re-invasion by antigens (streptococcal exo- and endotoxms, autoantigens, and complexes consisting of streptococcal toxins and components of tissue and blood proteins of sick people) is an important factor in the pathogenesis of the disease. It is known that blood of individuals who have suffered from a streptococcal infection contains antibodies against beta-haemolytic streptococci. In 1 -3 per cent of these cases the formation of antibodies does not produce immunity, and a secondary invasion of the body by specific and non-specific antigens leads to the development of hyperergia. Experiments have shown that streptococci bring about the formation of autoantigens which cause the production of autoantibodies in the body. These autoantibodies are responsible for lesions in certain tissues and organs.

Studies of high-molecular gamma-globulins and their complexes in rheumatic fever have shown that the normal human gamma-globulin contains two fractions (7S and 19S) which differ in their pre cipitate constant. The majority of the common antibodies are associated with the 7S gamma-globulin fraction, while isoagglutinins, Rh-agglutinins, and complement-fixing antibodies are contained in the 19S fraction. The gamma-globulin fraction, rich in 19S, is found to contain the rheumatoid factor. An interaction has been demonstrated between the rheumatoid factor and the antigen-antibody precipitate, the latter possessing antigenic properties. Thus, in its reaction with the antigen-antibody complex the rheumatoid factor behaves as the complement. Alternatively, the rheumatoid factor may act as an antibody to gamma-globulin or to the antigen-antibody complex, the latter acting as an antigen.

Antigen-antibody reactions result in the injury of the interstitial connective tissue, release of histamine, and inflammation. Disturbances of coordination in the hypophysis-adrenal system are encountered in rheumatic fever. For this reason supporters of the Selye theory consider rheumatic fever to be an adaptational disease. However, the above mentioned aspects on the mechanism of rheumatic fever can by no means cover all the complex processes involved in the pathogenesis of this disease.

According to its clinical course, rheumatic fever is differentiated into active and inactive phases. The active phase is characterized by acute rheumocarditis without valvular defects and relapsing rheumocarditis accompanied by valvular defects, polyarthritis, chorea, pleuritis, peritonitis, nephritis, he patitis, pneumonia, lesions in the skin and subcutaneous tissue, eyes, and other systems. The inactive phase develops in the form of rheumatic myocardiosclerosis, heart defects, and conditions following extracardial affections.

Three periods can be distinguished during the development of rheumatic fever: (1) period of acute streptococcal infection and initial sensitization; (2) penod ofhyperergic reactions, resulting frominteraction between antigens and antibodies, which are accompanied by pnmary rheumatic polyarthritis or carditis; (3) period of stable allergic reacts ity accompanied by pronounced manifestations of parallergy and autosensitization, profound and stable immunogenic disturbances, and relapses.

Laboratory diagnosis is made on the basis of determination of an increase in antistreptolysin, antifibrinolysin, and antihyaluronidase titres and detection of C-reactive protein.

Treatment of rheumatic patients is accomplished by several measures aimed at desensitization of the body, abatement of inflammatory conditions, recovery of normal body reactivity, condition of the nervous system, and disturbed processes, and control of local infections.

Prophylaxis includes prevention of streptococcal infections, strengthening of general resistance, and creation of favourable conditions for everyday life and work. In addition, all people suffering from rheumatic fever and those susceptible to the disease should be given prophylactic treatment with penicillin and tetracycline preparations in spring and autumn.

Additional materials about laboratory diagnosis

STAPHYLOCOCCAL INFECTION. In diseases caused by pathogenic staphylococci (Staphylococcus aureus) the materials to be examined include pus, mucosal secretions, blood, sputum, urine, and cerebrospinal fluid. In cases of food intoxications, vomit, faeces and food remnants are also. studied.

In open suppurative lesions the material is taken with a cotton wool swab after removing the superficial layer of the pus, which may contain non-pathogenic staphylococci and other microorganisms usually present on the skin and in the air. When purulent foci are unruptured, they are punctured and the pus from the syringe is poured out into a sterile test tube. Mucosal secretion is obtained with a tampon. Urine and sputum are collected into sterile test tubes and jars. Blood withdrawn from a patient's ulnar vein with a syringe as well as aseptically obtained cerebrospinal fluid are inoculated at the patient's bedside into a vessel containing 100-200 ml of sugar broth (pH 7,2–7,4). Staphylococci propagate quite readily in simple media too but the use of sugar broth is preferable since septicaemia may be secondary not only to staphylococci but also to other microorganisms which are more demanding with regard to nutrient media.

Bacterioscopic examination. Pus, sputum, faucial secretion, and other biological samples to be studied, with the exception of blood. are examined microscopically. Place a specimen of high density pus into a drop of sterile water; make smears with the help of a swab or loop and stain them by Gram's method. Staphylococci are Gram-positive and tend to be arranged in small clusters, in pairs, and in the form of short chains. Differentiation between staphylococci and streptococci by their distribution and tinctorial properties proves rather difficult. Consequently, the examination is not confined to microscopy, but includes cultivation of the tested material.

Bacteriological examination. On the first day of investigation with the help of a loop or spatula, inoculate the specimen into dishes with 3-5 per cent of blood, milk-salt, and yolk-salt agar and place it into an incubator at 37 °C for 18-24 hrs.

To isolate staphylococci, it is advisable to use dry elective medium, representing a mixture of hydrolysin and aminopeptide (1:1), because this medium facilitates faster growth of staphylococci as compared with other nutrient media. It may be used as a base for preparing milk-yolk-salt agar for the purpose of determining pigment formation and lecithinase activity.

On the second day the colonies are examined. On solid media staphylococci appear as convex, nontransparent medium-sized colonies of a homogeneous or fine-grain structure. Pathogenic strains on a blood agar with 0,25-0,5 per cent of glucose form a haemolytic zone around the colonies. On the yolk-salt agar most pathogenic staphylococci induce lecithovitellin (yolk) reaction manifested in the formation of a turbulent zone with an opalescent halo on the periphery around the colony.

Using dishes with milk-salt agar, pigment formation is determined in strains displaying this ability. The pigment may be golden, white or of a lemon-yellow colour.

Microscopic examination reveals typical Gram-positive staphylococci in such smears.

Further examination is aimed at isolating pure staphylococcal culture, for which purpose the colonies are transferred onto an agar slant.

Apart from haemolytic and lecithinase activity, pathogenetic staphylococci possess the ability to coagulate plasma, induce skin necrosis in rabbits, and to destroy DNA.

On the third day of bacteriological examination, the isolated culture is introduced into a test tube with rabbit citrate plasma to identify plasmocoagulase. For this purpose 10 ml of blood obtained from the rabbit heart are poured into a test tube with 1 ml of 5 per cent solution of sodium citrate; the plasma separated by centrifugation or sedimentation is diluted with isotonic saline (1:4) and poured into sterile test tubes by 0.5-ml portions. At present, there is dry citrate plasma which is diluted with isotonic saline before use. The inoculated cultures are placed into an incubator at 37 °C to determine the time when plasma coagulation manifests itself. Milliard suspension is prepared from the same culture (1 milliard of microorganisms per ml) and 0.2 ml of this suspension is injected intra-cutaneously to a light-coated rabbit. One-two days later skin necrosis develops at the site of injection.

Some differential signs of staphylococci are present in table.

Test	S. aureus	S. epidermidis	S. saprophyticus
Hemolysis	usually beta	usually none	usually none
Pigment	often creamy gold	usually white	usually white
Novobiocin test	sensitive	sensitive	resistant

To perform the plasmoagglutination test, plasma is poured into a test tube and pure staphylococcal culture is ground with a loop at a distance of 0,5 cm from the plasma surface. Then, using a loop, the plasma is transferred to the culture. A positive result is recognized by the appearance of agglutinate flakes on the tube wall.

To demonstrate DNase activity, a 24-bour staphylococcal culture is inoculated in the form of small plaques into a Petri dish containing Hottinger's agar and DNA[1][1].

Twenty-four hours later 5 ml of 1N HCl solution is introduced into the dish and zones of clearing indicating the presence of DNase are counted in 3-5 min.

Lysozyme activity of staphylococci is considered to be an additional indicator of pathogenicity. To demonstrate it, solid nutrient medium with bacterial suspension of Micrococcus lysodeikticus is inoculated in a patch-like manner with 24-hour agar culture of staphylococcus. Zones of lysis form around the colonies when lysozyme is produced. Under anaerobic conditions pathogenic staphylococci break mannitol to acid.

Examination of cultural phagovar is of great epidemiological significance with regard to identifying the source of infection. The main international set of staphylococcal bacteriophages consists of 21 types divided into four groups, Staphylococcal bacteriophages in two working dilutions 1 TD (test-dilution), which should be no less than 10 ^-3, and 100 TD, not less than 10^–1, are the ones most commonly used for typing. Phagotyping is started with 1 TD, then, if the result has been negative, 100 TD is employed.

To diagnose staphylococcal diseases, one can use the IHA test with an erythrocytic diagnosticum (red blood cells are sensitized with alpha-toxin of the staphylococcus), which allows the detection of antibodies.

Determination of sensitivity of the isolated staphylococci to antibiotics is essential for the purposeful and effective treatment of a given patient.

To isolate a haemoculture of staphylococci, sugar broth is inoculated with blood and incubated at 37 °C for 18-24 hrs. The staphylococcus causes uniform cloudiness of the medium. On the second day of the study, prepare smears from the blood culture and streak the latter onto a meat-peptone agar slant and a blood agar plate to evaluate the haemolytic activity of staphylococci. On the third day, examine both the staphylococcal culture, which has grown on the agar slant, and the culture that has been isolated from the pus or other materials.

In inoculation of exudate, pus from unruptured abscesses and phlegmons, meat-peptone agar or 5 per cent blood agar is employed. The inoculated cultures are placed into an incubator at 37 °C for 18-24 hrs, pure culture is isolated and then identified by the aforementioned methods.

In food intoxications the material to be studied is streaked onto plates with milk-salt and yolk-salt agar and also onto broth containing 1 per cent of glucose (for enrichment). Material contaminated with extraneous microorganisms is inoculated onto blood agar containing 6-7 per cent of sodium chloride. Staphylococci demonstrate good growth and haemolytic action (they may propagate in the presence of 12 per cent of salt and 50 per cent of sugar) in this. medium; the development of enterobacteria and bacilli is inhibited, while Proteus is incapable of uninterrupted growth in the form of a film.

The cultures are placed into an incubator at 37 °C. On the next day, the colonies are examined, pure cultures are isolated, and staphylococci are identified. Pathogenic staphylococci responsible for food intoxications produce a golden, less commonly, white pigment, liquify gelatin, induce haemolysis, and coagulate plasma.

To establish the production of enterotoxin, the staphylococcal culture is streaked onto a special nutrient medium[2][2]. The inoculated cultures are placed into an exsiccator with 20 per cent of CO₂ and incubated at 37 °C for 3-4 days, and then filtered through Nos 3 and 4 membrane filters. The obtained filtrate (10-15 ml in total) is mixed with the equal amount of warm milk and fed to 1-2-month-old kittens or alternatively the filtrate is introduced into their stomach via a catheter. If enterotoxin is present, the kittens develop vomiting (the key sign of poisoning) in 30-60 min and diarrhoea in 2-3 hours, which persists for 2-3 days and may -result in death in grave cases. The filtrate may also be injected intraperitoneally, following its heating at 100 °C for 30 min to inactivate thermolabile fractions of a toxin. Currently, toxigenicity of isolated staphylococcal cultures is determined in vitro, making use of the diffuse precipitation test in gel.

Pathogenic staphylococci may harbour in the air of operating-theatres, dressing rooms, post-delivery wards, and other hospital premises. To demonstrate them, air samples are inoculated on yolk-salt agar[3][3].

Determination of the staphylococcal antitoxin in the blood serum becomes of great importance in diagnosing chronic staphylococcal infections (osteomyelitis, septicopyaemia, etc.) when bacteriological studies conducted together with aggressive antibiotic therapy yield no results. The reaction is based on suppression of the haemolytic activity of the staphylococcal toxin by the patient's serum antitoxin. For this purpose, add the patient's serum diluted 1:5; 1:10; 1:15; 1:20, etc. to the staphylococcal toxin taken in a definite dose, mix thoroughly, and add 0.05 ml of rabbit erythrocytes to the resultant mixture. Incubate the test tubes for 1 h at 37 °C and for 1 h at room temperature, and then read the results.

The amount of serum with which the tested dose of toxin either retards or inhibits haemolysis is taken as the serum titre.

In clinically healthy children and individuals with a history of staphylococcal skin infection or staphylococcal infection that complicated surgical disease, the titre of the staphylococcal antitoxin does not exceed 0,5-4 AU/ml. Patients with chronic staphylococcal infections usually demonstrate higher titres.

Summary. GRAM-POSITIVE COCCI. The Gram-positive cocci are grouped together based on their Gram-stain reaction, thick cell wall composition, and spherical shape. Most of the organisms in these groups are members of the Micrococcaceae family. All of the organisms in these groups are non-endospore forming chemosynthetic heterotrophs.

MICROCOCCUS. Micrococcus is a Gram-positive, aerobic bacterium which is a member of the Micrococcaceae family. Micrococcus cells can be observed under the microscope as spherical cells forming pairs or clusters. If cultured in broth or on nutrient agar, the colonies may be red or yellow when observed unstained. Although these bacteria are a common human skin contaminant, they are relatively harmless to humans because they maintain a saprophytic lifestyle. They can also be found in freshwater environments or in soil. Three common species of Micrococcus are M. luteus, M. roseus, and M. varians.

LABORATORY INDICATIONS:

Catalase +

Oxidative action on glucose

Growth on mannitol

STAPHYLOCOCCUS. Clinically, the most important genus of the Micrococcaceae family is Staphylococcus. The Staphylococcus genus is classified into two major groups: aureus and non-aureus. S. aureus is a the cause of soft tissue infections, as well as toxic shock syndrome (TSS). It can be distinguished from other species of Staphylococcus by a positive result in a coagulase test (all other species are negative).

The pathogenic effects of Staphylococcus are mainly asssociated with the toxins it produces. Most of these toxins are produced in the stationary phase of the bacterial growth curve. In fact, it is not uncommon for an infected site to contain no viable Staphylococcus cells. The S. aureus enterotoxin causes quick onset food poisoning which can lead to cramps and severe vomiting. Infection can be traced to contaminated meats which have not been fully cooked. These microbes also secrete leukocidin, a toxin which destroys white blood cells and leads to the formation of puss and acne. Particularly, S. aureus has been found to be the causative agent in such ailments as pneumonia, meningitis, boils, arthritis, and osteomyelitis (chronic bone infection). Most S. aureus are penicillin resistant, but vancomycin and nafcillin are known to be effective against most strains.

Of the non-aureus species, S. epidermidis is the most clinically significant. This bacterium is an opportunistic pathogen which is a normal resident of human skin. Those susceptible to infection by the bacterium are IV drug users, newborns, elderly, and those using catheters or other artificial appliances. Infection is easily treatable with vancomycin or rifampin.

LABORATORY INDICATIONS:

Anaerobic glucose fermentation with acid production

Catalase +

Nitrate +

Coagulase +

STREPTOCOCCAL INFECTION. In diseases caused by pathogenic streptococci (Streptococcus pyogenes}, the material subjected to the study is pus, blood, mucosal secretions, urine, sputum, and cerebrospinal fluid. The procedure of obtaining the material to be examined is the same as in diseases caused by staphylococci.

Bacterioscopic examination. In Gram-stained smears from pus and mucosal secretions streptococci appear as short chains, less commonly as pairs or individual cocci. In the latter case streptococci cannot be distinguished from staphylococci, and they should be studied for other attributes.

Bacteriological study. Samples of pus, mucosal secretions, urine, sputum, and cerebrospinal fluid are inoculated onto Petri dishes with 5 per cent blood agar (defibrinated blood should be preferably used) and into a test tube with sugar broth. After an overnight incubation at 37 °C, the colonies and growth in the sugar broth are studied. Streptococcus pyogenes develops with the formation of small flat rather dry granular colonies.

Streptococci producing beta-haemotoxin (streptolysin) form a zone of haemolysis around the colonies, while alpha-haemotoxin-producing streptococci are characterized by the appearance of green zones around the colonies as a result of methaemoglobin formation. There is no haemolysis in the absence of haemotoxin. In sugar broth streptococcal growth appears as flakes or granules on the bottom or walls whereas the medium remains transparent.

Smears from streptococcal colonies display no typical pattern of cocci in the form of chains. Cells are arranged singly, in pairs or in small aggregates. In liquid medium, a cultural smear exhibits typical chains of streptococci.

At further stages of examination the group and serovar of the streptococcus are determined.

Streptococcal groups (by Lancefield) are classified by the presence of a polysaccharide antigen which is revealed using the precipitation test with group sera (A, B, C, etc.).

Serovars (serotypes) of streptococci (by Griffith) are characterized by the presence of a specific protein antigen which is determined with the help of the slide agglutination test performed with typical agglutinating sera. For this purpose, a 24-hour broth culture of the isolated streptococcus is centrifuged and the sediment is diluted in 0,5-1 ml of isotonic sodium chloride solution. A drop of serum is mixed with a drop of the tested culture on a glass slide. The majority of pathogenic streptococci belong to group A.

To recover b-haemolytic streptococcus of group A, the immuno-fluorescence test may be employed. In this case a smear is prepared from the isolated streptococcal culture, fixed for 15 min with 95 per cent alcohol, and then stained with fluorescent serum for 15 min. After that, it is washed in running water, dried, and examined under the luminescent microscope.

Blood examination. The procedure of blood inoculation into sugar broth is the same as that employed in staphylococcal diseases. The presence of streptococci is indicated by the formation of floccular sediment on the bottom and haemolysis. Smears are characterized by long chains of streptococci. Microscopic examination allows a preliminary conclusion as to the presence or absence of streptococci. To detect haemotoxin, the culture is transferred onto a blood agar plate. Typical small colonies surrounded by a zone of haemolysis or a greenish halo develop in 24 hours (the third day of investigation). These findings permit the conclusion that the culture harbours S. pyogenes.

To isolate anaerobic streptococci (Peptestreptococcus anaerobius), which harbour the genital mucosa and may cause postpartal sepsis, blood is introduced into the Kitt-Tarozzi medium. Some strains of anaerobic streptococci form gas when they get into the liquid media.

Pathogenic streptococci show good growth in Garrod's medium. It presents meat-peptone agar (pH 7,4) to which 5 per cent of blood and 0,1 per cent aqueous solution of gentian violet are added (0,2 ml per every 100 ml of meat-peptone agar). Growth of saprophytic air microflora and enterococci on Garrod's medium is suppressed.

In order to determine pathogenic properties of streptococci, formation of toxins, in particular of fibrinolysin (streptokinase), is investigated. Human blood plasma is used for this purpose, which is obtained by adding 1 ml of 2 per cent citrate sodium solution to 10 ml of blood. Following sedimentation, the unstained plasma is separated and diluted in a 1 to 3 ratio. At the next stage, 0,5 ml of an 18–20-hour culture of the tested streptococcus and 0,5 ml of 0,25 per cent solution of calcium chloride are added. The test tubes are carefully shaken and placed in a water bath at 42 °C for 20–30 min.

A fibrin clot is formed during this period. The test tubes are left in the water bath for another 20 min. If the streptococcal culture produces fibrinolysin, the clot is dissolved within 20 min.

As some strains of streptococci dissolve fibrin slowly, the test tubes are transferred from the water bath to an incubator two hours later, and the results are read on the following day.

Determination of the amount of superficial M-protein, which is observed only in pathogenic strains, may be considered as a method evaluating the virulence of streptococcal cultures. Young cultures are utilized to obtain hydrochloric extracts, and the content of M-protein is determined in the latter by the precipitation test.

No laboratory studies are usually used in the diagnosis of scarlet fever. Occasionally, secretion of the faucial mucosa is cultivated, and streptococci of various serogroups are isolated.

Haemolytic streptococci releasing a- and P-toxins may harbour the air of various hospital rooms. To demonstrate them, air samples are inoculated onto Garrod's medium for the subsequent isolation and identification of pure culture.

In laboratory diagnosis, streptococci should be differentiated from enterococci (Streptococcus faecalis} which are characterized by the following specific features: ability to grow at temperatures varying from 10° to 45 °C, resistance to high concentrations of sodium chloride, penicillin, and alkaline medium (pH 9.6). Enterococci occur in diseases affecting the duodenum, gallbladder, and urinary tract. Their presence in the environment serves as a criterion of faecal contamination of drinking water, sewage waters, and food-stuffs.

Serological diagnosis of streptococcal infections is conducted in patients with chronic diseases treated with large doses of antibiotic and sulphanilamide drugs, i.e., when isolation of the causative agent by bacteriological methods proves to be very difficult. It envisages identification in the blood of the streptococcal antigen and specific streptococcal antibodies to toxins, in particular to steptolysin 0.

The streptococcal antigen in the patient's blood serum is demonstrated by complement fixation in the cold. For this test antistreptococcal immune serum is utilized, which is obtained through hyperim-munizing rabbits with streptococcal culture of serological group A. The antigen titre is assumed to be the maximum dilution of the serum tested, which induces the haemolytic inhibition of at least ++ intensity.

Isolation of antistreptolysin O contained in patients' sera is based on its capacity to neutralize the haemolytic activity of streptolysin O. For this purpose the patient's serum is diluted and streptolysin O (commercial drug) is added to the obtained dilutions. The mixture is incubated at 37 °C for 15 min and then 0,2 ml portions of rabbit erythrocytic suspension are added to all tubes. The tubes are reincubated for 1 h, and then the results are read. Positive cases are witnessed by the absence of haemolysis.

Techniques of identifying antibodies to other toxins released by streptococci, for example, to antistreptohyaluronidase, are also used for serological diagnosis.

INFECTION CAUSED BY STREPTOCOCCI OF PNEUMONIA (PNEUMOCOCCI). Streptococci of pneumonia (pneumococci), Streptococcus pneumoniae, are the causative agents responsible for croupous pneumonia, focal pneumonia, other respiratory diseases, creeping cornea! ulcer, suppurative processes in the middle ear and maxillary sinus, and also for sepsis and meningitis.

Material to be studied includes sputum, pus, cerebrospinal fluid, blood, and post-mortem organs.

Bacterioscopic examination. Two smears are made of the tested material (with the exception of blood); one is stained by the Gram method, the other by the Burri or Kozlovsky technique. Microorganisms and Indian ink placed on a glass slide are mixed by circular movements to prepare the conventional smear. Indian ink is diluted with isotonic saline in a ratio of' 1 to 4. The smear is dried in the air, left unfixed and stained for 2–3 min with formol-gentian violet (10 ml of 40 per cent formalin and 1,5 g of gentian violet). Formol-gentian violet may be substituted with 0,33 per cent aqueous fuchsine solution and 3 per cent alkaline solution of methylene blue. Microscopic examination reveals unstained capsules which contain violet bacterial cells against a black-gray background. Pneumococci are arranged in pairs, their cells are elongated, resembling a candle flame, and are surrounded with a capsule. Hence, microscopic findings seem to suggest the presence of the causative agent. Yet, it is bacteriological investigation that provides most reliable data.

Bacteriological examination. To obtain pure culture, 5-10 ml of blood is inoculated into a serum broth (1 part of serum and 3 parts of meat-peptone broth, pH 7,2–7,4) or into sugar broth, or into a special medium which contains (in 100 ml): 1,8–2,0 ml of agar-agar, 70–75 ml of Hottinger's hydrolysate or casein hydrolysate (1.8–2.0 g/l of amine nitrogen), 20-25 ml of bovine heart hydrolysate (1,40–1,60 g/I of amine nitrogen), 4-5 ml of defibrinated horse blood 0,5-0,7 ml of baking yeast extract. After 18–24-hour incubation in a heating block, the culture is transferred onto a plate with 10 per cent blood agar. Cerebrospinal fluid is centrifuged and the deposit is inoculated onto a blood agar. Colonies of the pneumococcus resemble those of the streptococcus: they are small, almost flat, non transparent, with a halo of a green colour or, less commonly, of haemolysis. Another characteristic sign is an impression in the centre of the colony.

As a rule, direct inoculation of the material onto nutrient media (pus, sputum) does not give a positive result since saprophytes, especially saprogenous microorganisms present in them, inhibit the growth of pneumococci. For this reason, pus and sputum are treated before cultivation: clumps are collected and comminuted in a porcelain mortar, then 1 ml of isotonic saline is added, and the resultant mixture is injected intraperitoneally to albino mice. Mice are very susceptible to the pneumococcus and die of pneumococcal septicaemia in 18-72 hrs. The mouse's carcass is dissected and the blood from the heart, pieces of the internal organs, and peritoneal fluid are inoculated into a blood agar plate and a test tube with serum broth.

Morphological and cultural characteristics do not allow any clear-cut differentiation between the pneumococcus and Streptococcus viridans. To achieve this, one can employ the reaction of pneumococcal lysis by bile: 1 ml of broth culture is introduced into a sterile test tube and then 0.5 ml of bovine bile is added. Ten-fifteen minutes of incubation in a heating block is enough to bring about complete lysis of the pneumococci. A tube containing bile-free broth culture serves as a control. Streptococcus viridans whose colonies resemble those of the pneumococcus are not dissolved by bile. If lysis is present, the tested material is inoculated onto the Hiss medium. In contrast to the streptococcus, the pneumococcus splits inulin with the formation of oxygen and forms ammonia from arginine.

The conducted study allows the final identification of the isolated microorganism. The factors to be taken into consideration are: a lancet shape of diplococci, the presence of the capsule in the native material, high virulence for albino mice, dissolvement by bile, and inulin splitting.

Summary. STREPTOCOCCUS. The Streptococcus genus consists of Gram-positive bacteria which appear as chains under microscopic observation. Members of Streptococcus can be aerobic, anaerobic, or microaerophilic. The organisms in this genus are characterized by a coccus appearance, a thick cell wall, and aerobic action on glucose. Four different classification systems exist:

CLINICAL

Pyogenic Streptococci

Oral Streptococci

Enteric Streptococci

HEMOLYSYS

alpha-hemolysis

beta-hemolysis

gamma-hemolysis

SEROLOGICAL-Lancefield (A-H), (K-U)

BIOCHEMICAL(physiological)

GROUP A. The first group in the Lancefield classification system includes only one species of Streptococcus, S. pyogenes. This particular opportunistic pathogen is responsible for about 90% of all cases of pharyngitis. A common form of pharyngitis is "Strep throat" which is characterized by inflamation and swelling of the throat, as well as development of pus-filled regions on the tonsils. Penicillin is usually administered to patients as soon as possible to quell the possibility of the infection spreading from the upper respiratory system into the lungs. Once in the lungs, the infection could give rise to pneumonia. Some cases also develop into rheumatic fever if left untreated. Other diseases linked to S. pyogenes are skin infections such as impetigo, cellulitis, and erysipelas.

LABORATORY INDICATIONS:

· Catalase -

· Beta-hemolysis

· Bacitracin sensitive

GROUP B. The B classification of Lancefield also includes only one bacterium, S. agalactiae. For years this bacterium has been the causative agent in mastitis in cows. Currently, it has been found to be a cause of sexually transmitted urogenital infections in females. Although infection is easily treated with penicillin, proper diagnosis is necessary for women nearing labor because the infection can easily spread to the child via the birth canal.

LABORATORY INDICATIONS:

· CAMP +

· Beta-hemolysis

GROUP D. Type D Streptococcus is the next clinically important bacterium because of the multitude of diseases it is known to cause. Although many are harmless, the pathogenic strains cause complications of the human digestive tract. This group has recently been reclassified into two divisions: Enterococcus and non-Enterococcus. The Enterococci include E. faecalis, a cause of urinary tract infections, and E. faecium, a bacterium resistant to many common antibiotics. Diseases such as septicemia, endocarditis, and appendicitis have also been attributed to group D Strep. Steptococcus is part of he normal human fecal flora . Once identified, Group D Strep can be treated with ampicillin alone or in combination with gentamicin.

LABORATORY INDICATIONS:

· Hydrolysis of bile esculin (dark brown medium) -this indicates the ability of the bacteria to tolerate bile

· Growth in high salt concentration.

Gram-negative cocci

Meningococci. The meningococcus (Neisseria meningitidis) was isolated from the cerebrospinal fluid of patients with meningitis and studied in detail in 1887 by A. Weichselbaum. At present the organism is classified in the genus Neisseria, family Neisseriaceae.

Morphology. The meningococcus is a coccus 0.6-1 mcm in diameter, resembling a coffee bean, and is found in pairs. The organism is Gram-negative. As distinct from pneumococci, meningococci are joined longitudinally by their concave edges while their external sides are convex.Spores, capsules and flagella are not formed. In pure cultures meningococci occur as tetrads (in fours) and in pus they are usually found within and less frequently outside the leukocytes. The G+C content in DNA ranges from 50.5 to 51.3 per cent.

Meningococci in cerebrospinal fluid. The leukocytes have engulfed (phagocytized) large number of the diplococci

In culture smears, small or very large cocci are seen singly, in pairs, or in fours. Meningococci may vary not only in shape but also in their Gram reaction. Gram-positive diplococci appear among the Gram-negative cells in smears.

Cultivation. The meningococcus is an aerobe or facultative anaerobe and does not grow on common media. It grows readily at pH 7.2-7.4 on media to which serum or ascitic fluid has been added. Optimum temperature for growth is 36-37 °C and there is no growth at 22° C. On solid media the organisms form fine transparent colonies measuring 2-3 mm in diameter. In serum broth they produce turbidity and a precipitate at the bottom of the test tube, and after 3-4 day's, a pellicle is formed on the surface of the medium.

Meningococci can be adapted to simple media by repeated subculture on media with a gradual change from the optimum protein concentration to media containing a minimal concentration of proteins.

Fermentative properties. Meningococci do not liquefy gelatin, cause no change in milk, and ferment glucose and maltose, with acid formation.

Toxin production. Meningococci produce toxic substances which possess properties of exo- and endotoxins. Disintegration of bacterial cells leads to the release of a highly toxic endotoxin. Meningococci readily undergo autolysis which is accompanied by accumulation of toxins in the medium. The meningococcal toxin is obtained by treating the bacterial cells with distilled water, or 10 N solution of soda, by heat autolysis, by exposure to ultraviolet rays.

Antigenic structure and classification. Meningococci were found to contain three fractions: carbohydrate (C) which is common to all meningococci, protein (P) which is found in gonococci and type III S. pneumoniae, and a third fraction with which the specificity of meningococci is associated. According to the International Classification, four groups of meningococci are distinguished, groups A, B, C, and D. Recently the number of types has increased to seven, but only the first two are dominant.

The organisms are characterized by intraspecies variability. A change of types takes place at certain times.

Resistance. The meningococcus is a microbe of low stability, and is destroyed by drying in a few hours. By heating to a temperature of 60° C it is killed in 10 minutes, and to 80 °C, in 2 minutes. When treated with 1 per cent phenol, the culture dies in 1 minute. The organism is very sensitive to low temperatures. Bearing this in mind, test material should be transported under conditions which protect the meningococcus against cooling.

Pathogenicity for animals. Animals are not susceptible to the meningococcus in natural conditions. The disease can be produced experimentally in monkeys and rabbits by subdural injections of meningococci. Intrapleural and intraperitoneal infection of guinea pigs and mice results in lethal intoxication. Septicaemia develops in experimental animals only when large doses are injected.

Pathogenesis and diseases in man. People suffering from meningococcal infection and carriers are sources of diseases. The infection is transmitted by the air-droplet route. The causative agent is localized primarily in the nasopharynx. From here it invades the lymph vessels and blood and causes the development of bacteriemia. Then as a result of metastasis the meningococci pass into the meninges and produce acute pyogenic inflammation in the membranes of the brain and spinal cord (nasopharyngitis, meningococcaemia, meningitis).

The disease usually arises suddenly with high temperature, vomiting, rigidity of the occipital muscles, severe headache, and increased skin sensitivity. Later paresis of the cranial nerves develops due to an increase in the intracranial pressure. Dilatation of the pupils, disturbances of accommodation, as well as other symptoms appear. A large number of leukocytes are present in the cerebrospinal fluid, and the latter after puncture escapes with a spurt because of the high pressure.

In some cases meningococcal sepsis develops. In such conditions the organisms are found in the blood, joints, and lungs. The disease mainly attacks children from 1 to 5 years of age. Before the use of antibiotics and sulphonamides the death rate was very high (30-60 per cent).

The population density plays an important part in the spread of meningitis. During epidemic outbreaks there is a large number of carriers for every individual affected by the disease. In non-epidemic periods the carrier rate increases in the spring and autumn. Body resistance and the amount and virulence of the causative agent are significant. Depending on these factors, the spread of infection is either sporadic or epidemic.

Meningitis can also be caused by other pathogenic microbes (streptococci, E. coli, staphylococci, bacteria of influenza, mycobacteria of tuberculosis, and certain viruses). These organisms, however, cause sporadic outbreaks of the disease, while meningococci may cause epidemic meningitis.

Immunity. There is a well-developed natural immunity in humans. Acquired immunity is obtained not only as a result of the disease but also as the result of natural immunity developed during the meningococcal carrier state. In the course of the disease agglutinins, precipitins, opsonins, and complement-fixing antibodies are produced. Recurring infections are rare.

Laboratory diagnosis. Specimens of cerebrospinal fluid, nasopharyngeal discharge, blood, and organs obtained at autopsy are used for examination.

The following methods of investigation are employed: (1) microscopic examination of cerebrospinal fluid precipitate; (2) inoculation of this precipitate, blood or nasopharyngeal discharge into ascitic broth, blood agar, or ascitic agar; identification of the isolated cultures by their fermentative and serologic properties; differentiation of meningococci from the catarrhal micrococcus (Branhamella catarrhalis) and saprophytes normally present in the throat. The meningococcus ferments glucose and maltose, whereas Branhamella catarrhalis does not ferment carbohydrates, and Neisseria sicca ferments glucose, levulose, and maltose; (3) performance of the precipitin reaction with the cerebrospinal fluid.

Treatment. Antibiotics (penicillin, oxytetracycline, etc.) and sulphonamides (streptocid, methylsulphazine) are prescribed.

Prophylaxis is ensured by general sanitary procedures and epidemic control measures (early diagnosis, transference of patients to hospital), appropriate sanitary measures in relation to carriers, quarantine in children's institutions. Observance of hygiene in factories, institutions public premises, and lodgings, and prevention of crowded condition are also obligatory. An antimeningococcal vaccine derived from the C/B serogroup is now under test. It contains specific polysaccharides.

The incidence of meningitis has grown recently. The disease follows a severe course and sometimes terminates in death.

Gonococci. The causative agent of gonorrhoea and blennorrhoea (Neisseria gonorrhoeae) was discovered in 1879 by A. Neisser in suppurative discharges. In 1885 E. Bumm isolated a pure culture of the organism and studied it in detail. Gonococci belong to the genus Neisseria, familyNeisseriaceae.

Morphology. Gonococci are morphologically similar to meningococci. The organism is a paired, bean-shaped coccus, measuring 0.6-1 mcm in diameter. It is Gram-negative and occurs inside and outside of the cells. Neither spores nor flagella are formed. Under the electron microscope a cell wall, 0.3-0.4 mcm in thickness, surrounding the gonococci is visible. The G+C content in DNA is 49.5 to 49.6 per cent.

Drawing of doughnut-shaped diplococci of Neisseria gonorrhoeae as they sometimes appear under the microscope.

Pleomorphism of the gonococci is a characteristic property. They readily change their form under the effect of medicines, losing their typical shape, and growing larger, sometimes turning Gram-positive, and are found outside the cells.

In chronic forms of the disease autolysis of the gonococci takes place with formation of variant types (Asch types). Usually gonococcal cells varying in size and shape are formed. The tendency toward morphological variability among the gonococci should be taken into account in laboratory diagnosis. L-forms occur under the effect of penicillin.

Cultivation. The gonococcus is an aerobe or facultative anaerobe which does not grow on ordinary media, but can be cultivated readily on media containing human proteins (blood, serum, ascitic fluid) when the pH of the media is in the range of 7.2-7.6. The optimum temperature for growth is 37° C, and the organism does not grow at 25 and 42° C. It also requires an adequate degree of humidity. Ascitic agar, ascitic broth, and egg-yolk medium are the most suitable media. On solid media gonococci produce transparent, circular colonies, 1-3 mm in diameter. Cultures of gonococci form a pellicle in ascitic broth, which in a few days settles at the bottom of the test tube.

Fermentative properties. The gonococcus possesses low biochemical activity and no proteolytic activity. It ferments only glucose, with acid formation.

Toxin production. The gonococci do not produce soluble toxin (exotoxin) An endotoxin is released as a result of disintegration of the bacterial cells. This endotoxin is also toxic for experimental animals.

Antigenic structure and classification. The antigenic structure of gonococci is associated with the protein (O-antigen) and polysaccharide (K-antigen) fractions. No group specific or international types of gonococci have been revealed. Gonococci and meningococci share some antigens in common.

Resistance. Gonococci are very sensitive to cooling. They do not survive drying, although they may live as long as 24 hours in a thick layer of pus or on moist objects. They are killed in 5 minutes at a temperature of 56 °C, and in several minutes after treatment with a 1 : 1000 silver nitrate solution or 1 per cent phenol.

Pathogenicity for animals. Gonococcus is not pathogenic for animals. An intraperitoneal injection of the culture into white mice results in fatal intoxication but does not produce typical gonorrhoea.

Pathogenesis and diseases in man. Patients with gonorrhoea are sources of the infection. The disease is transmitted via the genital organs and by articles of domestic use (diapers, sponges, towels, etc). The causative agent enters the body via the urethral mucous membranes and, in women, via the urethra and cervix uteri. Gonorrhoea is accompanied by acute pyogenic inflammation of the urethra, cervix uteri, and glands in the lower genital tract. Often, however, the upper genito-urinary organs are also involved. Inflammations of the uterus, uterine tubes, and ovaries occur in women, vulvovaginitis occurs in girls, and inflammation of the seminal vesicles and prostata in men. The disease may assume a chronic course. From the cervix uteri the gonococci can penetrate into the rectum. Inefficient treatment leads to affections of the joints and endocardium, and to septicaemia. Gonococci and Trichomonas vaginalis are often found at the same time in sick females. The trichomonads contain (in the phagosomes) gonococci protected by membranes against the effect of therapeutic agents. Gonococcus is responsible for gonorrhoeal conjunctivitis and blennorrhea in adults and newborn infants.

Immunity. The disease does not produce insusceptibility and there is no congenital immunity. Antibodies (agglutinins, precipitins, opsonins, and complement-fixing bodies) are present in patients' sera, but they do not protect the body from reinfection and recurrence of symptoms. Phagocytosis in gonorrhoea is incomplete. The phagocytic and humoral immunity produced in gonorrhoea is incapable of providing complete protection, so, in view of this fact, treatment includes measures which increase body reactivity. This is achieved by raising the patient's temperature artificially.

Laboratory diagnosis.

Specimens for microscopic examination are obtained from the discharge of the urethra, vagina, vulva, cervix uteri, prostate, rectal mucous membrane, and conjunctiva. The sperm and urine precipitates and filaments are also studied microscopically, Smears are stained by Gram's method and with methylene blue by Loeffler's method). Microscopy is quite frequently an unreliable diagnostic method since other Gram-negative bacteria, identical to the gonococci, may be present in the material under test. Most specific are the immuno-fluorescence methods (both direct and indirect). In the direct method the organisms under test are exposed to the action of fluorescent antibodies specific to gonococci. In the indirect method, the known organisms (gonococci) are treated with patient's serum. The combination of the antibody with the antigen becomes visible when fluorescent antiserum is added.

If diagnosis cannot be made by microscopic examination, isolation of the culture is carried out. For this purpose the test material (pus, conjunctival discharge, urine precipitate, etc.) is inoculated onto media. The Bordeux-Gengou complement-fixation reaction and the allergic test are employed in chronic and complicated cases of gonorrhea.

Treatment. Patients with gonorrhoea are prescribed antibiotics (bicillin-6, ampicillin, monomycin, kanamycin) and sulphonamides of a prolonged action. Injections of polyvalent vaccine and autovaccine as well as pyrotherapy (introduction of heterologous proteins) are applied in complicated cases.

Improper treatment renders the gonococci drug-resistant, and this may lead to the development of complications and to a chronic course of the disease.

Prophylaxis includes systematic precautions for establishing normal conditions of everyday and family life, health education and improvement of the general cultural and hygienic standards of the population.

In the control of gonorrhoea great importance is assigned to early exposure of sources of infection and contacts and to successful treatment of patients.

The prevention of blennorrhea is effected by introducing one or two drops of a 2 per cent silver nitrate solution into the conjunctival sac of all newborn infants. In certain cases (in prematurely born infants) silver nitrate gives no positive result. Good results are obtained by introducing two drops of a 3 per cent penicillin solution in oil into the conjunctival sac. The gonococci are killed in 15-30 minutes.

In spite of the use of effective antibiotics the incidence of gonorrhoea tends to be on the increase in all countries (Africa, America, South-Eastern Asia, Europe, etc.). The number of complications has also increased: gonococcal ophthalmia of newborn infants (blennorrhea), vulvovaginitis in children, and inflammation of the pelvic organs (salpingitis) and sterility in women. The rise in the incidence of gonorrhoea is caused by social habits (prostitution, homosexualism, etc.), inefficient registration of individuals harbouring the disease, deficient treatment, and the appearance of gonococci resistant to the drugs used.

The WHO expert committee has recommended listing the gonococcal infection among infectious diseases with compulsory registration and making a profound study of the cause of the epidemic character of gonococcal diseases in certain African countries. Stricter blennorrhea control measures, and elaboration of uniform criteria of clinical and laboratory diagnosis, and treatment of gonococcal infection and more efficient methods for determining the sensitivity of circulating gonococci to various drugs are also recommended by the committee.

Additional material about diagnosis of Neisseria diseases

MENINGOCOCCAL INFECTION. Meningococcal infection is caused by meningococci (Neisseria meningitidis). The material to be tested is secretions from the nasal portion of the throat, cerebrospinal fluid, blood, and scrapings from elements of the haemorrhagic rash on the skin.

Cerebrospinal fluid is collected into a sterile tube to be inoculated onto nutrient media or to be promptly sent (without allowing it to cool down) to the laboratory. This requirement is necessitated by the fact that meningococci are very sensitive to temperature fluctuations.

Mucosal secretions in the nasal portion of the throat are collected with a special swab bent at a definite angle. The best results are obtained when the nasopharyngeal mucus is immediately streaked onto solid nutrient media. To achieve the maximal separation of bacterial cells, 2-3 plates with medium are utilized. If the material is to be studied 3-5 hrs after the collection, it is inoculated onto a liquid nutrient medium (casein hydrolysate of fermentative splitting, which contains 1.5 g/1 of amine nitrogen and 250 U/ml of ristomycin) and then placed in a 37 °C water bath. Thereafter, it is streaked onto serum agar and placed into an incubator.

Bacterioscopic examination of cerebrospinal fluid and blood permits detection of the causative agent. If the cerebrospinal fluid looks like pus, smears are prepared without its preliminary treatment whereas in the presence of only mild turbidity the cerebrospinal fluid is centrifuged and the deposit is used to make smears. The latter are stained with aniline dyes (aqueous solution of basic fuchsine, methylene blue) since the Gram staining method is associated with alteration in the formed elements of the cerebrospinal fluid and a large number of artefacts. Meningococci appear as bean-shaped diplococci situated within the leukocyte cytoplasm and touching each other with concave edges. A tender capsule is quite a frequent finding. In meningococcaemia meningococci may be demonstrated in blood smears. A thick-drop (film) preparation is made, stained for 2-3 min with aqueous solution of methylene blue without fixation, washed in tap water, and dried in the air. On a light blue background of the preparation one can see dark blue leukocytes with numerous small dark-blue cocci arranged in clusters, pairs, and singly in and around leukocytes.

Rapid diagnosis is performed by means of gel precipitation, counter-immunoelectrophoresis with group precipitating antisera or radioimmunoassay and based on the detection in the patient's cerebrospinal fluid or blood of the specific meningococcal antigen.

Bacteriological examination. The cerebrospinal fluid or its sediment is cultured simultaneously with conducting bacterioscopic study. The meningococcus grows on special nutrient media containing native protein (serum broth and agar). One can also use Hottinger's agar containing 0.15 per cent of insoluble starch, which does not change the cultural, fermentative, and agglutinating properties of the causative agent. It is preferable that the cerebrospinal fluid be cultured after centrifugation at 3500 X g for five minutes. Some 0.3-0.5 ml of the material is taken from the bottom and 2-3 drops are placed on the surface of heated nutrient medium. The inoculated culture is incubated at 37 °C and in conditions of elevated CO₂ contents. To do it, place onto the lid of a sterile Petri dish a sheet of filter paper soaked with 1.5-2.0 ml of 10 per cent pyrogallic acid and then cover it with a second sheet moistened with 1.5-2.0 ml of 20 per cent solution of sodium hydrocarbonate. The inoculated dish is covered with the lid containing the paper sheets and inverted (lid downward). The remainder of the cerebrospinal fluid is utilized for counter-immunoelectrophoresis.

On the second day of incubation at 37 °C, the growth is studied for its cultural properties. Meningococci form small, round, convex, and transparent colonies. Smears made of these colonies display polymorphic diplococci and tetracocci. The microscopic picture is so diverse that it creates the impression of unpure culture. The colonies are subcultured onto a serum agar slant.

On the third day of investigation, the isolated culture is agglutinated with meningococcal sera.

Prior to the use of sulpha nil amide drugs and antibiotics, it is necessary to determine the serovar of the meningococcus responsible for the disease since treatment is based on specific meningococcal sera. The agglutination test in Noble's modification is currently employed for determining the meningococcal serovar with an epidemiological purpose. Three-drop portions of thick suspension of microorganisms are poured into three test tubes, then three-drop aliquots of undiluted or diluted 1:10 meningococcal serum of A, B, and C serovars are added to them. The mixture is shaken for 2-4 min, then 10-20 drops of isotonic sodium chloride solution are added to each test tube, and the results are read.

To assay the fermentative activity of pure culture, it is transferred to media with lactose, glucose, maltose, sucrose, and fructose. Meningococci ferment glucose and maltose with the production of acid. The culture is also streaked onto a 5 per cent yolk agar and serum agar containing 5 per cent sugar. After a 48-hour incubation, 1 drop of Lugol's solution is put on the surface of the grown colonies. The appearance of brownish staining indicates polysaccharide splitting. Neisseria are identified by the oxidase test which consists in the following. On the colony formed on the serum agar place a drop of the freshly-prepared 1 per cent solution of hydrochloric paradiethylphenylendiamine. As a result, colonies possessing oxidase activity turn pink and then black. Such colonies are transferred to a serum agar for further investigation.

To differentiate between the meningococcus and non-pathogenic Neisseria (Neisseria catarrhalis), the ability of the latter to grow on simple nutrient media and to form colonies at room temperature (22 °C) is utilized.

To demonstrate the meningococcus in the blood, introduce 5-10 ml of blood obtained from a vein under sterile conditions into vials with 50 ml of broth containing 0.1 per cent of agar-agar. Subculture onto a serum agar 24 hours later. The procedures of isolation and identification of the cultures are the same as in the examination of cerebrospinal fluid.

Indirect haemagglutination with erythrocytes sensitized with group-specific polysaccharides is employed for serological diagnosis.

GONOCOCCAL INFECTION. The causative agent of gonorrhoea is the gonococcus (Neisseria gonorrhoeae} which is morphologically similar to the meningococcus. Bacterioscopic, bacteriological, and serological techniques are employed for the diagnosis of this disease.

Bacterioscopic examination is the main method for diagnosing acute gonorrhoea and blennorrhea. The material for examination is taken from the urethra in the following manner: wipe the urethral opening with cotton wool moistened with sterile physiological salt solution, press with your finger onto the posterior wall of the urethra in the outward direction (in females the forefinger is inserted into the vagina for this purpose), and express a drop of pus. The secretion from the prostate is obtained by prostatic massage. The secretion of the cervical mucosa is collected with a swab, following intravaginal introduction of Cusco's speculum. In patients with blennorrhea conjunctival secretion is removed with a loop and spread over a glass slide. The preparation is stained with alkaline solution of methylene blue and with the Gram stain (two smears). Upon microscopic examination gonococci appear as bean-shaped Gram-negative diplococci positioned outside or inside the cells (neutrophilic granulocytes) similar to meningococci.

Gram's staining allows differentiation of the gonococci from other bacteria. To ensure a more distinct outline of the gonococci, smears should be fixed by dimethylsulphoxide (dimexide). Pour dimexide on the smear until it is completely dry and then stain it.

Since the examined material may also contain other Gram-negative bacteria resembling the gonococci, both direct and indirect immunofluorescence methods are employed. In the direct immunofluorescence test the smears are treated with fluorescent antibodies against gonococci, in the indirect one, gonococci and the patient's serum are used. Conjugation between the antibody and the antigen becomes evident when a fluorescent serum against human globulins is added.

Bacteriological examination is carried out when the study of smears reveals either no gonococci or only their atypical, altered forms. In view of extreme sensitivity-of the gonococcus to temperature the material tested should not be transported. Moreover, the gonococcus is very sensitive to disinfectants, so it is advisable that 1 to 2 days before culturing the patients should temporarily discontinue the use of disinfectants and antibacterial drugs.

The material is inoculated immediately after its collection onto plates with a protein-containing meat-peptone agar. Ascitic-free media with casein digest, yeast autolysate, and native cattle serum are widely utilized for this purpose. Inclusion into the nutrient medium of ristomycin and poIymixin M (10 U/ml) significantly enhances gonococcal growth. Prior to inoculation, the nutrient medium should be heated in an incubator. To facilitate better growth of the gonococci, the inoculated plates are placed into an exsiccator with a CO₂ concentration amounting to 10 per cent.

A 24-hour incubation at 37 °C brings about the formation of transparent, with smooth edges, convex, mucoid colonies of the gonococcus, which resemble drops of dew. Pure culture is isolated and identified. Biochemically, the gonococcus shows weak activity and breaks down only glucose with the formation of acid. To determine oxidase activity, the culture is introduced into yolk medium (to 100 ml of protein-containing meat-peptone agar add 1.5 g of glucose, 6 ml of phenol red solution, and 15 ml of egg yolk). Agglutination with specific serum does not always yield positive results because the gonococcus has many serovars and the serum may contain only low titres of the appropriate agglutinins.

Serological diagnosis is resorted to in chronic gonorrhoea when the patient has no discharge, and bacterioscopic and bacteriological examinations are impossible. In such cases the complement-fixation test with the patient's blood serum or indirect immunofluorescence is used. A gonococcal vaccine or a special antigen prepared of killed (by variable methods, with antiformin being the most common one) gonococci is employed as the antigen.

Students’ practical activities

1. To carry out precipitation test with a liquor from a patient with an epidemic cerebrospinal meningitis.

In narrow test-tube pour 0.9 ml of cerebrospinal fluid and carefully, holding under an angle 45⁰, with Pasteur pipette slowly layer on the wall (separately for everyone) 0.1 ml of diagnostic antimeningococcal serum, which precipitated antigens of meningococci. The appearance of precipitate as the white ring on limit of two fluids the response is considered positive.

2. To carry out Bordeux – Gengou complement fixation test for diagnosis of chronic gonorrhea.

The scheme of Bordeux – Gengou complement fixation test

Ingredient, ml	Number of the test tubes
	1	2	3	4	5	6	7	8
Isotonic sodium chloride solution	0,5	0,5	0,5	0,5	0,5	0,5	0,5	0,5
Patient’s serum diluted 1:5	0,5	®	®	®	®	¯	–	0,5
Serum dilution	1:10	1:20	1:40	1:80	1:160	1:320	–	–
Gonococcal diagnosticum	0,5	0,5	0,5	0,5	0,5	0,5	0,5	–
Complement	0,5	0,5	0,5	0,5	0,5	0,5	0,5	0,5
Incubation for 45 min, temperature 37 °C
Hemolytic system (Hemolytic serum and 3 % sheep erythrocytes suspension )	1,0	1,0	1,0	1,0	1,0	1,0	1,0	1,0
Incubation for 30-60 min, temperature 37 °C
Results

In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction characterized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (+++ , ++), positive reaction manifested by the intensification of the liquid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythrocytes collected on the bottom of the tube). If the reaction is negative (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).

The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.

References:

1. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 1982, P197-216, 255-263.

2. Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 76–84.

3. Wesley A.Volk et al. Essentials of Medical Microbiology. Lippincott – Raven Publishers, Inc., Philadelphia–New York.–1995, p. 349-358.

---

---

[1][1] Into distilled water place some nucleic acid (10 mg/ml of DNA), add 1 ml of 0.8 per cent solution of sodium hydroxide to pach 20 ml of water, aiid heat the mixture until it is completely solved. To melted meat-peptone agar, which is cooled to 50 °C, add this solution of DNA (1 nig/nil) (1 ml of DNA solution per 9 ml of the meat-peptone agar). Following sterilization with nowing steam, this medium may be preserved for up to two months. When conducting an experiment, melt the agar medium with DNA, add 0.1 ml of sterile 10 per cent solution of calcium chloride to 10 ml of the medium, stir the resultant mixture, and pour it out onto a layer of well dried nutrient agar in a Petri dish.

[2][2] To 1000 ml of distilled water add 20 g of peptone, 1 g of sodium dihydro-phosphate, 1 ff of potassium dihydrophosphate. 5 g of sodium chloride, 0.1 g of calcium chloride, 0.2 g of magnesium chloride, and 0.8 g of agar-agar (pH 7.0-7.2). Sterilize for 20 min at 115 °C.

[3][3] To 100 ml of melted meat-peptone agar containing 7,5 g of sodium chloride add 20 ml of sterile yolk suspension, mix, and pour into plates. To prepare yolk suspension, remove the yolk from an egg, wash it from the white of the egg, place into a vial with beads, add 200 ml of isotonic saline, and shake Until it is completely mixed. After that keep it in a refrigerator.