Microbiology Virology and Immunology Department Pathogenic cocci Staphylococci

Microbiology, Virology, and Immunology Department Pathogenic cocci Staphylococci and streptococci. Meningococci and gonococci. Lecturer As. Prof. Pokryshko

Staphylococci

Classification. Staphylococci are included in the Firmicutes Bacteria, family Micrococcaceae, genus Staphylococcus. According to the contemporary classification, staphylococci are subdivided into more then 30 species. Among them: S. aureus, S. epidermidis, and S. saprophyticus, S. haemolyticus, S. capitis, S. hominis, S. warneri, S. xylosus etc.

Morphology. Staphylococci are spherical in shape, 0. 8 -1 mcm in diameter, and form irregular clusters resembling bunches of grapes. In smears from cultures and pus the organisms occur in short chains, in pairs, or as single cocci. Large spherical (Lforms) 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.

Electron micrograph showing Staphylococcus aureus morphology.

Staphylococci are Gram-positive organisms which possess no flagella and do not form spores.

Cultivation. Staphylococci are facultative-anaerobes. They grow well on ordinary nutrient media with a p. H 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.

Cultivation. On meat peptone agar Staphylococci produce well defined colonies with smooth edges, measuring from 1 -2 to 2. 5 mm in diameter. 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.

Antigenic structure. 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.

Virulence factors Staphylococci express many cell surface-associated and extracellular proteins that are potential virulence factors. For the majority of diseases caused by this organism, pathogenesis is multifactorial. Thus it is difficult to determine precisely the role of any given factor. This also reflects the inadequacies of many animal models for staphylococcal diseases.

The Virulence factors of Staphylococcus aureus

Protein A. Protein A is a surface protein of S aureus which binds immunoglobulin G molecules by the Fc region. In serum, bacteria will bind Ig. G molecules the wrong way round by this non-immune mechanism. In principle this will disrupt opsonization and phagocytosis. Indeed mutants of S aureus lacking protein A are more efficiently phagocytozed in vitro, and studies with mutants in infection models suggest that protein A enhances virulence.

Leukocidin. S aureus can express a toxin that specifically acts on polymorphonuclear leukocytes. Phagocytosis is an important defense against staphylococcal infection so leukocidin should be a virulence factor.

Membrane Damaging Toxins. a-toxin. The best characterized and most potent membrane-damaging toxin of S aureus is a-toxin. Susceptible cells have a specific receptor for a-toxin which allows low concentrations of toxin to bind, causing small pores through which monovalent cations can pass. At higher concentrations, the toxin reacts non-specifically with membrane lipids, causing larger pores through which divalent cations and small molecules can pass. However, it is doubtful if this is relevant under normal physiological conditions. In humans, platelets and monocytes are particularly sensitive to a-toxin

ß-toxin. ß-toxin is a sphingomyelinase which damages membranes rich in this lipid. The classical test for ß-toxin is lysis of sheep erythrocytes. The majority of human isolates of S aureus do not express ß-toxin. A lysogenic bacteriophage is inserted into the gene that encodes the toxin. This phenomenon is called negative phage conversion. Some of the phages that inactivate the ßtoxin gene carry the determinant for an enterotoxin and staphylokinase.

d-toxin. The d-toxin is a very small peptide toxin produced by most strains of S aureus. It is also produced by S epidermidis and S lugdunensis. The role of d-toxin in disease is unknown. g-toxin and leukocidin. The g-toxin and the leukocidins are two-component protein toxins that damage membranes of susceptible cells. The proteins are expressed separately but act together to damage membranes. There is no evidence that they form multimers prior to insertion into membranes. The g-toxin locus expresses three proteins. The B and C components form a leukotoxin with poor hemolytic activity, whereas the A and B components are hemolytic and weakly leukotoxic.

Superantigens: enterotoxins and toxic shock syndrome toxin. S aureus can express two different types of toxin with superantigen activity, enterotoxins, of which there are six serotypes (A, B, C, D, E and G) and toxic shock syndrome toxin (TSST-1). Enterotoxins cause diarrhea and vomiting when ingested and are responsible for staphylococcal food poisoning. When expressed systemically, enterotoxins can cause toxic shock syndrome (TSS) - indeed enterotoxins B and C cause 50% of non-menstrual TSS.

Epidermolytic (exfoliative) toxin (ET). This toxin causes the scalded skin syndrome in neonates, with widespread blistering and loss of the epidermis and have protease activity. It is not clear how the latter causes epidermal splitting. It is possible that the toxins target a very specific protein which is involved in maintaining the integrity of the epidermis.

Other Extracellular Proteins. Coagulase is an extracellular protein which binds to prothrombin in the host to form a complex called staphylothrombin. The protease activity characteristic of thrombin is activated in the complex, resulting in the conversion of fibrinogen to fibrin. This is the basis of the tube coagulase test, in which a clot is formed in plasma after incubation with the S aureus broth-culture supernatant. Coagulase is a traditional marker for identifying S aureus in the clinical microbiology laboratory.

Staphylokinase. Many strains of S aureus express a plasminogen activator called staphylokinase. The genetic determinant is associated with lysogenic bacteriophages. A complex formed between staphylokinase and plasminogen activates plasmin-like proteolytic activity which causes dissolution of fibrin clots. The mechanism is identical to streptokinase, which is used in medicine to treat patients suffering from coronary thrombosis. As with coagulase there is no evidence that staphylokinase is a virulence factor, although it seems reasonable to imagine that localized fibrinolysis might aid in bacterial spreading.

Enzymes. S aureus can express proteases, a lipase, a deoxyribonuclease (DNase) and a fatty acid modifying enzyme (FAME). The FAME enzyme may be important in abscesses, where it could modify anti-bacterial lipids and prolong bacterial survival. The thermostable DNase is an important diagnostic test for identification of S aureus.

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

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.

Pathogenesis and diseases May cause infection if the skin or mucous membranes are broken or damaged. Staphylococci are responsible for a number of local lesions in humans: humans hidradenitis, abscess, paronychia, blepharitis, furuncle, carbuncle, periostitis, osteomyelitis, folliculitis, sycosis, dermatitis, eczema, chronic pyodermia, peritonitis, meningitis, appendicitis, and cholecystitis. Staphylococcus aureus is considered the most pathogenic species, causing abscesses, boils, carbuncles, acne, impetego, and less commonly, pneumonia, osteomyelitis, endocarditis, cystitis, pyelonephritis, and food poisoning. 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.

Pathogenesis and diseases In some cases staphylococci may give rise to secondary infection in individuals suffering from smallpox, influenza, infection 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.

Pathogenesis and diseases 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 in 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. Intrauterine 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 postinfectional 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).

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.

Identification of Gram Positive Cocci: Staphylococcus p p Contains both pathogenic and nonpathogenic organisms Do not produce endospores, but are resistant to drying (desiccation) Found routinely on the surface of the skin Three major species: 1. 2. 3. p p Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus The three species can be distinguished from each other by various biochemical tests. In this lab we will perform some of these tests and observe the results.

Main characteristics S. aureus S. epider- S. sapromidis phyticus Plasmacoagulase + — — Phosphatase + + — Reductase + + — Protein A, superficial antigen + — — Mannitol + — + Trehalose + — + Production of alpha-toxin + – – Resistance to novobiocin S S R

Chemical and Biochemical Tests p p p The identification of organisms is based on cellular, cultural, and biochemical characteristics All species of Staphylococcus are Gram Positive Cocci (GPC) On nutrient agar they tend to be white (or cream colored), circular, entire, convex colonies. On Sheep Blood Agar Staphylococcus aureus may exhibit hemolysis of the agar in the area around the colonies. Tests to be performed: 1. Catalase test 2. Coagulase test 3. Growth and fermentation on Mannitol Salt Agar 4. Susceptibility to the antibiotic “Novobiocin”

Catalase Test p. The Catalase test determines if the organism produces the enzyme “Catalase”, which breaks down hydrogen peroxide (H 2 O 2) to water and oxygen (O 2). Catalase 2 H 2 O 2 p. Catalase 2 H 2 O + O 2 (g) allows organisms to break down harmful metabolites of aerobic respiration and may be seen in aerobic and facultatively anaerobic organisms. There are other enzymes produced by some organisms to handle other toxic end-products of metabolism, such as superoxide dismutase. Not all organisms produce catalase.

Coagulase Test Pathogenic organisms require mechanisms to help them overcome host defense systems. One mechanism involves coating the bacterial cells in a body substance, such as fibrin, to “hide” the bacterial cells from the immune system. This coating will not trigger an immune response by the host cells. The enzyme coagulase causes fibrin to be deposited on bacterial cells helping them to become “invisible” to the host immune system.

High Salt Tolerance p p p p p Some organisms cannot tolerate a high salt concentrations. Media containing higher than normal salt concentrations will inhibit the growth of these non-salt tolerant organisms. Mannitol salt agar contains a high salt concentration so only salt tolerant organisms will grow on it. Also, Mannitol salt agar contains the sugar Mannitol. Some organisms can utilize this sugar as a food source and will produce acidic by-products from this metabolism. The addition of acid to the medium by the fermentation of Mannitol changes the p. H. If a p. H indicator is present in the medium (such as Phenol red) a color change will occur dependant upon the p. H of the medium (agar or broth). Mannitol Salt Agar contains the p. H indicator “Phenol Red” This p. H indicator is red at neutral p. H (around 7. 0), but turns yellow under acidic conditions.

Antibiotic Susceptibility/Resistance p p p p Antibiotic susceptibility is another test that can be used to identify bacteria. A paper disc impregnated with the antibiotic, in this case Novobiocin, is placed on a lawn of bacteria following inoculation. The antibiotic in the disc diffuses into the surrounding agar. If the bacterial species is susceptible to the antibiotic there is a circle of “no-growth” around the disc where bacterial growth is inhibited by the antibiotic. If the bacteria is resistant to the antibiotic the cells grow right up the antibiotic disc. The bacterial species or strain is reported as being resistant to the antibiotic (R) or susceptible to the antibiotic (S) depending on the observations made. The diameter of the area of “no-growth” around the disc may determine the susceptibility or resistance of the organism to the antibiotic.

Interpretation of Results Catalase p Bubbling indicates a positive test for the presence of the catalase enzyme. Coagulase p Agglutination of the “Test” latex with no agglutination of the “Control” latex is considered a positive (+) test for the presence of this enzyme. All reactions occurring after 20 seconds should be ignored. p Agglutination of the “Test” latex with no agglutination of the “Control” latex is considered a positive (+) test for the presence of this enzyme.

Mannitol Salt Agar p p Two different characteristics of the organism are determined with this agar. The first is the organism’s ability to tolerate a high salt environment. Evidence of growth on the slant indicates the organism can grow in a high salt environment. Organisms that can ferment the sugar Mannitol produce an acid end-product that changes the red p. H indicator (Phenol red) in the media to yellow. Any yellow in the media is considered a positive test for Mannitol fermentation. It is possible to have growth, but no Mannitol fermentation.

Novobiocin Susceptibility p p A zone of growth inhibition 17 mm or less in diameter indicates resistance (R) to Novobiocin. If the zone is greater than 11 mm the organism is susceptible (S) to Novobiocin.

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

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. 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

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.

Cultivation. Streptococci are facultatively aerobic, and there 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 p. H 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. 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.

Cultivation. Some streptococcal strains cause haemolysis on blood agar, 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.

Types of hemolysis The Beta hemolysis:

The alpha hemolysis:

The Gamma ( non hemolytic)

Fermentative properties. Streptococci are nonproteolytic, 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;

Toxin production. (4) erythrogenic toxin produces inflammation in humans who have no antitoxins in their blood; (5) Streptococcus pneumoniae produces alpha-haemolysin secreted into the culture fluid and beta-haemolysin which is released after lysis of the streptococci.

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 (Psubstance). The M-substance is a protein which confers type specificity, virulence, and immunogenicity. The T-substance contains O-, 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 O 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 21 known Streptococcal species cannot be related to any antigenic group. Nine species are of interest for medical microbiology; The haemolytic streptococci, recovered from sick human beings, were subdivided 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.

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.

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. It is assumed that scarlet fever is caused by group A betahaemolytic 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 accompanied by septic and allergic conditions.

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 antiinfectious nature. It is associated with antitoxic and antibacterial factors. The antitoxins neutralize the streptococcal toxin and together with the opsonins facilitate phagocytosis.

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. 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.

Three periods can be distinguished during the development of rheumatic fever: (1) period of acute streptococcal infection and initial sensitization; (2) penod of hyperergic reactions, resulting frominteraction between antigens and antibodies, which are accompanied by pnmary rheumatic polyarthritis or carditis; (3) period of stable allergic reactivity accompanied by pronounced manifestations of parallergy and autosensitization, profound and stable immunogenic disturbances, and relapses. determination of an increase in antistreptolysin, antifibrinolysin, and antihyaluronidase titres and detection of C-reactive protein.

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 penicillinresistant 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 alphastreptococci (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 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.

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 (fig. 1). 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. 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 p. H 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. Microbiologists use a peptone-blood base medium in a moist chamber containing 5 -10 % CO 2. All media must be warmed to 37 degrees prior to inoculation as the organism is extremely susceptible to temperatures above or below 37 degrees. 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. Major toxin of N. meningitidis is its lipooligosaccharide, LOS, and its mechanism is endotoxic. The other important determinant of virulence of N. meningitidis is its antiphagocytic polysaccharide capsule. Fimbriae are factor of virulence

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 Twelve groups of meningococci are distinguished, groups A, B, C, D, H, I, K, L, X, Y, Z, 29 E, and W 135. Types A, B, C, Y, and W 135 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, family Neisseriaceae. Morphology. Gonococci are morphologically similar to meningococci. The organism is a paired, bean-shaped coccus, measuring 0. 6 -1 mcm in diameter. It is Gramnegative 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.

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 p. H 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 (Oantigen) 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.

The bacteria enter the epithelial cells by a process called parasite -directed endocytosis. During endocytosis the membrane of the mucosal cell retracts, pinching off a membrane-bound vacuole that contains the neisseriae; this vacuole is rapidly transported to the base of the cell, where bacteria are released by exocytosis into the subepithelial tissue. The bacteria are not necessarily destroyed within the phagocytic vacuole, but it is not clear whether they replicate in the vacuoles as intracellular parasites. The major porin protein, P. I (Por), in the outer membrane of the bacterium is thought to be the "invasin" that mediates penetration of a host cell. Each N. gonorrhoeae strain expresses only one type of Por; however, the Por of different strains may exhibit antigenic differences.

Neisseria gonorrhoeae can produce one or several outer membrane proteins called Opa (P. II) proteins. These proteins are subject to phase variation and are usually found on cells from colonies possessing a unique opaque phenotype called O+. At any particular time, the bacterium may express zero, one, or several different Opa proteins, and each strain has 10 or more genes for different Opas. Rmp (P. III) is an outer membrane protein found in all strains of N. gonorrhoeae. It does not undergo phase variation and is found in a complex with Por and LOS. It shares partial homology with the Omp. A protein of Escherichia coli. Antibodies to Rmp, induced either by a neisserial infection or by colonization with E. coli, block bactericidal antibodies directed against Por and LOS. In fact, anti-Rmp antibodies may increase susceptibility to infection by N. gonorrhoeae.

During infection, bacterial lipooligosaccharide (LOS) and peptidoglycan are released by autolysis of cells. Both soluble polysaccharides activate the host alternative complement pathway, while LOS also stimulates the production of tumor necrosis factor (TNF) that causes cell damage. Neutrophils are attracted to the site and feed sloppily on the bacteria. For unknown reasons, many gonococci are able to survive inside of the phagocytes, at least until the neutrophils themselves die and release the ingested bacteria.

Neisserial LOS has a profound effect on the virulence and pathogenesis of N. gonorrhoeae. The bacteria can express several antigenic types of LOS and can alter the type of LOS they express by some unknown mechanism. Gonococcal LOS produces mucosal damage in fallopian tube organ cultures and brings about the release of enzymes, such as proteases and phospholipases, that may be important in pathogenesis. Thus, gonococcal LOS appears to have an indirect role in mediating tissue damage. Gonococcal LOS is also involved in the resistance of N. gonorrhoeae to the bactericidal activity of normal human serum. Specific LOS oligosaccharide epitopes are known to be associated with a serum-resistant phenotypes of N. gonorrhoeae.

N. gonorrhoeae can utilize host-derived N-acetylneuraminic acid (sialic acid) to sialylate the oligosaccharide component of its LOS, converting a serum-sensitive organism to a serum-resistant one. Organisms with nonsialylated LOS are more invasive than those with sialylated LOS but organisms with sialylated LOS are more resistant to bactericidal effects of serum. There is also antigenic similarity between neisserial LOS and antigens present on human erthyrocytes. This similarity to "self" may preclude an effective immune response to these LOS antigens. N. gonorrhoeae is highly efficient at utilizing transferrin-bound iron for in vitro growth; many strains can also utilize lactoferrinbound iron. The bacteria bind only human transferrin and lactoferrin. This specificity is thought to be the reason these organisms are exclusively human pathogens.

Strains of N. gonorrhoeae produce two distinct extracellular Ig. A 1 proteases, which cleave the heavy chain of the human immunoglobulin at different points within the hinge region. Split products of Ig. A 1 have been found in the genital secretions of women with gonorrhea, suggesting that the neisserial Ig. A 1 protease is present and active during genital infection. It is thought that the Fab fragments of Ig. A 1 may bind to the bacterial cell surface and block the Fc-mediated functions other immunoglobulins.

Surface components of N. gonorrhoeae that may play a role in virulence Designatio n Location Contribution Pile Major fimbrial protein Initial cells P. II (Opa) Outer membrane protein Contributes to invasion P. I (Por) Outer membrane porin May prevent phagolysosome formation in neutrophils and/or reduce oxidative burst LOS Outer membrane lipooligosaccharide Elicits inflammatory response, triggers release of TNF P. III (Rmp) Outer membrane protein Elicits formation of ineffective antibodies that block bactercidal antibodies against P. I and LOS Tbp 1 Tbp 2 Outer membrane receptors for transferrin Iron acquisition for growth Outer membrane receptor for Iron acquisition for growth Lbp and binding to epithelial

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 immunofluorescence 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.

Laboratory diagnosis. 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.