Chair of Medical Biology, Microbiology, Virology, and Immunology

Staphylococci. Streptococci. Meningococci and Gonococci

Lecturer Prof. S.I. Klymnyuk

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 (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.

Main characteristics

S. aureus S. epider-midis

S. sapro-phyticus

Plasmacoagulase + — —

Phosphatase + + —

Reductase + + —

Protein A, super-ficial antigen

+ — —

Mannitol + — +

Trehalose + — +

Production of alpha-toxin

+ – –

Resistance to novobiocin

S S R

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.

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.

Enzymes. S aureus can express proteases, a lipase, a deoxyribonuclease (DNase) and a fatty acid modifying enzyme (FAME). The first three probably provide nutrients for the bacteria, and it is unlikely that they have anything but a minor role in pathogenesis. However, 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.

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.

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

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.

 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).

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 secreted into the culture fluid and beta-haemolysin which is released after lysis of the streptococci.

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.

The organisms grow at temperatures 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 pneumoniae

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.

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 beta-haemolytic streptococci which possess M-antigen and produce erythrogenic exotoxin.

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.

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

Cultivation. 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 % CO2.

All media must be warmed to 37 degrees prior to inoculation as the organism is extremely susceptible to temperatures above or below 37 degrees.

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

Toxin production. 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, 29E, and W135. Types A, B, C, Y, and W135 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.

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; (3) performance of the precipitin reaction with the cerebrospinal fluid.

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.

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.

Surface components of N. gonorrhoeae that may play a role in virulence

Designation Location Contribution

Pile Major fimbrial protein

Initial binding to epithelial cells

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 that block bactercidal antibodies against P.I and LOS

Tbp1 and Tbp2

Outer membrane receptors for transferrin

Iron acquisition for growth

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.